DNA Damage-induced Cell Cycle Checkpoints Research Articles

Syntaxin 18 regulates the DNA damage response and epithelial-to-mesenchymal transition to promote radiation resistance of lung cancer

Radiotherapy is an important modality in lung cancer treatment. Despite advances in treatment planning and dose delivery, patient benefit is still limited by in-field relapse and metastatic recurrence. Simultaneous application of cisplatinum-based chemotherapy leads to moderately improved outcomes, thus providing proof-of-concept for radiosensitization strategies in lung cancer. In an unbiased functional genetic screen for radiosensitization targets in lung cancer, we identified syntaxin 18, a protein involved in retrograde vesicular transport between the Golgi apparatus and endoplasmic reticulum, as mediator of radioresistance. Downregulation of endogenous syntaxin 18 specifically reduced clonogenic survival of radioresistant and radiosensitive lung cancer cells following X-radiation. Gene expression programs regulating DNA repair, mitotic checkpoints and mitosis were altered in isogenic cells with reduced syntaxin 18 expression. Functionally, this translated into impaired DNA damage-induced cell cycle checkpoints leading to cell death by mitotic catastrophe. Interestingly, downregulation of syntaxin 18 in lung cancer cells also impaired expression of markers of epithelial-mesenchymal-transition, and reduced migration and invasion capacity. These findings suggest that syntaxin 18 is a key player regulating genes responsible for controlling the growth of the primary tumor as well as metastases upon radiotherapy of lung cancer. They provide a promising lead for biologically rational radiosensitization strategies impacting on radiation-induced cell death as well as metastasis.

BRCA2 associates with MCM10 to suppress PRIMPOL-mediated repriming and single-stranded gap formation after DNA damage

The BRCA2 tumor suppressor protects genome integrity by promoting homologous recombination-based repair of DNA breaks, stability of stalled DNA replication forks and DNA damage-induced cell cycle checkpoints. BRCA2 deficient cells display the radio-resistant DNA synthesis (RDS) phenotype, however the mechanism has remained elusive. Here we show that cells without BRCA2 are unable to sufficiently restrain DNA replication fork progression after DNA damage, and the underrestrained fork progression is due primarily to Primase-Polymerase (PRIMPOL)-mediated repriming of DNA synthesis downstream of lesions, leaving behind single-stranded DNA gaps. Moreover, we find that BRCA2 associates with the essential DNA replication factor MCM10 and this association suppresses PRIMPOL-mediated repriming and ssDNA gap formation, while having no impact on the stability of stalled replication forks. Our findings establish an important function for BRCA2, provide insights into replication fork control during the DNA damage response, and may have implications in tumor suppression and therapy response.

ATR/ATM-Mediated Phosphorylation of BRCA1 T1394 Promotes Homologous Recombinational Repair and G2-M Checkpoint Maintenance.

BRCA1 maintains genome integrity and suppresses tumorigenesis by promoting homologous recombination (HR)-mediated repair of DNA double-strand breaks (DSB) and DNA damage-induced cell-cycle checkpoints. Phosphorylation of BRCA1 by ATM, ATR, CHK2, CDK, and PLK1 kinases has been reported to regulate its functions. Here we show that ATR and ATM-mediated phosphorylation of BRCA1 on T1394, a highly conserved but functionally uncharacterized site, is a key modification for its function in the DNA damage response (DDR). Following DNA damage, T1394 phosphorylation ensured faithful repair of DSBs by promoting HR and preventing single-strand annealing, a deletion-generating repair process. BRCA1 T1394 phosphorylation further safeguarded chromosomal integrity by maintaining the G2-M checkpoint. Moreover, multiple patient-derived BRCA1 variants of unknown significance were shown to affect T1394 phosphorylation. These results establish an important regulatory mechanism of BRCA1 function in the DDR and may have implications in the development or prognosis of BRCA1-associated cancers. SIGNIFICANCE: This study identifies a BRCA1 phosphorylation event critical for its DNA repair function and reveals the functional defects of several BRCA1 variants of unknown significance.

