Activation of DNA damage response signaling in mouse embryonic stem cells.

Mouse embryonic stem cells (mESC) are characterized by high proliferation activity. mESC are highly sensitive to genotoxic stresses and do not undergo G(1)/S checkpoint upon DNA-damage. mESC are supposed to develop sensitive mechanisms to maintain genomic integrity provided by either DNA damage repair or elimination of defected cells by apoptosis. The issue of how mESC recognize the damages and execute DNA repair remains to be studied. We analyzed the kinetics of DNA repair foci marked by antibodies to phosphorylated ATM kinase and histone H2AX (gammaH2AX). We showed that mESC display non-induced DNA single-strand breaks (SSBs), as revealed by comet-assay, and a noticeable background of gammaH2AX staining. Exposure of mESC to gamma-irradiation induced the accumulation of phosphorylated ATM-kinase in the nucleus as well as the formation of additional gammaH2AX foci, which disappeared thereafter. To decrease the background of gammaH2AX staining in control non-irradiated cells, we pre-synchronized mESC at the G(2)/M by low concentration of nocodazol for a short time (6 h). The cells were then irradiated and stained for gammaH2AX. Irradiation induced the formation of gammaH2AX foci both in G(2)-phase and mitotic cells, which evidenced for the active state of DNA-damage signaling at these stages of the cell cycle in mESC. Due to the G(1)/S checkpoint is compromised in mESCs, we checked, whether wild-type p53, a target for ATM kinase, was phosphorylated in response to gamma-irradiation. The p53 was barely phosphorylated in response to irradiation, which correlated with a very low expression of p53-target p21/Waf1 gene. Thus, in spite of the dysfunction of the p53/Waf1 pathway and the lack of cell cycle checkpoints, the mESC are capable of activating ATM and inducing gammaH2AX foci formation, which are necessary for the activation of DNA damage response.