Radiation Dose-Dependent Maintenance of G2 Arrest Requires Retinoblastoma Protein

In response to ionizing radiation (IR), cell cycle checkpoints are activated to provide time for DNA repair. Several different checkpoint mechanisms have been elucidated. However, mechanisms that regulate the duration of cell cycle arrest are not understood. Previous studies have shown that the retinoblastoma tumor suppressor protein (RB) is required for radiation-induced G1 arrest. Working with primary fibroblasts derived from Rb+/+ and Rb-/- mouse embryos, we show that RB also regulates the duration of G2 arrest. The initial G2 checkpoint response is enhanced in Rb-/- cells due to a defect in G1 arrest. However, the permanent arrest in G2 induced by higher doses of IR does not occur in Rb-/- cells. Rb-/- cells either resumed proliferation or underwent apoptosis at IR doses that caused the majority of Rb+/+ cells to arrest permanently in G2. The prolongation of G2 arrest in Rb+/+ cells correlated with a gradual accumulation of hypophosphorylated RB. Thus, regulation of the RB function may be an important aspect in the maintenance of cell cycle checkpoints in DNA damage response.

Key Words:

RB phosphorylation, Ionizing radiation, DNA damage, G2 checkpoint, Mouse embryo fibroblasts     

DNA damage invokes mismatch repair-dependent cyclin D1 attenuation and retinoblastoma signaling pathways to inhibit CDK2.

DNA-damage evokes cell cycle checkpoints, which function to maintain genomic integrity. The retinoblastoma tumor suppressor (RB) and mismatch repair complexes are known to contribute to the appropriate cellular response to specific types of DNA damage. However, the signaling pathways through which these proteins impact the cell cycle machinery have not been explicitly determined. RB-deficient murine embryo fibroblasts continued a high degree of DNA replication following the induction of cisplatin damage, but were inhibited for G(2)/M progression. This damage led to RB dephosphorylation/activation and subsequent RB-dependent attenuation of cyclin A and CDK2 activity. In both Rb+/+ and Rb -/- cells, cyclin D1 expression was attenuated following DNA damage. As cyclin D1 is a critical determinant of RB phosphorylation and cell cycle progression, we probed the pathway through which cyclin D1 degradation occurs in response to DNA damage. We found that attenuation of endogenous cyclin D1 is dependent on multiple mismatch repair proteins. We demonstrate that the mismatch repair-dependent attenuation of endogenous cyclin D1 is critical for attenuation of CDK2 activity and induction of cell cycle checkpoints. Together, these studies couple the activity of the retinoblastoma and mismatch repair tumor suppressor pathways through the degradation of cyclin D1 and dual attenuation of CDK2 activity.     

Differential role of RB in response to UV and IR damage

The retinoblastoma tumor suppressor (RB) is functionally inactivated in the majority of cancers and is a critical mediator of DNA damage checkpoints. Despite the critical importance of RB function in tumor suppression, the coordinate impact of RB loss on the response to environmental and therapeutic sources of damage has remained largely unexplored. Here, we utilized a conditional knockout system to ablate RB in adult fibroblasts. This model system enabled us to investigate the temporal role of RB loss on cell cycle checkpoints and DNA damage repair following ultraviolet (UV) and ionizing radiation (IR) damage. We demonstrate that RB loss compromises rapid cell cycle arrest following UV and IR exposure in adult primary cells. Detailed kinetic analysis of the checkpoint response revealed that disruption of the checkpoint is concomitant with RB target gene deregulation, and is not simply a manifestation of chronic RB loss. RB loss had a differential effect upon repair of the major DNA lesions induced by IR and UV. Whereas RB did not affect resolution of DNA double-strand breaks, RB-deficient cells exhibited accelerated repair of pyrimidine pyrimidone photoproducts (6-4 PP). In parallel, this repair was coupled with enhanced expression of specific factors and the behavior of proliferating cell nuclear antigen (PCNA) recruitment to replication and repair foci. Thus, RB loss and target gene deregulation hastens the repair of specific lesions distinct from its ubiquitous role in checkpoint abrogation.     