β-TrCP1 facilitates cell cycle checkpoint activation, DNA repair, and cell survival through ablation of β-TrCP2 in response to genotoxic stress

F-box proteins β-TrCP1 and β-TrCP2 are paralogs present in the human genome. They control several cellular processes including cell cycle and DNA damage signaling. Moreover, it is reported that they facilitate DNA damage-induced accumulation of p53 by directing proteasomal degradation of MDM2, a protein that promotes p53 degradation. However, the individual roles of β-TrCP1 and β-TrCP2 in the genotoxic stress-induced activation of cell cycle checkpoints and DNA damage repair remain largely unknown. Here, using biochemical, molecular biology, flow cytometric, and immunofluorescence techniques, we show that β-TrCP1 and β-TrCP2 communicate during genotoxic stress. We found that expression levels of β-TrCP1 are significantly increased while levels of β-TrCP2 are markedly decreased upon induction of genotoxic stress. Further, our results revealed that DNA damage-induced activation of ATM kinase plays an important role in maintaining the reciprocal expression levels of β-TrCP1 and β-TrCP2 via the phosphorylation of β-TrCP1 at Ser158. Phosphorylated β-TrCP1 potently promotes the proteasomal degradation of β-TrCP2 and MDM2, resulting in the activation of p53. Additionally, β-TrCP1 impedes MDM2 accumulation via abrogation of its lysine 63-linked polyubiquitination by β-TrCP2. Thus, β-TrCP1 helps to arrest cells at the G2/M phase of the cell cycle and promotes DNA repair upon DNA damage through attenuation of β-TrCP2. Collectively, our findings elucidate an intriguing posttranslational regulatory mechanism of these two paralogs under genotoxic stress and revealed β-TrCP1 as a key player in maintaining the genome integrity through the attenuation of β-TrCP2 levels in response to genotoxic stress.

Enhancing chemotherapy response through augmented synthetic lethality by co-targeting nucleotide excision repair and cell-cycle checkpoints

In response to DNA damage, a synthetic lethal relationship exists between the cell cycle checkpoint kinase MK2 and the tumor suppressor p53. Here, we describe the concept of augmented synthetic lethality (ASL): depletion of a third gene product enhances a pre-existing synthetic lethal combination. We show that loss of the DNA repair protein XPA markedly augments the synthetic lethality between MK2 and p53, enhancing anti-tumor responses alone and in combination with cisplatin chemotherapy. Delivery of siRNA-peptide nanoplexes co-targeting MK2 and XPA to pre-existing p53-deficient tumors in a highly aggressive, immunocompetent mouse model of lung adenocarcinoma improves long-term survival and cisplatin response beyond those of the synthetic lethal p53 mutant/MK2 combination alone. These findings establish a mechanism for co-targeting DNA damage-induced cell cycle checkpoints in combination with repair of cisplatin-DNA lesions in vivo using RNAi nanocarriers, and motivate further exploration of ASL as a generalized strategy to improve cancer treatment.

A kinetochore-based ATM/ATR-independent DNA damage checkpoint maintains genomic integrity in trypanosomes.

DNA damage-induced cell cycle checkpoints serve as surveillance mechanisms to maintain genomic stability, and are regulated by ATM/ATR-mediated signaling pathways that are conserved from yeast to humans. Trypanosoma brucei, an early divergent microbial eukaryote, lacks key components of the conventional DNA damage-induced G2/M cell cycle checkpoint and the spindle assembly checkpoint, and nothing is known about how T. brucei controls its cell cycle checkpoints. Here we discover a kinetochore-based, DNA damage-induced metaphase checkpoint in T. brucei. MMS-induced DNA damage triggers a metaphase arrest by modulating the abundance of the outer kinetochore protein KKIP5 in an Aurora B kinase- and kinetochore-dependent, but ATM/ATR-independent manner. Overexpression of KKIP5 arrests cells at metaphase through stabilizing the mitotic cyclin CYC6 and the cohesin subunit SCC1, mimicking DNA damage-induced metaphase arrest, whereas depletion of KKIP5 alleviates the DNA damage-induced metaphase arrest and causes chromosome mis-segregation and aneuploidy. These findings suggest that trypanosomes employ a novel DNA damage-induced metaphase checkpoint to maintain genomic integrity.

