ATM is upstream of DNA‐PKcs in the activation of DNA damage response by sodium selenite

Abstract

The role of selenium in mitigating tumorigenesis is not fully understood. Selenium in supranutritional doses can induce ROS, which indirectly causes DNA breaks. Previous reports indicate an interplay between Ataxia‐telangiectasia mutated (ATM) and DNA‐dependent protein kinase catalytic subunit (DNA‐PKcs) in the early stages of DNA damage response. We recently found that ATM is required for a selenium‐induced senescence response in non‐cancerous cells at an early stage. Thus, we hypothesize that the selenium‐induced DNA damage response can modify this ATM‐DNA‐PKcs cross‐talk. Phospho‐specific antibodies against ATM and DNA‐PKcs were used to follow the phosphorylation events after selenium treatment in MRC‐5 normal fibroblasts and ATM shRNA and control shRNA U‐2 OS osteosarcoma cells. Results from immunofluorescence analysis showed that selenium treatment induces phosphorylation of DNA‐PKcs at T2647, an ATM‐dependent phosphorylation site, and phosphorylation of DNA‐PKcs at S2056, an autocatalytic site of DNA‐PKcs, in non‐cancerous MRC‐5 cells but not in U‐2 OS cancer cells. Further studies in MRC‐5 cells with an ATM kinase inhibitor, KU55933, showed attenuation of the selenium‐induced DNA‐PKcs phosphorylation at both foci, whereas pre‐treatment with a DNA‐PKcs kinase inhibitor, NU7026, does not prevent ATM phosphorylation at S1981, an event leading to ATM pathway activation. The selenium‐induced DNA‐PKcs phosphorylation at S2056 is decreased in S‐phase cells. Taken together, our results provide the first evidence of ATM and DNA‐PKcs activation as an early tumorigenesis barrier in response to selenium exposure in non‐cancerous cells, and places ATM upstream of DNA‐PKcs in the selenium‐induced DNA damage response process.Grant Funding Source: University of Maryland