Abstract
The human checkpoint kinase Chk1 has been suggested as a target for cancer treatment. Here, we show that a new inhibitor of Chk1 kinase, CEP-3891, efficiently abrogates both the ionizing radiation (IR)-induced S and G(2) checkpoints. When the checkpoints were abrogated by CEP-3891, the majority (64%) of cells showed fragmented nuclei at 24 hours after IR (6 Gy). The formation of nuclear fragmentation in IR-treated human cancer cells was directly visualized by time-lapse video microscopy of U2-OS cells expressing a green fluorescent protein-tagged histone H2B protein. Nuclear fragmentation occurred as a result of defective chromosome segregation when irradiated cells entered their first mitosis, either prematurely without S and G(2) checkpoint arrest in the presence of CEP-3891 or after a prolonged S and G(2) checkpoint arrest in the absence of CEP-3891. The nuclear fragmentation was clearly distinguishable from apoptosis because caspase activity and nuclear condensation were not induced. Finally, CEP-3891 not only accelerated IR-induced nuclear fragmentation, it also increased the overall cell killing after IR as measured in clonogenic survival assays. These results demonstrate that transient Chk1 inhibition by CEP-3891 allows premature mitotic entry of irradiated cells, thereby leading to accelerated onset of mitotic nuclear fragmentation and increased cell death.
Highlights
In response to DNA-damaging agents such as ionizing radiation (IR), cells activate cell cycle checkpoints to delay cell cycle progression
We show that CEP-3891 efficiently abrogates the IR-induced S and G2 checkpoints, which results in an accelerated onset of nuclear fragmentation when IR-treated cells progress prematurely through mitosis
When nocodazole was added to the culture medium, the CEP-3891–treated and CEP-3891 ϩ 6 Gy-treated cells accumulated at mitosis, demonstrating that CEP3891–treated cells were cycling over the time course of this experiment
Summary
In response to DNA-damaging agents such as ionizing radiation (IR), cells activate cell cycle checkpoints to delay cell cycle progression. Much progress has been made toward understanding the signaling cascades responsible for induction of these checkpoints. Recent data showed that human Chk is required for both the IR-induced rapid S and G2 checkpoints [1,2,3,4]. We and others found that Chk controls the S-phase checkpoint by directly phosphorylating Cdc25A [3, 4], a human phosphatase that activates the S-phase promoting cyclin-dependent kinase (Cdk) 2 [7]. When Chk is inhibited, the Cdc25A phosphatase and, Cdk kinase activity are not down-regulated after IR, and the S-phase checkpoint is impaired [3, 4]. Chk is believed to control the G2 checkpoint via phosphorylation-mediated negative regulation of Cdc25A, Cdc25C, and Cdc25B [4, 8]. All three members of the Cdc family of phosphatases contribute to activation of the M phase promoting Cdk (9 –11)
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
