The Antiproliferative Activities of Human Interferons

在OVCAR3细胞中，IFNAR1/IFNAR2-RNAi(80-90%下调)或IRF9-RNAi(80%下调)可完全抑制干扰素的抗增殖活性。另一方面，IRF9-RNAi不能抑制干扰素的抗增殖活性。这些结果表明，干扰素通过IFNAR1/IFNAR2传递信号，并利用IRF9在OVCAR3细胞中诱导抗增殖活性。相反，STAT1-RNAi(70%敲除)不抑制干扰素的抗增殖活性，但部分抑制干扰素的抗增殖活性，这表明STAT1蛋白表达的减少(70%下调)对干扰素的抗增殖活性没有直接影响。此外，IFNAR1-RNAi或IRF9-RNAi可抑制TRAIL基因的表达，但STAT1-RNAi不能抑制TRAIL基因的表达，提示TRAIL基因的诱导在诱导干扰素的抗增殖活性中起一定作用。此外，基因芯片分析还显示，IRF9-RNAi对干扰素-2c没有明显的抑制STAT1基因表达的作用。总之，这些发现表明，无论是上调STAT1基因表达还是下调STAT1蛋白表达，都不会影响干扰素在OVCAR3细胞中的抗增殖活性。为了进一步研究IRF9在干扰素抗增殖活性中的作用，用Puno载体在对干扰素-945耐药的A549(人肺癌)和T98G(人多形性胶质瘤)细胞中构建了IRF9基因的稳定表达载体，并在第5天检测其抗增殖活性。在这些细胞中，IRF9的过表达显示出剂量依赖性地增强干扰素的抗增殖活性，但不能增强干扰素的抗增殖活性。因此，IRF9可能在JAK-STAT途径中干扰素的抗增殖活性中起主要作用。然而，JAK-STAT信号下游的其他因素，如TRAIL，可能也是IFN抗增殖活性的关键。 人单核细胞与干扰素的结合及其对肿瘤细胞的影响的分析表明，经干扰素处理的单核细胞对人骨肉瘤细胞显示出增强的抗肿瘤作用，细胞接触对这种作用是重要的。还观察到，I型和II型IFN的组合具有增强的单核细胞抗肿瘤活性。

In OVCAR3 cells, IFNAR1/IFNAR2-RNAi (80-90% knock down) or IRF9-RNAi (80% knockdown) completely inhibited the antiproliferative activity of IFN-α. On the other hand, the IRF9-RNAi demonstrated no inhibition of the antiproliferative activity of IFN-γ. These results suggest that IFN-α signals through IFNAR1/IFNAR2 and utilize IRF9 to elicit the antiproliferative activity in OVCAR3 cells. Conversely, Stat1-RNAi (70%knockdown) demonstrated no inhibition of the antiproliferative activity of IFN-α, but partial inhibition of that of IFN-γ, suggesting that a decrease in constitutive Stat1 protein expression (≤ 70% reduction) have no influence directly on the antiproliferative activity of IFN-α. Furthermore, TRAIL gene expression (related to apoptosis) was inhibited by IFNAR1-RNAi or IRF9-RNAi, but not by Stat1-RNAi following IFN-α treatment, suggesting that induction of TRAIL gene plays a role in eliciting the antiproliferative activity of IFN-α. In addition, the microarray analysis also revealed that IRF9-RNAi demonstrated no significant inhibition of Stat1 gene expression in response to IFN-α2c. Collectively, these findings suggest that neither up-regulation of Stat1 gene expression nor down-regulation of Stat1 protein expression affects the antiproliferative activity of IFN-α in OVCAR3 cells. For further investigation of the role of IRF9 in the antiproliferative activities of IFNs, stable transfectants of the IRF9 gene were constructed using pUNO plasmids in A549 (human lung carcinoma) and T98G (human glioblastoma multiforme) cells that were resistant to IFN-α and then antiproliferative activity was determined on day 5. In these cells, the IRF9 overexpression showed dose-dependent enhancement of the antiproliferative activity of IFN-α, but not of IFN-γ. Thus, IRF9 may have a predominant role in the antiproliferative activity of IFN-α in the JAK-STAT pathway. However, other factors downstream of the JAK-STAT signaling, such as TRAIL, may also be essential for the antiproliferative activities of IFNs. Assays performed to explore how the combination of human monocytes and IFN and their effect on tumor cells showed that IFN-treated monocytes exhibit an enhanced antitumor effect on human osteosarcoma cells and that cell contact is important for this effect. It was also observed that there was enhanced monocyte antitumor activity with a combination of Type I and II IFNs.