Cdk2 Kinase Activity Is Not Abrogated after DNA Damage in Mouse Embryonic Stem Cells.

Abstract

Stem cells have gained special attention for their implication in medicine where stem-cell based therapies promise powerful approach to treat different disorders, and, on the other hand, cancer stem cells have been suggested as important novel targets for the treatment of cancer. To successfully develop new therapies, a deeper understanding of the biology of stem cells is necessary. Embryonic stem (ES) cells are naturally immortalized pluripotent cells derived from early mammalian embryos. ES cells are characterized by unique self-renewal and differentiation abilities, as well as by special features of cell cycle regulation and DNA damage response. In general, when DNA damage occurs, checkpoint pathways are activated, preventing replication of damaged DNA and/or division of cells with damaged DNA. Somatic cells employ checkpoints throughout the whole cell cycle; in ES cells functional checkpoints have been described in S and G2 phases only. In somatic cells the G1/S transition is governed by Cdk2-cyclin E complex. G1-checkpoint mechanisms lead to inhibition of Cdk2 activity via two parallel pathways: Chk1/Chk2-Cdc25A and p53-p21. It has been suggested proteins of these pathways are not functional in ES cells. We aimed to unravel the causes of G1 checkpoint non-functionality in mouse ES (mES) cells. To analyze the events after DNA damage in G1 phase, we synchronized mES cells (lines HM-1 and V6.5) in M phase by nocodazole treatment. After release from nocodazole arrest the cells were gamma-irradiated (γ) in early and late G1 phase. We observed activation of both Chk2-Cdc25A and p53-p21 pathways in mES cells after DNA damage by γ-irradiation. However, FACS cell cycle analysis revealed that after γ-induced DNA damage mES cells did not arrest in G1; instead, cell cycle arrest occurred only at the G2/M boundary. Measurements of Cdk2 kinase activity in γ-irradiated and mock-treated mES cells revealed that although Cdk2-activating phosphatase Cdc25A is degraded after γ-irradiation, Cdk2 activity is not diminished. Since it has been reported earlier that in mES cells Chk2 is mislocalized to centrosomes, we speculated that full function of other cell cycle regulatory proteins might be hampered by aberrant localization as well. Our immunolocalization studies showed that both Cdk2 and its phosphorylated, inactive form (P-Thr14/Tyr15-Cdk2) localize to centrosomes in mES cells. This could, at least partially, influence its accessibility by interacting factors such as Cdc25A and explain the lack of Cdk2 activity downregulation after DNA damage despite activated checkpoint pathways. In conclusion, DNA damage in mES cells (lines HM-1 and V6.5) elicits fast activation of both Chk2-Cdc25A and p53-p21 G1 checkpoint pathways. However, since Cdk2 activity is not reduced after DNA damage, mES cells do not arrest in G1 phase. Other factors than those identified in somatic cells, including aberrant localization of cell cycle regulatory proteins, could play important roles in the regulation of cell cycle progression in mES cells. These factors lead to sustained Cdk2 kinase activity even in the presence of DNA damage.