DNA Damage Checkpoint Activation Research Articles

Meiotic Checkpoints: Repair or Removal?

Defects in meiosis can produce different checkpoint responses in female and male animals, suggesting that meiotic checkpoints exhibit sexual dimorphism. A recent study in Caenorhabditis elegans indicates that meiotic checkpoint activation is similar between the sexes and the primary difference lies in the downstream consequences.

RNAi-based screening identifies the Mms22L–Nfkbil2 complex as a novel regulator of DNA replication in human cells

Cullin 4 (Cul4)-based ubiquitin ligases emerged as critical regulators of DNA replication and repair. Over 50 Cul4-specific adaptors (DNA damage-binding 1 (Ddb1)-Cul4-associated factors; DCAFs) have been identified and are thought to assemble functionally distinct Cul4 complexes. Using a live-cell imaging-based RNAi screen, we analysed the function of DCAFs and Cul4-linked proteins, and identified specific subsets required for progression through G1 and S phase. We discovered C6orf167/Mms22-like protein (Mms22L) as a putative human orthologue of budding yeast Mms22, which, together with cullin Rtt101, regulates genome stability by promoting DNA replication through natural pause sites and damaged templates. Loss of Mms22L function in human cells results in S phase-dependent genomic instability characterised by spontaneous double-strand breaks and DNA damage checkpoint activation. Unlike yeast Mms22, human Mms22L does not stably bind to Cul4, but is degraded in a Cul4-dependent manner and upon replication stress. Mms22L physically and functionally interacts with the scaffold-like protein Nfkbil2 that co-purifies with histones, several chromatin remodelling and DNA replication/repair factors. Together, our results strongly suggest that the Mms22L-Nfkbil2 complex contributes to genome stability by regulating the chromatin state at stalled replication forks.

Long-distance inflammatory and genotoxic impact of cancer in vivo

The relationship between cancer and DNA damage is intimate and multifaceted. Damage to DNA can cause cancer, major treatment modalities including radiation therapy and chemotherapy can cure or slow down cancer through their genotoxic effects, and germline and somatic defects in the DNA damage response (DDR) machinery may predispose to or promote tumorigenesis, respectively (1, 2). Furthermore, replication stress with the ensuing endogenous DNA damage elicited by oncogenes in nascent tumor cells leads to activation of DNA damage checkpoints and cellular senescence or cell death, thereby providing an intrinsic biological barrier against tumor progression (3–5). The PNAS article by Redon et al. (6) adds yet another dimension to the interplay between DNA damage and cancer: a surprising discovery that cancer can elicit long-range signals that evoke serious types of DNA damage in otherwise healthy tissues in various parts of the body distant to sites of the growing tumor.

Mechanism of the ATP-dependent DNA end-resection machinery from Saccharomyces cerevisiae

If not properly processed and repaired, DNA double-strand breaks (DSBs) can give rise to deleterious chromosome rearrangements, which could ultimately lead to the tumour phenotype. DSB ends are resected in a 5' to 3' fashion in cells, to yield single-stranded DNA (ssDNA) for the recruitment of factors critical for DNA damage checkpoint activation and repair by homologous recombination. The resection process involves redundant pathways consisting of nucleases, DNA helicases and associated proteins. Being guided by recent genetic studies, we have reconstituted the first eukaryotic ATP-dependent DNA end-resection machinery comprising the Saccharomyces cerevisiae Mre11-Rad50-Xrs2 (MRX) complex, the Sgs1-Top3-Rmi1 complex, Dna2 protein and the heterotrimeric ssDNA-binding protein RPA. Here we show that DNA strand separation during end resection is mediated by the Sgs1 helicase function, in a manner that is enhanced by Top3-Rmi1 and MRX. In congruence with genetic observations, although the Dna2 nuclease activity is critical for resection, the Mre11 nuclease activity is dispensable. By examining the top3 Y356F allele and its encoded protein, we provide evidence that the topoisomerase activity of Top3, although critical for the suppression of crossover recombination, is not needed for resection either in cells or in the reconstituted system. Our results also unveil a multifaceted role of RPA, in the sequestration of ssDNA generated by DNA unwinding, enhancement of 5' strand incision, and protection of the 3' strand. Our reconstituted system should serve as a useful model for delineating the mechanistic intricacy of the DNA break resection process in eukaryotes.

