Inactivation of DNA-Dependent Protein Kinase Leads to Spindle Disruption and Mitotic Catastrophe with Attenuated Checkpoint Protein 2 Phosphorylation in Response to DNA Damage

Abstract

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 ATMefficient 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. Cancer Res; 70(9); 3657-66. ©2010 AACR.

Involvement of DNA-dependent Protein Kinase in Normal Cell Cycle Progression through Mitosis

“…Furthermore, Shang et al (30) recently reported that inactivation of DNA-PKcs resulted in spindle disruption and centrosome instability.…”

Steroidogenic Factor 1 (NR5A1) resides in centrosomes and maintains genomic stability by controlling centrosome homeostasis

“…Our observation of apoptosis and centrosome amplification caused by SF-1 deficiency may explain why Sf-1-null adrenogenital primordial cells die, as centrosome amplification causes aberrant mitosis and subsequent cell cycle arrest or cell death. 13,21 It would be interesting to examine whether the adrenogenital primordial cells of these Sf-1-null mice contain multiple centrosomes before they die. This experiment will not be easy because one has to catch the short time window at around E11.5 when these progenitor cells are dying.…”

Harnessing the complexity of DNA-damage response pathways to improve cancer treatment outcomes

“…Inactivation of DNA-PK increases polyploidy and mitotic catastrophe after irradiation and attenuates Chk2 phosphorylation in an ATM-independent manner (Shang et al, 2010). Despite the ability of DNA-PK inhibitors to potentiate chemo-and radiotherapy in vitro and in vivo, the underlying factors that regulate the contribution of cell cycle arrest and cell death to the overall loss of clonogenicity remain unclear.…”