Defective control of mitotic and post-mitotic checkpoints in poly(ADP-ribose) polymerase-1(-/-) fibroblasts after mitotic spindle disruption

Poly(ADP-ribose) polymerase-1 (PARP), a DNA damage-responsive nuclear enzyme present in higher eukaryotes, is well-known for its roles in protecting the genome after DNA damage. However, even without exogenous DNA damage, PARP may play a role in stabilizing the genome because cells or mice deficient in PARP exhibit various signs of genomic instability, such as tetraploidy, aneuploidy, chromosomal abnormalities and susceptibility to spontaneous carcinogenesis. Normally, cell cycle checkpoints ensure elimination of cells with genomic abnormalities. Therefore, we examined efficiency of mitotic and post-mitotic checkpoints in PARP-/- and PARP+/+ mouse embryonic fibroblasts treated with mitotic spindle disrupting agent colcemid. PARP+/+ cells, like most mammalian cells, eventually escaped from spindle disruption-induced mitotic checkpoint arrest by 60 h. In contrast, PARP-/- cells rapidly escaped from mitotic arrest within 24 h by downregulation of cyclin B1/CDK-1 kinase activity. After escaping from mitotic arrest; both the PARP genotypes arrive in G1 tetraploid state, where they face post-mitotic checkpoints which either induce apoptosis or prevent DNA endoreduplication. While all the G1 tetraploid PARP+/+ cells were eliminated by apoptosis, the majority of the G1 tetraploid PARP-/- cells became polyploid by resisting apoptosis and carrying out DNA endoreduplication. Introduction of PARP in PARP-/- fibroblasts partially increased the stringency of mitotic checkpoint arrest and fully restored susceptibility to G1 tetraploidy checkpoint-induced apoptosis; and thus prevented formation of polyploid cells. Our results suggest that PARP may serve as a guardian angel of the genome even without exogenous DNA damage through its role in mitotic and post-mitotic G1 tetraploidy checkpoints.     

Two Molecularly Distinct G2/M Checkpoints Are Induced by Ionizing Irradiation

Cell cycle checkpoints are among the multiple mechanisms that eukaryotic cells possess to maintain genomic integrity and minimize tumorigenesis. Ionizing irradiation (IR) induces measurable arrests in the G(1), S, and G(2) phases of the mammalian cell cycle, and the ATM (ataxia telangiectasia mutated) protein plays a role in initiating checkpoint pathways in all three of these cell cycle phases. However, cells lacking ATM function exhibit both a defective G(2) checkpoint and a prolonged G(2) arrest after IR, suggesting the existence of different types of G(2) arrest. Two molecularly distinct G(2)/M checkpoints were identified, and the critical importance of the choice of G(2)/M checkpoint assay was demonstrated. The first of these G(2)/M checkpoints occurs early after IR, is very transient, is ATM dependent and dose independent (between 1 and 10 Gy), and represents the failure of cells which had been in G(2) at the time of irradiation to progress into mitosis. Cell cycle assays that can distinguish mitotic cells from G(2) cells must be used to assess this arrest. In contrast, G(2)/M accumulation, typically assessed by propidium iodide staining, begins to be measurable only several hours after IR, is ATM independent, is dose dependent, and represents the accumulation of cells that had been in earlier phases of the cell cycle at the time of exposure to radiation. G(2)/M accumulation after IR is not affected by the early G(2)/M checkpoint and is enhanced in cells lacking the IR-induced S-phase checkpoint, such as those lacking Nbs1 or Brca1 function, because of a prolonged G(2) arrest of cells that had been in S phase at the time of irradiation. Finally, neither the S-phase checkpoint nor the G(2) checkpoints appear to affect survival following irradiation. Thus, two different G(2) arrest mechanisms are present in mammalian cells, and the type of cell cycle checkpoint assay to be used in experimental investigation must be thoughtfully selected.     

A Stronger DNA Damage-Induced G2 Checkpoint Due to Over-Activated CHK1 in the Absence of PARP-1