Akt/PKB suppresses DNA damage processing and checkpoint activation in late G2

Using chemical genetics to reversibly inhibit Cdk1, we find that cells arrested in late G2 are unable to delay mitotic entry after irradiation. Late G2 cells detect DNA damage lesions and form gamma-H2AX foci but fail to activate Chk1. This reflects a lack of DNA double-strand break processing because late G2 cells fail to recruit RPA (replication protein A), ATR (ataxia telangiectasia and Rad3 related), Rad51, or CtIP (C-terminal interacting protein) to sites of radiation-induced damage, events essential for both checkpoint activation and initiation of DNA repair by homologous recombination. Remarkably, inhibition of Akt/PKB (protein kinase B) restores DNA damage processing and Chk1 activation after irradiation in late G2. These data demonstrate a previously unrecognized role for Akt in cell cycle regulation of DNA repair and checkpoint activation. Because Akt/PKB is frequently activated in many tumor types, these findings have important implications for the evolution and therapy of such cancers.

Sgs1 and Exo1 Redundantly Inhibit Break-Induced Replication and De Novo Telomere Addition at Broken Chromosome Ends

In budding yeast, an HO endonuclease-inducible double-strand break (DSB) is efficiently repaired by several homologous recombination (HR) pathways. In contrast to gene conversion (GC), where both ends of the DSB can recombine with the same template, break-induced replication (BIR) occurs when only the centromere-proximal end of the DSB can locate homologous sequences. Whereas GC results in a small patch of new DNA synthesis, BIR leads to a nonreciprocal translocation. The requirements for completing BIR are significantly different from those of GC, but both processes require 5′ to 3′ resection of DSB ends to create single-stranded DNA that leads to formation of a Rad51 filament required to initiate HR. Resection proceeds by two pathways dependent on Exo1 or the BLM homolog, Sgs1. We report that Exo1 and Sgs1 each inhibit BIR but have little effect on GC, while overexpression of either protein severely inhibits BIR. In contrast, overexpression of Rad51 markedly increases the efficiency of BIR, again with little effect on GC. In sgs1Δ exo1Δ strains, where there is little 5′ to 3′ resection, the level of BIR is not different from either single mutant; surprisingly, there is a two-fold increase in cell viability after HO induction whereby 40% of all cells survive by formation of a new telomere within a few kb of the site of DNA cleavage. De novo telomere addition is rare in wild-type, sgs1Δ, or exo1Δ cells. In sgs1Δ exo1Δ, repair by GC is severely inhibited, but cell viaiblity remains high because of new telomere formation. These data suggest that the extensive 5′ to 3′ resection that occurs before the initiation of new DNA synthesis in BIR may prevent efficient maintenance of a Rad51 filament near the DSB end. The severe constraint on 5′ to 3′ resection, which also abrogates activation of the Mec1-dependent DNA damage checkpoint, permits an unprecedented level of new telomere addition.

Tau Ser262 phosphorylation is critical for A 42-induced tau toxicity in a transgenic Drosophila model of Alzheimer's disease

The amyloid-beta 42 (Abeta42) peptide has been suggested to promote tau phosphorylation and toxicity in Alzheimer's disease (AD) pathogenesis; however, the underlying mechanisms are not fully understood. Using transgenic Drosophila expressing both human Abeta42 and tau, we show here that tau phosphorylation at Ser262 plays a critical role in Abeta42-induced tau toxicity. Co-expression of Abeta42 increased tau phosphorylation at AD-related sites including Ser262, and enhanced tau-induced neurodegeneration. In contrast, formation of either sarkosyl-insoluble tau or paired helical filaments was not induced by Abeta42. Co-expression of Abeta42 and tau carrying the non-phosphorylatable Ser262Ala mutation did not cause neurodegeneration, suggesting that the Ser262 phosphorylation site is required for the pathogenic interaction between Abeta42 and tau. We have recently reported that the DNA damage-activated Checkpoint kinase 2 (Chk2) phosphorylates tau at Ser262 and enhances tau toxicity in a transgenic Drosophila model. We detected that expression of Chk2, as well as a number of genes involved in DNA repair pathways, was increased in the Abeta42 fly brains. The induction of a DNA repair response is protective against Abeta42 toxicity, since blocking the function of the tumor suppressor p53, a key transcription factor for the induction of DNA repair genes, in neurons exacerbated Abeta42-induced neuronal dysfunction. Our results demonstrate that tau phosphorylation at Ser262 is crucial for Abeta42-induced tau toxicity in vivo, and suggest a new model of AD progression in which activation of DNA repair pathways is protective against Abeta42 toxicity but may trigger tau phosphorylation and toxicity in AD pathogenesis.

