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.

Department of Cell Cycle and Cancer and Apoptosis Laboratory Institute of Cancer Biology Danish Cancer Society Copenhagen Denmark