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Vol. 14, Issue 10, 3989-4002, October 2003

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DNA Damage during the Spindle-Assembly Checkpoint Degrades CDC25A, Inhibits Cyclin–CDC2 Complexes, and Reverses Cells to Interphase

Jeremy P.H. Chow ^* , Wai Yi Siu ^* , Tsz Kan Fung ^*, Wan Mui Chan ^*, Anita Lau ^*, Talha Arooz ^*, Chuen-Pei Ng ^*, Katsumi Yamashita , and Randy Y.C. Poon ^* 

^* Department of Biochemistry, Hong Kong University of Science and Technology, Clear Water Bay, Hong Kong; Division of Life Science, Graduate School of Natural Science and Technology, Kanazawa University, Kakuma-machi, Kanazawa 920-1192, Japan

Submitted March 23, 2003; Revised June 13, 2003; Accepted June 17, 2003
Monitoring Editor: Douglas Koshland

Cell cycle checkpoints that monitor DNA damage and spindle assembly are essential for the maintenance of genetic integrity, and drugs that target these checkpoints are important chemotherapeutic agents. We have examined how cells respond to DNA damage while the spindle-assembly checkpoint is activated. Single cell electrophoresis and phosphorylation of histone H2AX indicated that several chemotherapeutic agents could induce DNA damage during mitotic block. DNA damage during mitotic block triggered CDC2 inactivation, histone H3 dephosphorylation, and chromosome decondensation. Cells did not progress into G₁ but seemed to retract to a G₂-like state containing 4N DNA content, with stabilized cyclin A and cyclin B1 binding to Thr14/Tyr15-phosphorylated CDC2. The loss of mitotic cells was not due to cell death because there was no discernible effect on caspase-3 activation, DNA fragmentation, or viability. Extensive DNA damage during mitotic block inactivated cyclin B1-CDC2 and prevented G₁ entry when the block was removed. The mitotic DNA damage responses were independent of p53 and pRb, but they were dependent on ATM. CDC25A that accumulated during mitosis was rapidly destroyed after DNA damage in an ATM-dependent manner. Ectopic expression of CDC25A or nonphosphorylatable CDC2 effectively inhibited the dephosphorylation of histone H3 after DNA damage. Hence, although spindle disruption and DNA damage provide conflicting signals to regulate CDC2, the negative regulation by the DNA damage checkpoint could overcome the positive regulation by the spindle-assembly checkpoint.

Abbreviations used: ADR, adriamycin; AT, ataxia-telangiectasia; CMP, camptothecin; CIS, cisplatin; ETP, etoposide; NOC, nocodazole; Topo, topoisomerase.

These authors contributed equally to this work.

Corresponding author. E-mail address: bcrandy{at}ust.hk.

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