Transfer of Ku86 RNA antisense decreases the radioresistance of human fibroblasts

Ku86 has been shown to be involved in DNA double-strand break (DSB) repair and radiosensitivity in rodents, but its role in human cells is still under investigation. The purpose of this study was to evaluate the radiosensitiv ity and DSB repair after transfection of a Ku86-antisense in a human fibrob last cell line. Simian virus 40-transformed MRC5V1 human fibroblasts were t ransfected with a vector (pcDNA3) containing a Ku86-antisense cDNA. The mai n endpoints were Ku86 protein level, Ku DNA end-binding and DNA protein kin ase activity, clonogenic survival, and DSB repair kinetics, After transfect ion of the Ku86-antisense, decreased Ku86 protein expression, Ku DNA end-bi nding activity, and DNA protein kinase activity were observed in the unclon ed cellular population. The fibroblasts transfected with the Ku86-antisense showed also a radiosensitive phenotype, with a surviving fraction at 2 Gy of 0.29 compared with 0.75 for the control and 20% of unrepaired DSB observ ed at 24 hours after irradiation compared with 0% for the control. Several clones were also isolated with a decreased level of Ku86 protein, a survivi ng fraction at 2 Gy between 0.05 and 0.40, and 10-20% of unrepaired DSB at 24 hours. This study is the first to show the implication of Ku86 in DSB re pair and in the radiosensitivity of human cells. This investigation strongl y suggests that Ku86 could constitute an appealing target for combining gen e therapy and radiation therapy.