Abstract 3874: Single-agent Chk1 inhibition is anti-proliferative in leukemia cells in vitro and in vivo

Abstract Chk1 is a serine/threonine kinase that plays important roles in the cellular response to genotoxic stress. For this reason, there is a great deal of interest in using inhibitors of Chk1 to potentiate the effects of DNA-damaging chemotherapeutics. In addition, multiple studies have demonstrated that Chk1 activity is essential during an unperturbed cell cycle to ensure proper DNA replication and maintain genomic integrity. Therefore, it is plausible that a Chk1 inhibitor could also be efficacious as a single-agent therapeutic for human cancer. Here we show that treatment with Chk1-A, a potent and selective inhibitor of Chk1, alone is anti-proliferative against a wide array of cancer cell lines with varying degrees of potency. We sought to understand the mechanisms by which Chk1 inhibition derives the observed anti-proliferative effect. Employing the human leukemia cell line HEL92.1.7, a line particularly sensitive to Chk1 inhibition in terms of proliferation, we characterized the biochemical and functional effects of Chk1-A treatment. We observed concentration-dependent increases in phosphorylation of H2A. X, Chk1, and Chk2, which are markers of DNA damage and cell-cycle checkpoint activation. These biochemical events correlated with S-phase accumulation and eventual apoptosis. In vivo, we found that HEL92.1.7 tumor xenografts were sensitive to oral administration of Chk1-A at a dose that was well tolerated. Together, these studies suggest that inhibition of Chk1 results in DNA damage that induces apoptosis and that use of a Chk1 inhibitor as a single-agent could be an effective strategy to treat certain types of human cancers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3874.

Abstract 3948: Identification of phosphorylated NF45 (T388) as a potential pharmacodynamic biomarker for ARQ 501 (β-lapachone)

Abstract ARQ 501, a fully synthetic version of β-Lapachone, has demonstrated anti-cancer activity and entered multiple phase II trials. Although the mechanism of the cytotoxic activity of ARQ 501 towards cancer cells is not completely understood, it appears that both DNA damage and DNA checkpoint activation play a role in cell death caused by this compound. We used a proteomic approach to search for pharmacodynamic (PD) biomarkers for ARQ 501 by profiling phosphoproteins. ARQ 501 treatment strongly induced threonine phosphorylation of certain proteins around 45 kD in MIA PaCa2 cells. One of the proteins was identified by mass spectrometry analysis as NF45, also known as ILF2. Immunoprecipitation experiments using a customized antibody against pNF45 confirmed that NF45 is phosphorylated in response to ARQ 501 at threonine 388 in a TQ site (an ATM/ATR phosphorylation motif) near the carboxyl terminus. ARQ 501 increased pNF45 expression in a time- and concentration-dependent manner accompanied by activation of ATM and DNAPK. Inhibition of ATM and DNAPK suppressed ARQ 501-induced pNF45 (T388) elevation. Evidence of ARQ 501-induced DNA damage is supported by the observation of CHK2 phosphorylation and γ-H2AX elevation. We therefore examined the effects of several DNA damaging agents on NF45 phosphorylation and found that only ARQ 501 was able to increase pNF45 (T388) levels, suggesting that induction of pNF45 is not a general response to DNA damage, and that ARQ 501 may inhibit cell proliferation by an alternate mechanism. Immunofluorescent staining using pNF45 (T388) antibody showed there is a significant increase in pNF45 (T388) levels predominantly in nuclei. In order to validate pNF45 (T388) as a PD biomarker, we examined pNF45 (T388) levels in human tumor mouse xenograft models treated with ARQ 501. The ARQ 501 treated tumors treated with ARQ 501 showed increased pNF45 (T388) nuclear staining by immunohistochemistry compared to the vehicle group. Therefore, phosphorylation of NF45 at threonine 388 may serve as a PD biomarker for ARQ 501. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3948.

