WORTMANNIN IS A POTENT INHIBITOR OF DNA DOUBLE-STRAND BREAK BUT NOT SINGLE-STRAND BREAK REPAIR IN CHINESE-HAMSTER OVARY CELLS

Wortmannin, an inhibitor of p110 PI 3-kinase, also inhibits DNA-depend ent protein kinase, which is known to mediate DNA double strand break repair, It was recently demonstrated that wortmannin sensitized cells to ionizing radiation (IR) (Price and Youmell, Cancer Res., 56, 246-25 0, 1996). Wortmannin was used to determine if the potentiation of IR-i nduced cytotoxicity in Chinese hamster ovary cells could be accounted for by an inhibition of DNA double strand break (DSB) repair, Wortmann in, at concentrations which were non-toxic per se (5 and 20 mu M), inc reased IR cytotoxicity with dose enhancement factors at 10% survival o f 2.7+/-0.28 (5 mu M) and 5.3+/-0.86 (20 mu M). The effects of wortman nin on DSB levels were assessed by neutral elution, The effects of wor tmannin on the kinetics of DSB repair were evaluated over a 3 h time c ourse, Wortmannin (50 mu M) completely inhibited DSB repair over this period, without having any effect on DSB levels itself, The concentrat ion-dependent effects of wortmannin on DSB levels showed that inhibiti on of DSB repair was significant at 1 mu M, and near-maximal at 20 mu M. In marked contrast, it exerted no effect on the kinetics of single strand break (SSB) repair as assessed by alkaline elution, even at con centrations as high as 50 mu M. There was an excellent correlation bet ween the concentration-dependence and exposure time of wortmannin requ ired to enhance IR cytotoxicity and inhibit DSB repair, These data imp licate inhibition of DNA-dependent protein kinase, and the consequent inhibition of DSB repair, as the mechanism whereby wortmannin potentia tes the cytotoxicity of IR.