Hypoxia-induced alterations of G2 checkpoint regulators

Hypoxia promotes an aggressive tumor phenotype with increased genomic instability, partially due to downregulation of DNA repair pathways. However, genome stability is also surveilled by cell cycle checkpoints. An important issue is therefore whether hypoxia also can influence the DNA damage-induced cell cycle checkpoints. Here, we show that hypoxia (24 h 0.2% O2) alters the expression of several G2 checkpoint regulators, as examined by microarray gene expression analysis and immunoblotting of U2OS cells. While some of the changes reflected hypoxia-induced inhibition of cell cycle progression, the levels of several G2 checkpoint regulators, in particular Cyclin B, were reduced in G2 phase cells after hypoxic exposure, as shown by flow cytometric barcoding analysis of individual cells. These effects were accompanied by decreased phosphorylation of a Cyclin dependent kinase (CDK) target in G2 phase cells after hypoxia, suggesting decreased CDK activity. Furthermore, cells pre-exposed to hypoxia showed increased G2 checkpoint arrest upon treatment with ionizing radiation. Similar results were found following other hypoxic conditions (∼0.03% O2 20 h and 0.2% O2 72 h). These results demonstrate that the DNA damage-induced G2 checkpoint can be altered as a consequence of hypoxia, and we propose that such alterations may influence the genome stability of hypoxic tumors.

Regulation of mismatch repair by histone code and posttranslational modifications in eukaryotic cells.

DNA mismatch repair (MMR) protects genome integrity by correcting DNA replication-associated mispairs, modulating DNA damage-induced cell cycle checkpoints and regulating homeologous recombination. Loss of MMR function leads to cancer development. This review describes progress in understanding how MMR is carried out in the context of chromatin and how chromatin organization/compaction, epigenetic mechanisms and posttranslational modifications of MMR proteins influence and regulate MMR in eukaryotic cells.

Regulation of NEIL1 protein abundance by RAD9 is important for efficient base excision repair

RAD9 participates in DNA damage-induced cell cycle checkpoints and DNA repair. As a member of the RAD9-HUS1-RAD1 (9-1-1) complex, it can sense DNA damage and recruit ATR to damage sites. RAD9 binding can enhance activities of members of different DNA repair pathways, including NEIL1 DNA glycosylase, which initiates base excision repair (BER) by removing damaged DNA bases. Moreover, RAD9 can act independently of 9-1-1 as a gene-specific transcription factor. Herein, we show that mouse Rad9−/− relative to Rad9+/+ embryonic stem (ES) cells have reduced levels of Neil1 protein. Also, human prostate cancer cells, DU145 and PC-3, knocked down for RAD9 demonstrate reduced NEIL1 abundance relative to controls. We found that Rad9 is required for Neil1 protein stability in mouse ES cells, whereas it regulates NEIL1 transcription in the human cells. RAD9 depletion enhances sensitivity to UV, gamma rays and menadione, but ectopic expression of RAD9 or NEIL1 restores resistance. Glycosylase/apurinic lyase activity was reduced in Rad9−/− mouse ES and RAD9 knocked-down human prostate cancer whole cell extracts, relative to controls. Neil1 or Rad9 addition restored this incision activity. Thus, we demonstrate that RAD9 regulates BER by controlling NEIL1 protein levels, albeit by different mechanisms in human prostate cancer versus mouse ES cells.

