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

Abstract

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.

Downregulation of Fer induces PP1 activation and cell-cycle arrest in malignant cells

“…Moreover, simultaneous knockdown of these two key cell-cycle regulators in PC3 cells led to the dephosphorylation of Ser795 in pRB, but did not profoundly affect Thr821, which is a preferential target for CDK2 (Zarkowska and Mittnacht, 1997). The unchanged phosphorylation level of Thr821 in CDK4/ CDK2-depleted PC3 cells, and of Ser780, Ser807 and Ser811 in CDK4/CDK2-depleted MDA-MB-231 cells, could result from the activity of the CDK1/cyclin A complex, which would compensate for the lack of CDK2/cyclin E and CDK2/cyclin A kinases in these treated cells (Chow et al, 2003).…”

An ATM- and ATR-dependent checkpoint inactivates spindle assembly by targeting CEP63

“…In agreement, there is growing evidence for Plk1 and Chk2 interaction in this mitotic checkpoint response (Tsvetkov et al, 2003;Seo et al, 2003;Jang et al, 2007). A report by Chow et al showed exposure of mitotic cells to DNA damage leads to ATM dependent destruction of Cdc25A phosphatase, inactivatation of Cdk1-Cyclin B and cell cycle reversal into G2 phase (Chow et al, 2003). An alternative mitotic DNA damage checkpoint mechanism was devised from studies in Drosophila embryos in which ATM (Mei-41 in the fly) induction is thought to cause a mitotic delay by stabilising Cyclin A, which prevents anaphase onset (Su and Jaklevic, 2001;Laurencon et al, 2003).…”

Generation of an indestructible cyclin B1 by caspase-6-dependent cleavage during mitotic catastrophe

“…To enrich the transfected cells, cells were subjected to a transient selection with blasticidin as outlined earlier (Fung et al, 2007). Double thymidine synchronization (Arooz et al, 2000), determination of mitotic index (Chow et al, 2003a), transfection (Chan et al, 2008b) and preparation of cellfree extracts were performed as described. Live cell imaging analysis was preformed as described (Chan et al, 2008a); images were taken at 5 min per frame.…”