Abstract 3875: Persimmon leaf extract inhibit gamma ray and heavy ion induced DNA damage checkpoint activity in A549 lung adenocarcinoma cells

Abstract Persimmon leaves have been commonly used as traditional medicines in Asia. They have variety kinds of health benefits in such prevention of cancer, hypertension, and inflammation. However, a few of scientific evidences are reported to date. Recent study reported that the cell cycle checkpoint system is playing a pivotal role in DNA damage response, and it is expected that the discovery of checkpoint inhibitor will be an effective assistance of anti-cancer therapy. Checkpoint signaling cascades are critically modulated by ATM (Ataxia telangiectasia-mutated) and ATR (ATM-Rad3-related) protein kinases. Generally, ATM primarily responds to ionizing radiation-induced DNA double-strand breaks, whereas ATR is activated by stalled replication resulting from UV and some genotoxic drugs. In the previous study, we demonstrated that a flavonoid-enriched persimmon leaf extract (FEPLE) promoted cytotoxic effect of cancer cells by chemotherapeutic agents inhibiting the checkpoint activity, especially in ATM dependent pathway. Here, we investigated whether FEPLE inhibits checkpoint activity during DNA damage response induced by radiation such as gamma irrradiation (γIR) and heavy ion (HI) treatment. Dried persimmon leaves were prepared by a decoction for 30 min. A soluble extract was subsequently partitioned into ethyl acetate layer which is flavonoid-enriched persimmon leaves extract (FEPLE). FEPLE included eight flavonol components, non-galloylated flavonoids (kaempferol 3-O-glucoside, kaempferol 3-O-galactoside, quercetin 3-O-glucoside, quercetin 3-O-galactoside) and galloylated flavonoids (their 2″-gallated flavonol glucoside). γIR (2.5 to 20 Gy) and HI (2.5 to 20 Gy) were performed in National Institute of Radiological Sciences (NIRS) and Heavy Ion Medical Accelerator in Chiba (HIMAC), respectively. Human lung adenocarcinoma A549 cells were incubated with FEPLE (0, 1, 10, 30 μg/mL) at one hour prior to γIR or HI treatment. Intracellular levels of phosphorylation in each checkpoint molecule such as p53, SMC1 and Chk1 were determined by western blot using phospho-specific antibodies of Ser15, Ser966 and Ser345, respectively. Phosphorylation on Ser10 of histone H3 was measured by flow-cytometry as a marker of cells in mitosis. The phosphorylation of p53, SMC1 and Chk1 were increased by the treatment of γIR and HI. FEPLE reduced the phosphorylation of checkpoint proteins in both γIR and HI. Furthermore, ATM activity, estimated by the phosphorylation of Ser1981, was inhibited by the treatment of FEPLE. The pre-treatment of FEPLE significantly prevented the decrease of mitotic cells in HI exposed cells. These results indicated that DNA damage checkpoint system in γIR or HI exposed cells were abrogated by FEPLE treatment through the inhibition of ATM-dependent signaling pathway. FEPLE might be useful for clinical application by combining γIR or HI cancer therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3875.

Abstract 3873: Squalene enhanced chemotherapy and radiation therapy sensitivity in mice through the inhibition of DNA damage checkpoint activity