PEA15 Regulates the DNA Damage-Induced Cell Cycle Checkpoint and Oncogene-Directed Transformation

Regulation of the DNA damage response and cell cycle progression is critical for maintaining genome integrity. Here, we report that in response to DNA damage, COPS5 deubiquitinates and stabilizes PEA15 in an ATM kinase-dependent manner. PEA15 expression oscillates throughout the cell cycle, and the loss of PEA15 accelerates cell cycle progression by activating CDK6 expression via the c-JUN transcription factor. Cells lacking PEA15 exhibit a DNA damage-induced G2/M checkpoint defect due to increased CDC25C activity and, consequentially, higher cyclin-dependent kinase 1 (CDK1)/cyclin B activity, and accordingly they have an increased rate of spontaneous mutagenesis. We find that oncogenic RAS inhibits PEA15 expression and that ectopic PEA15 expression blocks RAS-mediated transformation, which can be partially rescued by ectopic expression of CDK6. Finally, we show that PEA15 expression is downregulated in colon, breast, and lung cancer samples. Collectively, our results demonstrate that tumor suppressor PEA15 is a regulator of genome integrity and is an integral component of the DNA damage response pathway that regulates cell cycle progression, the DNA-damage-induced G2/M checkpoint, and cellular transformation.

A Cell Cycle-Dependent Regulatory Circuit Composed of 53BP1-RIF1 and BRCA1-CtIP Controls DNA Repair Pathway Choice

DNA double-strand break (DSB) repair pathway choice is governed by the opposing activities of 53BP1 and BRCA1. 53BP1 stimulates nonhomologous end joining (NHEJ), whereas BRCA1 promotes end resection and homologous recombination (HR). Here we show that 53BP1 is an inhibitor of BRCA1 accumulation at DSB sites, specifically in the G1 phase of the cell cycle. ATM-dependent phosphorylation of 53BP1 physically recruits RIF1 to DSB sites, and we identify RIF1 as the critical effector of 53BP1 during DSB repair. Remarkably, RIF1 accumulation at DSB sites is strongly antagonized by BRCA1 and its interacting partner CtIP. Lastly, we show that depletion of RIF1 is able to restore end resection and RAD51 loading in BRCA1-depleted cells. This work therefore identifies a cell cycle-regulated circuit, underpinned by RIF1 and BRCA1, that governs DSB repair pathway choice to ensure that NHEJ dominates in G1 and HR is favored from S phase onward.

Novel Functions of the Phosphatase SHP2 in the DNA Replication and Damage Checkpoints

Replication stress- and DNA damage-induced cell cycle checkpoints are critical for maintaining genome stability. To identify protein phosphatases involved in the activation and maintenance of the checkpoints, we have carried out RNA interference-based screens with a human phosphatome shRNA library. Several phosphatases, including SHP2 (also called PTPN11) were found to be required for cell survival upon hydroxyurea-induced replicative stress in HeLa cells. More detailed studies revealed that SHP2 was also important for the maintenance of the checkpoint after DNA damage induced by cisplatin or ionizing radiation in HeLa cells. Furthermore, SHP2 was activated after replicative stress and DNA damage. Although depletion of SHP2 resulted in a delay in cyclin E accumulation and an extension of G1 phase, these cell cycle impairments were not responsible for the increase in apoptosis after DNA damage. Depletion of SHP2 impaired CHK1 activation, checkpoint-mediated cell cycle arrest, and DNA repair. These effects could be rescued with a shRNA-resistant SHP2. These results underscore the importance of protein phosphatases in checkpoint control and revealed a novel link between SHP2 and cell cycle checkpoints.

