G1 checkpoint is compromised in mouse ESCs due to functional uncoupling of p53-p21Waf1 signaling.

Mouse embryonic stem cells (mESCs) lack of G1 checkpoint despite that irradiation (IR) activates ATM/ATR-mediated DDR signaling pathway. The IR-induced p53 localizes in the nuclei and up-regulates p21/Waf1 transcription but that does not lead to accumulation of p21/Waf1 protein. The negative control of the p21Waf1 expression appears to occur at 2 levels of regulation. First, both p21/Waf1 gene transcription and the p21/Waf1 protein content increase in mESCs treated with histone-deacetylase inhibitors, implying its epigenetic regulation. Second, proteasome inhibitors cause the p21/Waf1 accumulation, indicating that the protein is a subject of proteasome-dependent degradation in ESСs. Then, the dynamics of IR-induced p21Waf1 protein show its accumulation at long-term time points (3 and 5 days) that coincides with an increase in the proportion of G1-phase cells, down-regulation of Oct4 and Nanog pluripotent gene transcription and activation of endoderm-specific genes sox17 and afp. In addition, nutlin-dependent stabilization of p53 in mESC was also accompanied by the accumulation of p21/Waf1 as well as restoration of G1 checkpoint and an onset of differentiation. Thus, the lack of functional p21/Waf1 is indispensable for maintaining self-renewal and pluripotency of mESCs.

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.