DNA damage results in cell cycle arrest in G2. Centrosomes also separate in G2, raising the question of whether separation occurs during the DNA damage-induced G2 arrest. Nek2, the mammalian homologue of NIMA, is a cell cycle-regulated serine/threonine protein kinase that regulates centrosome separation during G2. Here we show that damaged cells fail to activate Nek2. Both Nek2 levels and activity are reduced after DNA damage. Radiation inhibits the premature centrosome splitting induced by overexpression of Nek2, indicating that Nek2 is involved in activation of the G2 checkpoint and is not secondary to cell cycle arrest. We confirm using siRNA that centrosome separation and cell growth are impaired in the absence of Nek2. These studies define a previously unreported DNA damage response of inhibition of centrosome separation mechanistically linked to Nek2.

X-irradiation-induced DNA damage perturbs the G 1 , S, and G 2 phases of the cell cycle. The behavior of cells after they have experienced a DNA damage checkpoint delay is poorly characterized. We therefore examined the fates of irradiated tumor cells that have overcome a prolonged G 2 checkpoint delay. Most irradiated cells progressed through mitosis without significant delay, but failed to complete cytokinesis as they remained tethered to each other at the midbody. We observed that the movement of centrioles at the time of cytokinesis was impaired in the irradiated, bridged cells. We attribute the perturbation of centriole dynamics to the presence of chromatin bridges that spanned the daughter cells. The bridged cells exhibited different fates that included death, fusion that formed multinucleated cells, or another round of mitosis with no noticeable cell cycle delays. The presence of ;H2AX foci in the bridge as well as in the separated nuclei indicated that cells were proliferating despite the presence of DNA damage. It seems that DNA damage checkpoints were not reactivated in cells that overrode a prolonged G 2 delay. Cells deficient in ATM, H2AX, XRCC3, or ligase 4 exhibited a higher frequency of radiation-induced bridges than controls, suggesting that the DNA bridges resulted from inadequate DNA repair. These data show a previously unappreciated cytologic hallmark of DNA damage in dividing cells. Chromatin bridges that interfere with cytokinesis are likely to contribute to the replication failure and clonogenic death of cells exposed to irradiation. [Cancer Res 2008;68(10):3724-32]

Two splice variants of Nek2 kinase, a member of the NIMA-related family, have been identified as Nek2A and Nek2B. Nek2A regulates centrosome disjunction, spindle formation checkpoint signaling, and faithful chromosome segregation. A specific role for Nek2B has not yet been identified. Here, we have examined the distinct roles of Nek2A and Nek2B using timelapse video microscopy to follow the fate of cells progressing through the cell cycle in the absence of either Nek2A or Nek2B. We show that the down-regulation of Nek2B leads to a mitotic delay in the majority of cells. Upon exiting mitosis, cells exhibit mitotic defects such as the formation of multinucleated cells. Such phenotypes are not observed in cells that exit mitosis in the absence of Nek2A. These observations suggest that Nek2B may be required for the execution of mitotic exit.

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Activation of the G 2 /M cell cycle checkpoint by DNA damage prevents cells from entering mitosis. Centrosome separation is initiated in G 2 phase and completed in M phase. This critical process for cell division is targeted by G 2 /M checkpoint. Here we show that Plk1 signaling plays an important role in regulation of centrosome separation after DNA damage. Constitutively active Plk1 overrides the inhibition of centrosome separation induced by DNA damage. This inhibition is dependent on ATM, but not on Chk2 or Chk1. Nek2 is a key regulator of centrosome separation and is a target of Plk1 in blocking centrosome separation. We found that Plk1 can phosphorylate Nek2 in vitro and interacts with Nek2 in vivo. Downregulation of Plk1 with RNA interference prevents Nek2-induced centrosome splitting. DNA damage is known to inhibit Plk1 activity. We propose that the DNA damage-induced inhibition of Plk1 leads to inhibition of Nek2 activity and thus prevents centrosome separation.To maintain the integrity of the genome, mammalian cells have developed surveillance mechanisms called checkpoints that arrest the cell cycle after activation by unreplicated or damaged DNA (1, 2). Damage by ionizing radiation may activate G 1 , S, and G 2 /M checkpoints (3). The G 2 /M checkpoint prevents mitotic entry after DNA is damaged (4). The activation of this checkpoint requires the functions of checkpoint kinases ATM and ATR (ATM-and Rad3-related). Upon activation, ATM or ATR can phosphorylate and activate many proteins involved in cell cycle regulation, such as checkpoint kinases Chk1 and Chk2, P53 and BRCA1, therefore elicit a subsequent cell cycle arrest (5-8).The centrosome is the major microtubule-organizing center of animal cells (9). It consists of a pair of centrioles surrounded by a protein matrix called pericentriolar material (10). During the cell cycle, centrosomes are duplicated and segregated along with the genome. The centrosome cycle has been divided into several steps including centrosome duplication, centrosome maturation, centrosome separation, and centriole disorientation (11). Centrosome separation is initiated in G 2 phase and is completed in mitosis, leading to the formation of bipolar mitotic spindle (12).The centrosomal cycle is closely integrated into the cell cycle, and the DNA damage checkpoints may regulate the centrosomal cycle. In Drosophila, a variety of DNA damaging agents trigger centrosome inactivation (13,14). We have demonstrated that centrosomal separation is inhibited after exposure to DNA-damaging agents including IR, 2 indicating that the centrosome may be implicated in a checkpoint function during cell cycle progression (15). However, the checkpoint pathways and genes involved in this cellular response to DNA damage are not known. Polo-like kinase 1 (Plk1) is a serine/threonine protein kinase that is involved at several points in mitotic progression (16 -18). Plk1 shows cell cycle dependent expression, accumulating to maximal levels during G 2 and M phases (19). At mitosis, Plk1 activates phosph...