USP4 inhibits p53 through deubiquitinating and stabilizing ARF-BP1

Tumour suppressor p53 levels in the cell are tightly regulated by controlled degradation through ubiquitin ligases including Mdm2, COP1, Pirh2, and ARF-BP1. The ubiquitination process is reversible via deubiquitinating enzymes, such as ubiquitin-specific peptidases (USPs). In this study, we identified ubiquitin-specific peptidase 4 (USP4) as an important regulator of p53. USP4 interacts directly with and deubiquitinates ARF-BP1, leading to the stabilization of ARF-BP1 and subsequent reduction of p53 levels. Usp4 knockout mice are viable and developmentally normal, but showed enhanced apoptosis in thymus and spleen in response to ionizing radiation. Compared with wild-type mouse embryonic fibroblasts (MEFs), Usp4-/- MEFs exhibited retarded growth, premature cellular senescence, resistance to oncogenic transformation, and hyperactive DNA damage checkpoints, consistent with upregulated levels and activity of p53 in the absence of USP4. Finally, we showed that USP4 is overexpressed in several types of human cancer, suggesting that USP4 is a potential oncogene.

Abstract 2990: Characterization of a mitotic interaction of ATM with AuroraB

Abstract Ataxia-telangiectasia mutated (ATM) is a protein kinase that plays a pivotal role in DNA damage-induced cell cycle checkpoints and DNA repair. Mutation of ATM leads to the autosomal recessive disorder ataxia-telangiectasia, characterized by comprehensive clinical phenotypes such as cancer predisposition and neurodegeneration. One of the cellular phenotypes of ATM-deficiency is the spindle assembly checkpoint (SAC) defect and aneuploidy, indicating that ATM has a functional role in mitosis. We have recently demonstrated that ATM is activated in mitosis and that ATM is required for SAC. The molecular mechanism of mitotic ATM activation has started to emerge. We show here using coimmunoprecipitation that ATM interacts with Aurora B in unsynchronized cells and that this interaction increases during mitosis. Aurora B is a protein kinase and mitotic chromosomal passenger involved in the progression of mitosis. Although regulation of the Aurora B activity in response to DNA damage is dependent on ATM, Aurora B appears to serve as an upstream element of the mitotic ATM activation pathway. Aurora B phosphorylates ATM at Ser1403 and this phosphorylation event is required for activation of the SAC. We also show that Ser1403 is dephosphorylated by Protein Phosphatase 1 (PP1), thus implicating PP1 in the regulation of mitosis and the SAC. We demonstrate that the interaction between ATM and Aurora B is partially dependent on the enzymatic activity of both proteins. We also characterize the protein domains essential for the interaction of ATM with Aurora B and study the functional significance of this interaction. These data expand to our understanding of the roles of ATM outside of DNA damage repair and link ATM to cell cycle control. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2990. doi:10.1158/1538-7445.AM2011-2990

Abstract 1999: ATR- and ATM-regulated H3K56 acetylation mediates recovery from UV-induced cell-cycle checkpoint arrest

