Department of Cancer Biology and Pharmacology, University of Illinois College of Medicine at Peoria, Peoria, IL 61605, USA.
Int J Oncol. 2010 Apr;36(4):809-16. doi: 10.3892/ijo_00000557.
Stereospecific radiation treatment offers a distinct opportunity for temporal and spatial regulation of gene expression at tumor sites by means of inducible promoters. To this end, a plasmid, pCArG-U2, was constructed by incorporating nine CArG elements (in tandem) of EGR1 gene upstream to uPA and uPAR siRNA oligonucleotides in a pCi-neo vector. Radiation-induced siRNA expression was detected in a meningioma cell line (IOMM-Lee). Immunoblotting and RT-PCR analyses confirmed downregulation of uPA and uPAR. A similar effect was observed in transfected cells followed by H2O2 treatment. Moreover, pre-treatment of transfected cells with N-acetyl L-cysteine blocked the silencing of uPA and uPAR, which further confirmed the oxidative damage-mediated downregulation. Cell proliferation assays and Western blot analysis for apoptotic molecules confirmed cell death in a radiation-inducible fashion. Migration and matrigel invasion assays also revealed a marked decrease in migration and invasion. Immunocytochemistry showed a marked decrease in uPA and uPAR levels in transfected and irradiated cells. H&E staining revealed a decrease in the pre-established tumor volume among the animals treated with pCArG-U2 and radiation. Immunohistochemistry of the brain sections established with intracranial tumors also revealed a marked decrease in uPA and uPAR in a radiation-inducible fashion. Taken together, our data suggest pCArG-U2 as a suitable candidate for radiation-inducible gene therapy.
立体定向放射治疗为通过诱导启动子在肿瘤部位对基因表达进行时空调节提供了一个独特的机会。为此，我们构建了一个质粒 pCArG-U2，该质粒通过在 pCi-neo 载体上将 EGR1 基因的九个 CArG 元件（串联）置于 uPA 和 uPAR siRNA 寡核苷酸的上游，从而实现了 siRNA 的表达诱导。在脑膜瘤细胞系（IOMM-Lee）中检测到放射诱导的 siRNA 表达。免疫印迹和 RT-PCR 分析证实 uPA 和 uPAR 的下调。在转染细胞中观察到类似的效果，随后进行 H2O2 处理。此外，用 N-乙酰-L-半胱氨酸预处理转染细胞可阻断 uPA 和 uPAR 的沉默，这进一步证实了氧化损伤介导的下调。细胞增殖实验和凋亡分子的 Western blot 分析证实了以辐射诱导的方式发生细胞死亡。迁移和基质胶侵袭实验也显示出迁移和侵袭的显著减少。免疫细胞化学显示转染和照射细胞中 uPA 和 uPAR 水平明显降低。H&E 染色显示接受 pCArG-U2 和放射治疗的动物中预先建立的肿瘤体积减少。对颅内肿瘤建立的脑切片进行免疫组织化学分析也显示，uPA 和 uPAR 以辐射诱导的方式显著减少。总之，我们的数据表明 pCArG-U2 是一种适合辐射诱导基因治疗的候选物。
