Overexpressed human RAD50 exhibits cell death in a p21 (WAF1/CIP1)-dependent manner: Its potential utility in local gene therapy of tumor

Previously, mouse RAD50, one of the mammalian DNA recombination repair gene s, was reported to have limited epitopic homology to p53, Here we report th e functional characteristics of overexpressed human RAD50 (hRAD50), Transie nt transfection of hRAD50 in several cultured cells caused cytotoxicity, We established tetracycline-regulated, stable hRAD50 expression systems in Sa OS-2 cells, which retain mutated p53, and in HeLa cells. After tetracycline withdrawal, cell death and multinucleated giant cells were observed with i ncreased hRAD50 expression, and p21(WAF1/CIP1) but not p53 was increased. T ransient transfection of hRAD50 in HCT116 p21(-/-) cells caused no cytotoxi city, but there was a significantly decreased survival rate in p21(+/+) cel ls. These cytotoxic effects of overexpressed hRAD50 in HeLa, SaOS-2, and HC T116 p21(+/+) cells were partially blocked by pretreatment of cells with N- benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone, a pan-caspase inhibitor, When the hRAD50 expression cDNA was injected intratumorally with liposomes , it regressed or delayed tumor development in the animal model and nitric oxide synthase expression was induced in the tumor tissues that had regress ed. Our results indicate that overexpressed hRAD50 has an antiproliferation activity in vitro and in vivo in a p21-dependent manner.