Complex Relationship Between Chk1and P53for Determining Cellular Responses To Induced DNA Damage In HeLa Cells

Chkl is an important kinase involved in the S phase DNA damage checkpoint. After being activated by DNA double-strand breaks, Chkl can phosphorylate Cdc25phosphatases, consequently block cyclin-dependent kinases activation and cause cell cycle arrest. Tumor suppressor p53is another key factor in DNA damage checkpoint pathways. There have been several studies suggesting that p53also plays an important role in S phase DNA damage checkpoint. In this study, we investigated the relationship between Chkl and p53in S phase DNA damage checkpoint and how they divide the work as well as cooperate with each other.HeLa cells, which are infected with HPV18virus, were chosen as our study model. Because HPV18E6protein binds to p53and targets it for ubiquitin-mediated degradation, the p53pathway is not functional in HeLa cells.18E6siRNA was applied to inhibit the expression of HPV18E6and restore the expression and function of p53in HeLa cells. We also combined siRNA technology with synchronized cells and got the S phase HeLa cells with functional p53, building a foundation for our following researches.Here, in MMS-treated S-phase HeLa cells, cyclinB1expression was up-regulated in p53-deficient cells when the phosphorylation of Chkl was inhibited by UCN-01, a specific Chkl inhibitor. Consequently, this led to a significant increase in the number of abnormal mitosis. However, under the same treatment conditions, HeLa cells with functional p53did not show an obvious increase in the expression level of cyclinB1, suggesting that p53prevents cyclinBl re-accumulation generated by UCN-01-induced inhibition of Chkl activity in MMS-induced S-phase arrest cells. Meanwhile, under the Chkl-inhibition condition, the restoration of functional p53did not allow the MMS-arrested HeLa cells into abnormal mitosis, but significantly promoted apoptosis of MMS-arrested HeLa cells. These results suggest that functional p53in the p53-proficient cells could play an assistant role in S-phase DNA damage checkpoint to prevent aberrant mitosis through the induction of apoptosis when the function of Chkl is impaired in DNA-damaged S-phase cells.Furthermore, we observed that the Chkl activation protected the MMS-arrested HeLa cells treated with18E6siRNA transfection from p53-induced apoptosis, and the cleaved caspase-3was not detected in the p53-proficent MMS-treated cells under the Chkl-activation condition. However, this activated capase-3was detected in the p53-proficent MMS-treated cells in the presence of UCN-01, accompanying with apoptosis. While, both mRNA level and protein level of Bax were up-regulated in p53-proficient HeLa cells arrested in S-phase by the MMS treatment either in the presence or absence of UCN-01, suggesting that the Bax up-regulation induced by p53is Chkl-independent. Taken together, these results suggest that Chkl inhibits p53-induced caspase-3-dependent apoptosis but did not suppress p53-mediated the Bax up-regulation.Further study showed that Chkl activation suppressed the down-regulation of Bcl-2in protein level in p53-proficient HeLa cells arrested in S-phase by the MMS treatment. Meanwhile, Chkl activation also prevented the down-regulation of XIAP in mRNA level which is p53-dependent and maintained the up-regulation of XIAP protein levels at the translational level, thereby inhibited the cytochrome c release from mitochondria and the activation of caspase-3after MMS treatment. These results suggest that Chkl inhibits p53-induced caspase-3-dependent apoptosis through maintaining the high levels of Bcl-2and XIAP to suppress the apoptotic signal of Bax.In conclusion, Chkl plays the major role in the S-phase DNA damage checkpoint to prevent aberrant mitosis by induction of cell-cycle arrest and to maintain the cell viability by inhibition of p53-induced apoptosis in mammalian cells in response to DNA damage stress. When Chkl is inhibited in the S-phase arrested mammalian cells with damaged DNA, p53could play an assistant role to inhibit aberrant mitosis by induction of caspase-3-dependent apoptosis.