To explore effects of DNA damage on cell-cycle progression in p53-deficient tumor cells, synchronized HeLa cells at G1, S and G2/M phases were treated with methyl methanesulfnate (MMS). The results showed that the MMS treatment resulted in the cell-cycle arrest or delay in all 3 phases, while the S-phase cells were the most sensitive to MMS. Further studies demonstrated that ATM-Chk2 and p38 MAPK signaling pathways were activated in all 3 phases when the cells were treated with MMS; whereas Chk1 was activated only in S phase under the drug treatment, indicating that Chk1 specifically participated in S-phase checkpoints. To analyze the role of Chk1 in S-phase checkpoints, we administered a specific Chk1 inhibitor, UCN-01, to the S-phase cells. The results showed that the S-phase cells treated with MMS+UCN-01 could enter aberrant mitosis without finishing DNA replication, indicating that Chk1 mainly functions in the DNA damage checkpoint rather than in the replication checkpoint. In addition, MMS treatment alone inhibited the accumulation of cyclin B1, a key component of M-phase CDK-cyclin complex, in the S-phase cells, whereas the inhibition of Chk1 activation resulted in the accumulation of cyclin B1 in the MMS-treated S-phase cells. This observation further supports the view that DNA-damaged S-phase cells enter abnormal mitosis when Chk1 activation is inhibited. Our results demonstrate that Chk1 is a specific kinase that plays an important role in the MMS-induced S-phase DNA damage checkpoint. As p53 is not involved in this process, Chk1 may be a potential target for p53-deficient tumor therapy.
microRNAs are single-stranded,non-coding RNAs that regulate gene expression.The microRNA-16 family has been reported to be involved in cell-cycle regulation,which could also downregulate expression of multiple pro-proliferation genes.The present results demonstrated that miR-16 expression in HeLa cells increased when the cells were arrested during S-phase under methyl methanesulfate (MMS) treatment.This further resulted in downregulation of a target protein CDC25A,whereas miR-16 expression did not increase when HeLa cells were arrested during the MMS-treated G0/G1 or G2/M phase.Furthermore,when HeLa cells were arrested during S-phase with hydroxyurea treatment,miR-16 expression did not increase.These results suggest that expression levels of microRNAs in mammalian cells are delicately regulated under variable cellular conditions.
