Transfection of human tumour cells with Mre11 siRNA and the increase in radiation sensitivity and the reduction in heat-induced radiosensitization

Double-strand DNA breaks (DSBs) are potentially lethal DNA lesions induced by ionizing radiation. In eukaryotes, DSBs can be repaired by homologous recombination (HR) or non-homologous end-joining (NHEJ). DNA repair protein Mre11 participates in both the NHEJ and HR DNA repair pathways. Hyperthermia has been used clinically as a radiosensitizer. However, the mechanisms by which radiosensitization is induced by hyperthermia, especially moderate hyperthermia (41degreesC) are not fully understood. Previous studies suggest that 41degreesC reduces the nuclear Mre11 protein level in a manner that correlates with heat-induced changes in radiation sensitivity. Therefore, siRNA technology was used in the present study to reduce Mre11 gene expression to determine if reduced Mre11 protein levels induced radiosensitization and if such radiosensitization is similar to that induced by moderate hyperthermia. The results show that (1) the cellular level of the Mre11 protein was reduced about 60 +/- 18% by a 24-h treatment with siRNA. Results from the Mre11 protein turnover assay showed a half-life of 11.6 +/- 0.5 h for the Mre11 protein, which is consistent with reduction in protein level in 24 h after Mre11 siRNA treatment assuming a delay of 4-8 h to reduce RNA levels. After 48 h in siRNA, cellular Mre11 protein levels increased to approximately pretreatment levels. NSY cells were sensitized to ionizing radiation after 24 h of treatment with Mre11 siRNA. Two hours at 41degreesC did not increase the radiation sensitivity of cells with a reduced Mre11 protein level following a 24-h siRNA treatment. These data support the conclusion that the DSB repair protein, Mre11, appears to be a target for radiosensitization by moderate hyperthermia.