Abstract DNA damage results in chromatin remodeling to allow prompt access and efficient loading of repair and checkpoint proteins at damage sites to initiate cell cycle arrest and successfully complete DNA repair. Subsequently, chromatin structure is restored and cells begin to recover from checkpoint arrest. This ‘access-repair-restore’ model of DNA repair entails a coordinated functional interaction among chromatin remodelers, histone chaperones and a variety of histone modifications. Recent studies have implicated a role for H3K56 acetylation in the DNA damage response and checkpoint recovery. In this study, we determined the role of H3K56 acetylation in response to UV-induced DNA damage. We show that H3K56 is deacetylated within 15 minutes of UV-irradiation of normal human fibroblasts (NHFs), and acetylation is promptly recovered with full restoration at 48 hours. In yeast, acetylated H3K56 is deposited at the sites of DNA damage in an ASF1-dependent manner and is involved in checkpoint recovery. Therefore, we tested whether the checkpoint kinases, ATR and ATM, regulate the acetylation of H3K56. Interestingly, H3K56 acetylation was significantly lower in Seckel cells compared to NHFs, indicating a role of ATR in regulating H3K56 acetylation even in the absence of DNA damage. On the other hand, in AT and ATM-depleted HeLa cells, the post-repair recovery of H3K56 acetylation was defective. These data indicate a contributory role of ATR and ATM in regulating H3K56 acetylation. Next, we assessed the role of histone chaperone ASF1A in deacetylation/acetylation of H3K56. shRNA-mediated knockdown of ASF1A did not show a discernable defect in the deacetylation of H3K56 in response to UV-induced DNA damage, but the restoration of H3K56 acetylation was impaired. Thus, ASF1A appears to be essential for the post-repair recovery of H3K56 acetylation. To further confirm the role of ASF1A-mediated H3K56 acetylation in checkpoint recovery, we determined the level of H2AX phosphorylation at various times following UV-irradiation in ASF1A-deficient and H3K56R mutant cells, which are defective in H3K56 acetylation. Both cell types were shown to retain phosphorylated H2AX for a longer time at damage sites as compared to the control wild-type cells, indicating the persistence of checkpoint arrest in the absence of H3K56 acetylation. Similarly, the phosphorylated state of checkpoint kinases, Chk1 and Chk2, was maintained for longer times in the absence of ASF1A and in H3K56R mutant cells. FACS analysis of cell cycle progression after UV-irradiation of both ASF1A-deficient and H3K56R mutant cells revealed a slower recovery from the checkpoint arrest. Taken together, our results demonstrate an important function of H3K56 acetylation in the recovery from DNA damage-induced cell cycle checkpoint arrest. (This work was supported by NIH grants ES2388, ES12991 and CA93413) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1999. doi:10.1158/1538-7445.AM2011-1999

Cohesin phosphorylation and mobility of SMC1 at ionizing radiation-induced DNA double-strand breaks in human cells

Cohesin, a hetero-tetrameric complex of SMC1, SMC3, Rad21 and Scc3, associates with chromatin after mitosis and holds sister chromatids together following DNA replication. Following DNA damage, cohesin accumulates at and promotes the repair of DNA double-strand breaks. In addition, phosphorylation of the SMC1/3 subunits contributes to DNA damage-induced cell cycle checkpoint regulation. The aim of this study was to determine the regulation and consequences of SMC1/3 phosphorylation as part of the cohesin complex. We show here that the ATM-dependent phosphorylation of SMC1 and SMC3 is mediated by H2AX, 53BP1 and MDC1. Depletion of RAD21 abolishes these phosphorylations, indicating that only the fully assembled complex is phosphorylated. Comparison of wild type SMC1 and SMC1S966A in fluorescence recovery after photo-bleaching experiments shows that phosphorylation of SMC1 is required for an increased mobility after DNA damage in G2-phase cells, suggesting that ATM-dependent phosphorylation facilitates mobilization of the cohesin complex after DNA damage.

Oncogenic Wip1 Phosphatase Is Inhibited by miR-16 in the DNA Damage Signaling Pathway

Wild-type p53-induced phosphatase 1 (Wip1) was identified as an oncogene amplified and overexpressed in several human cancers. Recent evidence suggested that Wip1 is a critical inhibitor in the ATM/ATR-p53 DNA damage signaling pathway. Wip1 dephosphorylates several key DNA damage-responsive proteins and reverses DNA damage-induced cell cycle checkpoints. Previous reports showed that Wip1 was transcriptionally induced by p53 at the early stage of the DNA damage response. To investigate the temporal and functional regulation of Wip1, we identified a microRNA, miR-16, that specifically targets the mRNA of Wip1 and thus negatively regulates the expression level of Wip1. miR-16 itself is induced immediately after DNA damage. Therefore, the increase in Wip1 protein level is significantly postponed compared with that of its mRNA level, preventing a premature inactivation of ATM/ATR signaling and allowing a functional completion of the early DNA damage response. To better understand miR-16 biological functions in the context of cancer cells, we examined its expression in mammary tumor stem cells and found it to be markedly downregulated in mammary tumor stem cells. Overexpression of miR-16 or inhibition of Wip1 suppresses the self-renewal and growth of mouse mammary tumor stem cells and sensitizes MCF-7 human breast cancer cells to the chemotherapeutic drug doxorubicin. Together, our results suggest an important role of miR-16 in the regulation of Wip1 phosphatase in the DNA damage response and mammary tumorigenesis.

