DNA damage during the spindle-assembly checkpoint degrades CDC25A, inhibits cyclin-CDC2 complexes, and reverses cells to interphase.
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.
D000818:Animals; D064007:Ataxia Telangiectasia Mutated Proteins; D042846:CDC2-CDC28 Kinases / Q000378:metabolism*; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D018797:Cell Cycle Proteins; D016923:Cell Death / Q000502:physiology; D002478:Cells, Cultured; D002875:Chromosomes / Q000502:physiology; D003001:Cloning, Molecular; D019925:Cyclin A / Q000378:metabolism; D019926:Cyclin B / Q000378:metabolism; D056744:Cyclin B1; D004249:DNA Damage / Q000502:physiology*; D026942:DNA Topoisomerases / Q000378:metabolism; D004268:DNA-Binding Proteins; D016193:G1 Phase / Q000502:physiology; D016195:G2 Phase / Q000502:physiology; D006367:HeLa Cells; D006657:Histones / Q000378:metabolism*; D006801:Humans; D051379:Mice; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D008941:Spindle Apparatus / Q000378:metabolism*; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism; D025521:Tumor Suppressor Proteins; D020687:cdc25 Phosphatases / Q000378:metabolism*
C534699:CCNB1 protein, human; C534700:Ccnb1 protein, mouse; D018797:Cell Cycle Proteins; D019925:Cyclin A; D019926:Cyclin B; D056744:Cyclin B1; D004268:DNA-Binding Proteins; C468783:H2AX protein, human; D006657:Histones; D016159:Tumor Suppressor Protein p53; D025521:Tumor Suppressor Proteins; C576324:ATM protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; C576325:Atm protein, mouse; D017346:Protein Serine-Threonine Kinases; D042846:CDC2-CDC28 Kinases; C517004:CDC25A protein, human; C517005:Cdc25a protein, mouse; D020687:cdc25 Phosphatases; C505407:CASP3 protein, human; C505409:Casp3 protein, mouse; D053148:Caspase 3; D020169:Caspases; D026942:DNA Topoisomerases