Activation of DNA damage response signaling in mouse embryonic stem cells

Abstract

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 (γH2AX). We showed that mESC display non-induced DNA single-strand breaks (SSBs), as revealed by comet-assay, and a noticeable background of γH2AX staining. Exposure of mESC to γ-irradiation induced the accumulation of phosphorylated ATM-kinase in the nucleus as well as the formation of additional γH2AX foci, which disappeared thereafter. To decrease the background of γH2AX 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 γH2AX. Irradiation induced the formation of γH2AX 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 γ-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 γH2AX foci formation, which are necessary for the activation of DNA damage response.

Homologous Recombination Conserves DNA Sequence Integrity Throughout the Cell Cycle in Embryonic Stem Cells

“…However, mammalian ESC exhibit the robust induction of a DNA damage response that facilitates the elimination of damaged cells by apoptosis [4,37,44,50]. Consistent with previous reports [43], our results demonstrate that DNA damage response mechanisms in mESC include the recruitment of DNA repair components and the activation of gH2AX foci. Moreover, our results indicate that an additional mechanism for the maintenance of mESC genomic integrity relies on the increased activation of HR throughout the cell cycle, in marked contrast to fibroblasts.…”

“…1B). Previous studies in mESC and in mouse embryonic teratocarcinoma cells (mECC) also showed a high proportion of noninduced single and DSBs [43]. Both mESC and mECC are very rapidly dividing cells with similar cell cycle profiles and cell cycle duration, estimated at 10-12 h. It has been suggested that the high levels of DNA damage observed in mECC may be due to their rapid replication rate, perhaps as a consequence of incomplete maturation of replication forks [44].…”

Cancer stem cells and escape from drug-induced premature senescence in human lung tumor cells: Implications for drug resistance and in vitro drug screening models

“…On the other hand, it has been shown that normal stem cells have only partially active DNA damage checkpoints. 24,32,33 This suggested that CSCs can actually be less prone to undergo irreversible growth arrest after exposure to DNA damaging agents. This, in turn, would result in an ineffective treatment.…”

DNA Repair in Embryonic Stem Cells

“…Notably, mESCs display an elevated basal level of -H2AX, even in the absence of DSBs (Banath et al, 2009;Chuykin et al, 2008). Nevertheless, when H2AX phosphorylation is followed over time, mESCs and differentiated cells show a similar behavior with respect to foci formation and dissolution (Adams et al, 2010a;Chuykin et al, 2008). The reason for the high basal level of phosphorylated H2AX in mESCs is as yet unclear.…”

“…In ESCs, ATM also becomes phosphorylated at serine 1981, relocates to DSBs and phosphorylates the histone www.intechopen.com variant protein H2AX within a few minutes post irradiation (Momcilovic et al, 2009). Notably, mESCs display an elevated basal level of -H2AX, even in the absence of DSBs (Banath et al, 2009;Chuykin et al, 2008). Nevertheless, when H2AX phosphorylation is followed over time, mESCs and differentiated cells show a similar behavior with respect to foci formation and dissolution (Adams et al, 2010a;Chuykin et al, 2008).…”