Abstract ATM (Ataxia Telangiectasia Mutated) protein kinase plays a crucial role in cellular DNA damage responses. The inhibition of ATM leads to an abolishment of one such signal pathways termed as “checkpoints”. It is expected that the discovery of checkpoint inhibitor will be an effective assistance of anti-cancer therapy. Recent study reported that Wip1, a magnesium dependent protein phosphatase, de-phosphorylates molecules involved DNA damage checkpoint response such ATM (Ser1981). On the other hand, Squalene (SQ) has been thought to assist anti-cancer therapies such as chemo and/or radiation therapy though there is few available data which refer to its detailed mechanisms. We previously showed that SQ enhances the γ-irradiation (IR) sensitivity through the induction of Wip1 in A549 adenocarcinoma cells. It was indicated that IR induced DNA damage checkpoint was abrogated by SQ pre-treatment, and led to the shrinkage of tumor in vivo. Here, we report SQ sensitizes chemotherapy and an advanced radiation therapy, heavy ion (HI) exposure, in mice using allo/xeno-graft model. Mouse Colon-26 and human MKN45 cells (5 × 106 cells/ mouse) were transplanted into Balb/c and SCID mice, respectively. The tumor volumes were estimated by measuring the growing diameters of tumor tissue every day. SQ (200μl, oral administration) was challenged after day-8 and later days. Chemotherapy using DOX (2 mg/kg, i.p.) was conducted in same days with SQ. One-time exposure of HI (Carbon, 290MeV) was performed on the day-8 in NIRS (National Institute of Radiological Sciences, Japan). Marked change of body weight was not observed between the treatments. DOX and HI prevented the tumor growth. SQ itself inhibited tumor growth slightly. The administration of SQ significantly improved the efficacy of DOX and HI on tumor growth. Wip1 protein expression in tumor tissue was detected by western blot analysis, and was elevated by the oral administration of SQ. These results were confirmed in xenograft model using MKN45 cells and SCID mice. Taken together these findings, SQ sensitized chemo/radio therapy through the inhibition of DNA damage checkpoint activity by Wip1. Further in-depth study is required to determine the clinical feasibility of SQ as a sensitizing agent for anti-cancer therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3873.

A DNA damage-activated checkpoint kinase phosphorylates tau and enhances tau-induced neurodegeneration

Hyperphosphorylation of the microtubule associated protein tau is detected in the brains of individuals with a range of neurodegenerative diseases including Alzheimer's disease (AD). An imbalance in phosphorylation and/or dephosphorylation of tau at disease-related sites has been suggested to initiate the abnormal metabolism and toxicity of tau in disease pathogenesis. However, the mechanisms underlying abnormal phosphorylation of tau in AD are not fully understood. Here, we show that the DNA damage-activated Checkpoint kinase 2 (Chk2) is a novel tau kinase and enhances tau toxicity in a transgenic Drosophila model. Overexpression of Drosophila Chk2 increases tau phosphorylation at Ser262 and enhances tau-induced neurodegeneration in transgenic flies expressing human tau. The non-phosphorylatable Ser262Ala mutation abolishes Chk2-induced enhancement of tau toxicity, suggesting that the Ser262 phosphorylation site is involved in the enhancement of tau toxicity by Chk2. In vitro kinase assays revealed that human Chk2 and a closely related checkpoint kinase 1 (Chk1) directly phosphorylate human tau at Ser262. We also demonstrate that Drosophila Chk2 does not modulate the activity of the fly homolog of microtubule affinity regulating kinase, which has been shown to be a physiological tau Ser262 kinase. Since accumulation of DNA damage has been detected in the brains of AD patients, our results suggest that the DNA damage-activated kinases Chk1 and Chk2 may be involved in tau phosphorylation and toxicity in the pathogenesis of AD.

Caffeine Confers Radiosensitization of PTEN-Deficient Malignant Glioma Cells by Enhancing Ionizing Radiation–Induced G1 Arrest and Negatively Regulating Akt Phosphorylation

PTEN mutations are frequently found in malignant glioma and can result in activated phosphatidylinositol-3-kinase/Akt survival signaling associated with resistance to radiotherapy. Strategies to interfere with aberrant PI3K/Akt activity are therefore being developed to improve the therapeutic efficacy of radiotherapy in patients with malignant glioma. The methylxanthine caffeine has been described as a PI3K inhibitor and is also known to sensitize cells to ionizing radiation. However, a direct association between these two caffeine-mediated effects has not been reported yet. Therefore, we asked whether caffeine or its derivative pentoxifylline differentially affect the radiosensitivity of malignant gliomas with different PTEN status. As models, we used the radiosensitive EA14 malignant glioma cell line containing wild-type PTEN and the radioresistant U87MG malignant glioma cell line harboring mutant PTEN. Our study revealed that caffeine and pentoxifylline radiosensitized PTEN-deficient but not PTEN-proficient glioma cells. Radiosensitization of PTEN-deficient U87MG cells by caffeine was significantly correlated with the activation of the G(1) DNA damage checkpoint that occurred independently of de novo synthesis of p53 and p21. The p53 independency was also confirmed by a significant caffeine-mediated radiosensitization of the glioma cell lines T98G and U373MG that are deficient for both PTEN and p53. Furthermore, caffeine-mediated radiosensitization was associated with the inhibition of Akt hyperphosphorylation in PTEN-deficient cells to a level comparable with PTEN-proficient cells. Our data suggest that the methylxanthine caffeine or its derivative pentoxifylline are promising candidate drugs for the radiosensitization of glioma cells particularly with PTEN mutations.

