Regulation of cell cycle checkpoints that monitor DNA damage and DNA replication block.

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Chkl regulates both DNA damage and replication checkpoints in mammals. Although Chk1 is crucial for embryonic cell survival, its function in normal cell cycle remains largely unknown. With the use of a Cre/lox-conditional mouse line, we demonstrate here the essential and indispensable role of Chk1 in regulation of S-phase progression and prevention of mitotic initiation in embryonic cell cycle. An analysis using laser scanning cytometer revealed that Chk1 deficiency resulted in premature mitosis with the premature chromosome condensation and the phosphorylation of histone H3 at serine10 due to abnormal activation of cyclin B-Cdc2, and led to the activation of caspases 3 and 9 triggered by cytoplasmic release of cytochrome c. This deficiency was associated with foci formation by the phosphorylated histone, H2AX (gH2AX), specifically at S phase. Ectopic expression of Cdc2AF, a mutant that cannot be phosphorylated at inhibitory sites, also induced premature mitosis and foci formation by gH2AX at S phase in both ES cells and HCT116 cells. Inhibition of ATM and ATR with caffeine protected against cell death from premature mitosis. p53-deficient cells were highly resistant to lethality from premature mitosis as well. Our results therefore suggest that ATM/ATR-p53 is required for mitotic catastrophe that eliminates cells escaping Chk1-dependent mitotic regulation. Loss of this function might be important in mammalian tumorigenesis.