An antisense construct of full-length human RAD50 cDNA confers sensitivity to ionizing radiation and alkylating agents on human cell lines.

In Saccharomyces cerevisiae, Rad50 is reported to participate in the repair of double-stranded DNA breaks, and most rad50 mutants are unable to repair gamma-ray-induced DNA damage. In this study, we examined whether human RAD50 is involved in the repair of DNA damage induced by gamma radiation, radiomimetic alkylating agents, or UVB radiation in cultured human cells. Because homozygous null RAD50 mutant cells could not be isolated, human 293 embryonic kidney cells and A431 epithelial tumor cells were transfected with antisense RAD50 cDNA to obtain viable cell lines which expressed reduced RAD50. Selected individual clones were subjected to PCR-Southern and Western blot analyses to confirm the integrity of the antisense RAD50 construct and the reduced RAD50 expression levels. The cells engineered to express reduced RAD50 levels showed significantly increased sensitivity to gamma radiation, mitomycin C and methylmethane sulfonate compared with control cells that were transfected with the vector alone. However, there were no differences in viability of cells with reduced RAD50 levels and control cells treated with UVB radiation. These results indicate that human RAD50 is involved in the repair of DNA damage induced by gamma radiation and alkylating agents in mammalian cells and suggest the possible application of antisense RAD50 cDNA transfection as a radiation sensitizer in radiation oncology.