Poly(ADP-ribose) polymerase-1 (PARP-1) is involved in multi-pathways to respond to DNA damage. Lack of or inhibition of PARP-1 activity leads to slow progress of cell cycle and sensitization of cells to different stresses. Recently, it was reported that besides the Ku- dependent main non-homologous end joining (NHEJ) pathway, there is a PARP-1-dependent complementary NHEJ pathway to repair DNA double strand break (DSB). Here we show that compared with PARP-1+/+ cells, PARP-1-/- cells display a much stronger G2 checkpoint response following ionizing radiation (IR). Treatment with Chk1 siRNA abolishes the stronger G2 checkpoint response and sensitizes PARP-1-/- cells to IR. These data indicate that the stronger G2 checkpoint response in PARP-1-/- cells is CHK1-dependent, which protects cells from IR-induced killing. We also show that 4-Amino-1,8-naphthalimide (4-AN, inhibitor of PARP) but not methoxyamine (inhibitor of base excision repair (BER)), affects IR-induced G2 arrest and cell sensitivity in PARP-1+/+ cells, resulting in the phenotypes similar to those of PARP-1-/- cells. These results indicate that DSB repair from the complementary NHEJ pathway of PARP-1, but not single strand break (SSB) repair from the BER function of PARP-1, may play an essential role in the over-activated CHK1 regulated G2 checkpoint response and radiosensitivity in PARP-1-/- cells.     

A Mitotic Phosphorylation Feedback Network Connects Cdk1, Plk1, 53BP1, and Chk2 to Inactivate the G2/M DNA Damage Checkpoint

DNA damage checkpoints arrest cell cycle progression to facilitate DNA repair. The ability to survive genotoxic insults depends not only on the initiation of cell cycle checkpoints but also on checkpoint maintenance. While activation of DNA damage checkpoints has been studied extensively, molecular mechanisms involved in sustaining and ultimately inactivating cell cycle checkpoints are largely unknown. Here, we explored feedback mechanisms that control the maintenance and termination of checkpoint function by computationally identifying an evolutionary conserved mitotic phosphorylation network within the DNA damage response. We demonstrate that the non-enzymatic checkpoint adaptor protein 53BP1 is an in vivo target of the cell cycle kinases Cyclin-dependent kinase-1 and Polo-like kinase-1 (Plk1). We show that Plk1 binds 53BP1 during mitosis and that this interaction is required for proper inactivation of the DNA damage checkpoint. 53BP1 mutants that are unable to bind Plk1 fail to restart the cell cycle after ionizing radiation-mediated cell cycle arrest. Importantly, we show that Plk1 also phosphorylates the 53BP1-binding checkpoint kinase Chk2 to inactivate its FHA domain and inhibit its kinase activity in mammalian cells. Thus, a mitotic kinase-mediated negative feedback loop regulates the ATM-Chk2 branch of the DNA damage signaling network by phosphorylating conserved sites in 53BP1 and Chk2 to inactivate checkpoint signaling and control checkpoint duration.     

The stress kinase MRK contributes to regulation of DNA damage checkpoints through a p38gamma-independent pathway

DNA damage induced by ionizing radiation (IR) activates a complex cellular response that includes checkpoints leading to cell cycle arrest. The stress-activated mitogen-activated protein kinase (MAPK) p38gamma has been implicated in the G(2) phase checkpoint induced by IR. We recently discovered MRK as a member of the MAPK kinase kinase family that activates p38gamma. Here we investigated the role of MRK in the checkpoint response to IR. We identified autophosphorylation sites on MRK that are important for its kinase activity. A phosphospecific antibody that recognizes these sites showed that MRK is activated upon IR in a rapid and sustained manner. MRK depletion by RNA interference resulted in defective S and G(2) checkpoints induced by IR that were accompanied by reduced Chk2 phosphorylation and delayed Cdc25A degradation. We also showed that Chk2 is a substrate for MRK in vitro and is phosphorylated at Thr(68) by active MRK in cells. MRK depletion also increased sensitivity to the killing effects of IR. In addition, MRK depletion reduced IR-induced activation of p38gamma but had no effect on p38alpha activation, indicating that MRK is a specific activator of p38gamma after IR. Inhibition of p38gamma by RNA interference, however, did not impair IR-induced checkpoints. Thus, in response to IR MRK controls two independent pathways: the Chk2-Cdc25A pathway leading to cell cycle arrest and the p38gamma MAPK pathway.     

RB-dependent S-phase response to DNA damage

The retinoblastoma tumor suppressor protein (RB) is a potent inhibitor of cell proliferation. RB is expressed throughout the cell cycle, but its antiproliferative activity is neutralized by phosphorylation during the G(1)/S transition. RB plays an essential role in the G(1) arrest induced by a variety of growth inhibitory signals. In this report, RB is shown to also be required for an intra-S-phase response to DNA damage. Treatment with cisplatin, etoposide, or mitomycin C inhibited S-phase progression in Rb(+/+) but not in Rb(-/-) mouse embryo fibroblasts. Dephosphorylation of RB in S-phase cells temporally preceded the inhibition of DNA synthesis. This S-phase dephosphorylation of RB and subsequent inhibition of DNA replication was observed in p21(Cip1)-deficient cells. The induction of the RB-dependent intra-S-phase arrest persisted for days and correlated with a protection against DNA damage-induced cell death. These results demonstrate that RB plays a protective role in response to genotoxic stress by inhibiting cell cycle progression in G(1) and in S phase.     

