Inactivation of DNA-dependent protein kinase leads to spindle disruption and mitotic catastrophe with attenuated checkpoint protein 2 Phosphorylation in response to DNA damage.
DNA-dependent protein kinase catalytic subunit (DNA-PKcs) is well known as a critical component involving the nonhomologous end joining pathway of DNA double-strand breaks repair. Here, we showed another important role of DNA-PKcs in stabilizing spindle formation and preventing mitotic catastrophe in response to DNA damage. Inactivation of DNA-PKcs by small interfering RNA or specific inhibitor NU7026 resulted in an increased outcome of polyploidy after 2-Gy or 4-Gy irradiation. Simultaneously, a high incidence of multinucleated cells and multipolar spindles was detected in DNA-PKcs-deficient cells. Time-lapse video microscopy revealed that depression of DNA-PKcs results in mitotic catastrophe associated with mitotic progression failure in response to DNA damage. Moreover, DNA-PKcs inhibition led to a prolonged G(2)-M arrest and increased the outcome of aberrant spindles and mitotic catastrophe in Ataxia-telangiectasia mutated kinase (ATM)-deficient AT5BIVA cells. We have also revealed the localizations of phosphorylated DNA-PKcs/T2609 at the centrosomes, kinetochores, and midbody during mitosis. We have found that the association of DNA-PKcs and checkpoint kinase 2 (Chk2) is driven by Ku70/80 heterodimer. Inactivation of DNA-PKcs strikingly attenuated the ionizing radiation-induced phosphorylation of Chk2/T68 in both ATM-efficient and ATM-deficient cells. Chk2/p-T68 was also shown to localize at the centrosomes and midbody. These results reveal an important role of DNA-PKcs on stabilizing spindle formation and preventing mitotic catastrophe in response to DNA damage and provide another prospect for understanding the mechanism coupling DNA repair and the regulation of mitotic progression.
Shang
Zeng-Fu
ZF
Department of Radiation Toxicology and Oncology, Beijing Institute of Radiation Medicine, Beijing, PR China.
Huang
Bo
B
Xu
Qin-Zhi
QZ
Zhang
Shi-Meng
SM
Fan
Rong
R
Liu
Xiao-Dan
XD
Wang
Yu
Y
Zhou
Ping-Kun
PK
D064007:Ataxia Telangiectasia Mutated Proteins; D018797:Cell Cycle Proteins / Q000378:metabolism; D018385:Centrosome / Q000201:enzymology; D064447:Checkpoint Kinase 2; D004249:DNA Damage / Q000502:physiology*; D051747:DNA-Activated Protein Kinase / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000172:deficiency / Q000378:metabolism; D004789:Enzyme Activation; D006367:HeLa Cells; D006801:Humans; D008938:Mitosis / Q000235:genetics / Q000502:physiology*; D010766:Phosphorylation; D011123:Polyploidy; D017346:Protein Serine-Threonine Kinases / Q000172:deficiency / Q000378:metabolism*; D008941:Spindle Apparatus / Q000201:enzymology*; D025521:Tumor Suppressor Proteins / Q000172:deficiency / Q000378:metabolism
D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D025521:Tumor Suppressor Proteins; D064447:Checkpoint Kinase 2; C576324:ATM protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; C578634:CHEK2 protein, human; D051747:DNA-Activated Protein Kinase; D017346:Protein Serine-Threonine Kinases