The role of BRCA1 in DNA damage response

BRCA1 is a well-established tumor suppressor gene, which is frequently mutated in familial breast and ovarian cancers. The gene product of BRCA1 functions in a number of cellular pathways that maintain genomic stability, including DNA damage-induced cell cycle checkpoint activation, DNA damage repair, protein ubiquitination, chromatin remodeling, as well as transcriptional regulation and apoptosis. In this review, we discuss recent advances regarding our understanding of the role of BRCA1 in tumor suppression and DNA damage response, including DNA damage-induced cell cycle checkpoint activation and DNA damage repair.

NEK11 regulates CDC25A degradation and the IR-induced G2/M checkpoint

DNA damage-induced cell-cycle checkpoints have a critical role in maintaining genomic stability. A key target of the checkpoints is the CDC25A (cell division cycle 25 homologue A) phosphatase, which is essential for the activation of cyclin-dependent kinases and cell-cycle progression. To identify new genes involved in the G2/M checkpoint we performed a large-scale short hairpin RNA (shRNA) library screen. We show that NIMA (never in mitosis gene A)-related kinase 11 (NEK11) is required for DNA damage-induced G2/M arrest. Depletion of NEK11 prevents proteasome-dependent degradation of CDC25A, both in unperturbed and DNA-damaged cells. We show that NEK11 directly phosphorylates CDC25A on residues whose phosphorylation is required for beta-TrCP (beta-transducin repeat-containing protein)-mediated polyubiquitylation and degradation of CDC25A. Furthermore, we demonstrate that CHK1 (checkpoint kinase 1) directly activates NEK11 by phosphorylating it on Ser 273, indicating that CHK1 and NEK11 operate in a single pathway that controls proteolysis of CDC25A. Taken together, these results demonstrate that NEK11 is an important component of the pathway enforcing the G2/M checkpoint, suggesting that genetic mutations in NEK11 may contribute to the development of human cancer.

FOXO Transcription Factors Enforce Cell Cycle Checkpoints and Promote Survival of Hematopoietic Cells after DNA Damage

The PI3K/AKT signaling pathway contributes to cell cycle progression of cytokine-dependent hematopoietic cells under normal conditions, and it is absolutely required to override DNA damage-induced cell cycle arrest checkpoints in these cells. Phosphatidylinositol-3-kinase (PI3K)/AKT activity also correlates with Cdk2 activity in hematopoietic cells, suggesting that Cdk2 activation may be a relevant end point for this signaling pathway. However, mediators downstream of AKT in this pathway have not been defined. The forkhead transcription factor O (FOXO) family are negatively regulated by AKT-dependent phosphorylation and are known regulators of genes affecting cell cycle progression. We show that enhanced FOXO activity replicates the effect of PI3K inhibitors in enforcing G(1) and G(2) phase arrest after DNA damage. Conversely, knockdown of endogenous FOXO proteins increased Cdk2 activity and overrode DNA damage checkpoints in cells lacking PI3K activity. Moreover, loss of FOXO activity caused an increase in sensitivity to cisplatin-induced cell death, which was associated with failure to arrest cell cycle progression in the face of DNA damage caused by this chemotherapeutic agent. These cell cycle arrests were dependent on p27 expression when mediated by FOXO3a alone, but also involve p27-independent mechanisms when promoted by endogenous FOXO proteins. Together, these observations show that FOXO proteins enforce DNA damage-induced cell cycle arrest in hematopoietic cells. Inhibition of FOXO activity by cytokine-induced PI3K/AKT signaling is sufficient to override these DNA damage-induced cell cycle checkpoints, but may negatively impact hematopoietic cell viability.