RB signaling prevents replication-dependent DNA double-strand breaks following genotoxic insult

Cell cycle checkpoints induced by DNA damage play an integral role in preservation of genomic stability by allowing cells to limit the propagation of deleterious mutations. The retinoblastoma tumor suppressor (RB) is crucial for the maintenance of the DNA damage checkpoint function because it elicits cell cycle arrest in response to a variety of genotoxic stresses. Although sporadic loss of RB is characteristic of most cancers and results in the bypass of the DNA damage checkpoint, the consequence of RB loss upon chemotherapeutic responsiveness has been largely uninvestigated. Here, we employed a conditional knockout approach to ablate RB in adult fibroblasts. This system enabled us to examine the DNA damage response of adult cells following acute RB deletion. Using this system, we demonstrated that loss of RB disrupted the DNA damage checkpoint elicited by either cisplatin or camptothecin exposure. Strikingly, this bypass was not associated with enhanced repair, but rather the accumulation of phosphorylated H2AX (γH2AX) foci, which indicate DNA double-strand breaks. The formation of γH2AX foci was due to ongoing replication following chemotherapeutic treatment in the RB-deficient cells. Additionally, peak γH2AX accumulation occurred in S-phase cells undergoing DNA replication in the presence of damage, and these γH2AX foci co-localized with replication foci. These results demonstrate that acute RB loss abrogates DNA damage-induced cell cycle arrest to induce γH2AX foci formation. Thus, secondary genetic lesions induced by RB loss have implications for the chemotherapeutic response and the development of genetic instability.     

The nuclear death domain protein p84N5 activates a G2/M cell cycle checkpoint prior to the onset of apoptosis

In contrast to extracellular signals, the mechanisms utilized to transduce nuclear apoptotic signals are not well understood. Characterizing these mechanisms is important for predicting how tumors will respond to genotoxic radiation or chemotherapy. The retinoblastoma (Rb) tumor suppressor protein can regulate apoptosis triggered by DNA damage through an unknown mechanism. The nuclear death domain-containing protein p84N5 can induce apoptosis that is inhibited by association with Rb. The pattern of caspase and NF-kappaB activation during p84N5-induced apoptosis is similar to p53-independent cellular responses to DNA damage. One hallmark of this response is the activation of a G(2)/M cell cycle checkpoint. In this report, we characterize the effects of p84N5 on the cell cycle. Expression of p84N5 induces changes in cell cycle distribution and kinetics that are consistent with the activation of a G(2)/M cell cycle checkpoint. Like the radiation-induced checkpoint, caffeine blocks p84N5-induced G(2)/M arrest but not subsequent apoptotic cell death. The p84N5-induced checkpoint is functional in ataxia telangiectasia-mutated kinase-deficient cells. We conclude that p84N5 induces an ataxia telangiectasia-mutated kinase (ATM)-independent, caffeine-sensitive G(2)/M cell cycle arrest prior to the onset of apoptosis. This conclusion is consistent with the hypotheses that p84N5 functions in an Rb-regulated cellular response that is similar to that triggered by DNA damage.     

Role of a DNA damage checkpoint pathway in ionizing radiation-induced glioblastoma cell migration and invasion

Ionizing radiation (IR) induces a DNA damage response that includes activation of cell cycle checkpoints, leading to cell cycle arrest. In addition, IR enhances cell invasiveness of glioblastoma cells, among other tumor cell types. Using RNA interference, we found that the protein kinase MRK, previously implicated in the DNA damage response to IR, also inhibits IR-induced cell migration and invasion of glioblastoma cells. We showed that MRK activation by IR requires the checkpoint protein Nbs1 and that Nbs1 is also required for IR-stimulated migration. In addition, we show that MRK acts upstream of Chk2 and that Chk2 is also required for IR-stimulated migration and invasion. Thus, we have identified Nbs1, MRK, and Chk2 as elements of a novel signaling pathway that mediates IR-stimulated cell migration and invasion. Interestingly, we found that inhibition of cell cycle progression, either with the CDK1/2 inhibitor CGP74514A or by downregulation of the CDC25A protein phosphatase, restores IR-induced migration and invasion in cells depleted of MRK or Chk2. These data indicate that cell cycle progression, at least in the context of IR, exerts a negative control on the invasive properties of glioblastoma cells and that checkpoint proteins mediate IR-induced invasive behavior by controlling cell cycle arrest.     

