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hES say G1-yeS!

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Cells utilise multiple DNA damage checkpoints during the cell cycle in order to prevent unwanted genetic changes being passed onto daughter cells. The cell cycle can pause at the G1/S phase checkpoint to repair any DNA damage before DNA replication and cell division, allowing for proper propagation of genetic information. However, a long pause can allow the cells to differentiate and if the DNA damage is beyond repair, the cells can also initiate apoptosis, which mainly occurs in the G2 phase of the cell cycle. Strict regulation of the cell cycle and its checkpoints is of key importance in hESC biology and results from various groups are now beginning to reveal the complexities of this system.

The G1/S phase checkpoint of the cell cycle is active in normal human somatic cells, but previous data had suggested that this important checkpoint did not function in hESC, allowing for rapid progression through the G1 phase (Becker et al, 2006, Becker et al, 2007 and Filion et al, 2009). However, an article in Stem Cells from the lab of AleŇ° Hampl reveals that hESC are indeed able to execute G1/S checkpoint activation, therefore guarding against the accumulation of deleterious genomic changes. In this study, hESCs were UVC irradiated in order to induce DNA damage, and the authors were able to demonstrate cell cycle arrest before DNA synthesis (S phase). Further, it was found that this checkpoint was under the control of CDK2, which was regulated by CDC25A but not the classical p21/p53 pathway.

In detail, their results show that UVC irradiation of hESC (CCTL12 and 14) resulted in fewer cells progressing to S-phase, as compared with controls. This was accompanied by a decrease in CDK2 activity which, along with CCNE (Cyclin E), normally regulates G1/S transit. Whilst a high level of active p53 protein was found in irradiated cells, protein expression of the CDK-inhibitor p21, remained unchanged, suggesting that this pathway does not regulate CDK2 activity. However, levels of CDC25A protein, a phosphatase known to remove inhibitory CDK2 phosphorylation was decreased, suggesting this may be a key regulator of CDK2 activity in the G1/S phase checkpoint in hESC. Further proof of this was shown by siRNA-mediated down-regulation of CDC25A which led to an accumulation of hESC in G1 phase. Cdc25A is itself regulated by phosphorylation, by p38 MAP kinase and the checkpoint kinases CHK1 and 2. Phosphorylation of CDC25A allows for its degradation and analysis using chemical inhibitors and siRNA found that CDC25A levels remained unchanged only when the activity of the checkpoint kinases Chk1 and 2 was eliminated. Further, UVC-irradiated cells under Chk1 and 2 activity suppression passed through the cell cycle normally.

This data shows that hESC do exhibit a G1/S checkpoint and that this is regulated by Chk1/2-Cdc25A-mediated control of CDK2. Previous studies have implicated both Cdc25A (Zhang et al, 2009) and CDK2 (Neganova et al, 2009) as being vitally important to cell cycle regulation in hESC and the results from this study reinforce the role of these proteins in the cell cycle and in the importance of the G1/S phase checkpoint in hESC.

 

References

Becker KA, Ghule PN, Therrien JA et al. Self-renewal of human embryonic stem cells is supported by a shortened G1 cell cycle phase. J Cell Physiol 2006; 209: 883-893.

Becker KA, Stein JL, Lian JB et al. Establishment of histone gene regulation and cell cycle checkpoint control in human embryonic stem cells. J Cell Physiol 2007; 210: 517-526.

Filion TM, Qiao M, Ghule PN et al. Survival responses of human embryonic stem cells to DNA damage. J Cell Physiol 2009; 220: 586-592.

Neganova I, Zhang X, Atkinson S et al. Expression and functional analysis of G1 to S regulatory components reveals an important role for CDK2 in cell cycle regulation in human embryonic stem cells. Oncogene 2009; 28: 20-30.

Zhang X, Neganova I, Przyborski S et al. A role for NANOG in G1 to S transition in human embryonic stem cells through direct binding of CDK6 and Cdc25A. J Cell Biol 2009; 184: 67-82.