Checkpoint kinase 1 (CHK1) is a key mediator that links the machineries that monitor DNA integrity to components of the cell cycle engine. In this work, I have studied the functions and regulation of CHK1, as well as explored the effects of inhibition of CHK1. I found that following genotoxic stresses, phosphorylation of CHK1 on Ser317 and Ser345 was required for checkpoint activation. Moreover, phosphorylation on these two major sites was interdependent. The intramolecular interaction between the N-terminal kinase domain and C-terminal regulatory domain of CHK1 was also investigated. The interaction resulted in the inhibition of the kinase activity of CHK1, and was disrupted by the stress-induced phosphorylation. The cell cycle arrest induced by the auto-activated C-terminally truncate...[ Read more ]

Checkpoint kinase 1 (CHK1) is a key mediator that links the machineries that monitor DNA integrity to components of the cell cycle engine. In this work, I have studied the functions and regulation of CHK1, as well as explored the effects of inhibition of CHK1. I found that following genotoxic stresses, phosphorylation of CHK1 on Ser317 and Ser345 was required for checkpoint activation. Moreover, phosphorylation on these two major sites was interdependent. The intramolecular interaction between the N-terminal kinase domain and C-terminal regulatory domain of CHK1 was also investigated. The interaction resulted in the inhibition of the kinase activity of CHK1, and was disrupted by the stress-induced phosphorylation. The cell cycle arrest induced by the auto-activated C-terminally truncated CHK1 was also investigated. Cells were delayed in early G₁ phase at day 3 after transfection, and were arrested in S phase at day 4. Despite the decrease in expression of the N-terminal kinase domain, the cell cycle arrest could be maintained until day 6 of transfection when significant apoptosis was observed.

To study the effects of CHK1 disruption on the G₂ DNA damage checkpoint, I have tracked the fates of individual cells, in particular focusing on the mitosis immediately after checkpoint abrogation with the CHK1 inhibitor UCN-01. The checkpoint bypass resulted in either mitotic catastrophe or survival into G₁ after defective cytokinesis. Mitotic catastrophe was promoted by an extension of mitosis, as HeLa cells that were trapped in mitosis for longer than ~4 h were unable to recover and exit mitosis. Further supporting this idea, weakening the spindle-assembly checkpoint, by either depleting MAD2 or overexpressing the MAD2 binding protein p31^comet, suppressed mitotic catastrophe. Conversely, delaying of mitotic exit by depleting either p31^comet or CDC20 tipped the balance towards mitotic catastrophe. However, the susceptibility of mitotic catastrophe induced by ionizing radiation and UCN-01 treatment was cell line-specific. To sensitize checkpoint-abrogated cells to mitotic catastrophe, the KIF11 inhibitor monastrol was used to activate the spindle-assembly checkpoint. These studies indicated that more effective mitotic catastrophe could be triggered by ionizing radiation and UCN-01 in conjunction with the inhibition of KIF11. Importantly, similar sensitizing of mitotic catastrophe could be achieved by the same procedure in different cell lines. Taken together, the studies of this thesis reveal the complexity of the regulation CHK1 as well as the potential use of CHK1 in cancer therapies.