Cell cycle re-entry mechanisms after DNA damage checkpoints: Giving it some gas to shut off the breaks!

In order to maintain genetic integrity, cells are equipped with cell cycle checkpoints that detect DNA damage, orchestrate repair, and if necessary, eliminate severely damaged cells by inducing apoptotic cell death. The mitotic machinery is now emerging as an important determinant of the cellular responses to DNA damage where it functions as both the downstream target and the upstream regulator of the G2/M checkpoint. Cell cycle kinases and the DNA damage checkpoint kinases appear to reciprocally control each other. Specifically, cell cycle kinases control the inactivation of DNA damage checkpoint signaling. Termination of a DNA damage response by mitotic kinases appears to be an evolutionary conserved mechanism that allows resumption of cell cycle progression. Here we review recent reports in which molecular mechanisms underlying checkpoint silencing at the G2/M transition are elucidated.     

Radiosensitization by Chir-124, a selective Chk1 inhibitor: Effects of p53 and cell cycle checkpoints

Checkpoint kinase-1 (CHK1) is a key regulator of the DNA damage-elicited G2-M checkpoints. The aim of the present study was to investigate the effects of a selective CHK1 inhibitor, Chir124, on cell survival and cell cycle progression following ionizing radiation (IR). Treatment with Chir-124 resulted in reduced clonogenic survival and abrogated the IR- induced G2-M arrest in a panel of isogenic HCT116 cell lines after IR. This radiosensitizing effect was relatively similar between p53-/- and p53-sufficient wild type (WT) HCT116 cells. However, the number of mitotic cells (as measured by assessing the phosphorylation of mitotic proteins) increased dramatically in p53-/- HCT116 cells after concomitant Chir-124 exposure, compared to IR alone, while no such effect was observed in p53-sufficient WT HCT116 cells. In p53-/- cells, Chir-124 treatment induced a marked accumulation of polyploid cells that were characterized by micronucleation or multinucleation. p21-/- HCT116 cells displayed a similar pattern of response as p53-/- cells. Chir-124 was able to radiosensitize HCT116 cells that lack checkpoint kinase-2 (CHK2) or that were deficient for the spindle checkpoint protein Mad2. Finally, Chir-124 could radiosensitize tetraploid cell lines, which were relatively resistant against DNA damaging agents. Altogether these results suggest that Chir-124-mediated radiosensitization is profoundly influenced by the p53 and cell cycle checkpoint system.     

Characterization of G(1) Checkpoint Control in the Yeast Saccharomyces Cerevisiae following Exposure to DNA-Damaging Agents

The delay of S-phase following treatment of yeast cells with DNA-damaging agents is an actively regulated response that requires functional RAD9 and RAD24 genes. An analysis of cell cycle arrest indicates the existence of (at least) two checkpoints for damaged DNA prior to S-phase; one at START (a G(1) checkpoint characterized by pheromone sensitivity of arrested cells) and one between the CDC4- and CDC7-mediated steps (termed the G(1)/S checkpoint). When a dna1-1 mutant (that affects early events of replicon initiation) also carries a rad9 deletion mutation, it manifests a failure to arrest in G(1)/S following incubation at the restrictive temperature. This failure to execute regulated G(1)/S arrest is correlated with enhanced thermosensitivity of colony-forming ability. In an attempt to characterize the signal for RAD9 gene-dependent G(1) and G(1)/S cell cycle arrest, we examined the influence of the continued presence of unexcised photoproducts. In mutants defective in nucleotide excision repair, cessation of S-phase was observed at much lower doses of UV radiation compared to excision-proficient cells. However, this response was not RAD9-dependent. We suggest that an intermediate of nucleotide excision repair, such as DNA strand breaks or single-stranded DNA tracts, is required to activate RAD9-dependent G(1) and G(1)/S checkpoint controls.     

p53-dependent Chk1 phosphorylation is required for maintenance of prolonged G2 Arrest

