Development of gene-mediated radiotherapy for tumors

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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.