Development of gene-mediated radiotherapy for tumors

Rad51是同源重组修复途径的关键组分。先前用反义寡核苷酸进行的实验，旨在下调Rad51基因的表达，结果导致小鼠胶质瘤细胞系在高剂量辐射后的放射敏感性增加，并且在辐射前用Rad51反义分子治疗胶质瘤小鼠的存活率增加。作为我们努力设计提高放射治疗有效性策略的一部分，我们探索了在基因治疗背景下使用RNAi（RNA干扰）作为降低小鼠畸胎癌F9细胞中Rad51表达的手段。Rad51 siRNA（短干扰RNA）转染后，细胞培养2天，然后用2 Gy剂量的x射线照射。用MTT法分析细胞的放射敏感性。Rad51 siRNA转染细胞中Rad51蛋白的表达量约为对照细胞表达量的30-40%。与模拟转染的细胞相比，转染了Rad51 siRNA的细胞对x射线照射的敏感性明显增加。此外，用抗肿瘤药物顺铂治疗细胞时，也观察到Rad51 siRNA转染的增敏效果，顺铂结合并破坏DNA。将细胞毒性降低50%的顺铂浓度从0.4μM变为0.2μM。x射线照射与顺铂同时照射对F9细胞的生长抑制具有叠加效应。这些结果表明Rad51在细胞对辐射和抗癌药物顺铂的反应中起关键作用，并表明Rad51靶向RNAi在肿瘤放射增敏中的潜在应用。

Rad51 is a key component of the homologous recombination repair pathway. Previous experiments with the antisense oligonucleotides, designed to down-regulate Rad51 gene expression, resulted in leading to increased radiosensitivity of mouse glioma cell lines after high doses of radiation and an increased survival rate for glioma-bearing mice treated with the Rad51 antisense molecule prior to irradiation. As part of our efforts to devise strategies for enhancing the effectiveness of radiation therapy, we explored the use of RNAi(RNA interference) as a means of attenuating Rad51 expression in mouse teratocarcinoraa F9 cells in a gene therapy context. After transfection of the Rad51 siRNA(short interfering RNA), cells were cultured for two days, then irradiated by X-ray at a dose of 2 Gy. Radiosensitivity of the cells was analyzed,by MTT assay. The amount of Rad51 protein in Rad51 siRNA transfected cells was shown to approximately 30-40% of the level s expressed in control cells. The increased sensitivity of the cells to X-ray irradiation was evident when the cells were transfected with Rad51 siRNA, compared to mock-transfected cells. Furthermore, the effect of sensitization with Rad51 siRNA transfection was also observed when the cells were treated with an anti-tumor drug, cisplatin, which bind and break DNA. The concentration of cisplatin, which reduced cytotoxysicity to 50%, became 0.2μM from 0.4μM. Simultaneous exposure of the cells to X-ray irradiation and cisplatin exerted additive effect to the growth inhibition of F9 cells.These results show the critical role of Rad51 in cellular responses to radiation as well as an anti-cancer drug, cisplatin, and indicate the potential use of Rad51-targeted RNAi in tumor radiosensitization.