Targeting checkpoint kinases has been shown to have a potential chemosensitizing effect in cancer treatment. However, inhibitors of such kinases preferentially abrogate the DNA damage-induced G2 checkpoint in p53-/- as opposed to p53+/+ cells. The mechanisms by which p53 (TP53) can prevent abrogation of the G2 checkpoint are unclear. Using normal human diploid p53+/+ and p53-/- fibroblasts as model systems, we have compared the effects of three checkpoint inhibitors, caffeine, staurosporine and UCN-01, on gamma-radiation-induced G2 arrest. The G2 arrest in p53+/+ cells was abrogated by caffeine, but not by staurosporine and UCN-01, whereas the G2 arrest in p53-/- cells was sensitive to all three inhibitors. Chk2 (CHEK1) phosphorylation was maintained in the presence of all three inhibitors in both p53+/+ and p53-/- cells. Chk1 phosphorylation was maintained only in the presence of staurosporine and UCN-01 in p53+/+ cells. In the presence of caffeine Chk1 phosphorylation was inhibited regardless of p53 status. The pathway of Chk1 phosphorylation --> Cdc25A degradation --> inhibition of cyclin B1/Cdk1 activity --> G2 arrest is accordingly resistant to staurosporine and UCN-01 in p53+/+ cells. Moreover, sustained phosphorylation of Chk1 in the presence of staurosporine and UCN-01 is strongly related to phosphorylation of p53. The present study suggests the unique role of Chk1 in preventing abrogation of the G2 checkpoint in p53+/+ cells.     

Dissecting cellular responses to irradiation via targeted disruptions of the ATM-CHK1-PP2A circuit

Exposure of proliferating cells to genotoxic stresses activates a cascade of signaling events termed the DNA damage response (DDR). The DDR preserves genetic stability by detecting DNA lesions, activating cell cycle checkpoints and promoting DNA damage repair. The phosphoinositide 3-kinase-related kinases (PIKKs) ataxia telangiectasia-mutated (ATM), ATM and Rad 3-related kinase (ATR) and DNA-dependent protein kinase (DNA-PK) are crucial for sensing lesions and signal transduction. The checkpoint kinase 1 (CHK1) is a traditional ATR target involved in DDR and normal cell cycle progression and represents a pharmacological target for anticancer regimens. This study employed cell lines stably depleted for CHK1, ATM or both for dissecting cross-talk and compensatory effects on G?/M checkpoint in response to ionizing radiation (IR). We show that a 90% depletion of CHK1 renders cells radiosensitive without abrogating their IR-mediated G?/M checkpoint arrest. ATM phosphorylation is enhanced in CHK1-deficient cells compared with their wild-type counterparts. This correlates with lower nuclear abundance of the PP2A catalytic subunit in CHK1-depleted cells. Stable depletion of CHK1 in an ATM-deficient background showed only a 50% reduction from wild-type CHK1 protein expression levels and resulted in an additive attenuation of the G?/M checkpoint response compared with the individual knockdowns. ATM inhibition and 90% CHK1 depletion abrogated the early G?/M checkpoint and precluded the cells from mounting an efficient compensatory response to IR at later time points. Our data indicates that dual targeting of ATM and CHK1 functionalities disrupts the compensatory response to DNA damage and could be exploited for developing efficient anti-neoplastic treatments.     

Never-in-mitosis related Kinase 1 functions in DNA damage response and checkpoint control

Nek1, the first mammalian ortholog of the fungal protein kinase never in mitosis A, is involved early in the DNA damage sensing/repair pathway after ionizing radiation. Here we extend this finding by showing that Nek1 localizes to nuclear foci of DNA damage in response to many different types of damage in addition to IR. Untransformed cells established from kat2J/Nek1 -/- mice fail to arrest properly at G1/S and M-phase checkpoints in response to DNA damage. G1-S-phase checkpoint control can be rescued by ectopically overexpressing wild-type Nek1. In Nek1-/- murine cells and in human cells with Nek1 expression silenced by siRNA, the checkpoint kinases Chk1 and Chk2 fail to be activated properly in response to ionizing or UV radiation. In cells without functional Nek1, DNA is not repaired properly, double-stranded DNA breaks persist long after low dose IR, and excessive numbers of chromosome breaks are observed. These data show that Nek1 is important for efficient DNA damage checkpoint control and for proper DNA damage repair.