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

Author SummaryDNA is constantly damaged both by factors outside our bodies (such as ultraviolet rays from sunlight) and by factors from within (such as reactive oxygen species produced during metabolism). DNA damage can lead to malfunctioning of genes, and persistent DNA damage can result in developmental disorders or the development of cancer. To ensure proper DNA repair, cells are equipped with an evolutionarily conserved DNA damage checkpoint, which stops proliferation and activates DNA repair mechanisms. Intriguingly, this DNA damage checkpoint responds to DNA damage throughout the cell cycle, except during mitosis. In this work, we have addressed how cells dismantle their DNA damage checkpoint during mitosis to allow cell division to proceed even if there is damaged DNA present. Using the observation that kinases phosphorylate their substrates on evolutionarily conserved, kinase-specific sequence motifs, we have used a combined computational and experimental approach to predict and verify key proteins involved in mitotic checkpoint inactivation. We show that the checkpoint scaffold protein 53BP1 is phosphorylated by the mitotic kinases Cdk1 and Polo-like kinase-1 (Plk1). Furthermore, we find that Plk1 can inactivate the checkpoint kinase Chk2, which is downstream of 53BP1. Plk1 is shown to be a key mediator of mitotic checkpoint inactivation, as cells that cannot activate Plk1 fail to properly dismantle the DNA damage checkpoint during mitosis and instead show DNA damage-induced Chk2 kinase activation. Two related papers, published in PLoS Biology (Vidanes et al., doi:10.1371/journal.pbio.1000286) and PLoS Genetics (Donnianni et al., doi:10.1371/journal.pgen.1000763), similarly investigate the phenomenon of DNA damage checkpoint silencing.

The MCM-Binding Protein ETG1 Aids Sister Chromatid Cohesion Required for Postreplicative Homologous Recombination Repair

The DNA replication process represents a source of DNA stress that causes potentially spontaneous genome damage. This effect might be strengthened by mutations in crucial replication factors, requiring the activation of DNA damage checkpoints to enable DNA repair before anaphase onset. Here, we demonstrate that depletion of the evolutionarily conserved minichromosome maintenance helicase-binding protein ETG1 of Arabidopsis thaliana resulted in a stringent late G2 cell cycle arrest. This arrest correlated with a partial loss of sister chromatid cohesion. The lack-of-cohesion phenotype was intensified in plants without functional CTF18, a replication fork factor needed for cohesion establishment. The synergistic effect of the etg1 and ctf18 mutants on sister chromatid cohesion strengthened the impact on plant growth of the replication stress caused by ETG1 deficiency because of inefficient DNA repair. We conclude that the ETG1 replication factor is required for efficient cohesion and that cohesion establishment is essential for proper development of plants suffering from endogenous DNA stress. Cohesion defects observed upon knockdown of its human counterpart suggest an equally important developmental role for the orthologous mammalian ETG1 protein.

ATM- and ATR-mediated response to DNA damage induced by a novel camptothecin, ST1968

DNA damage response and checkpoint activation are expected to influence the sensitivity to DNA-damaging agents. This study was designed to investigate the DNA damage response to the novel camptothecin, ST1968, in two tumor cell lines with a different biological background (A2780 and KB), which underwent distinct cell cycle perturbations and cell death modalities. Following treatment with the camptothecin or ionizing radiation, both inducing double-strand DNA breaks, the ovarian carcinoma A2780 cells exhibited activation of the ATM-Chk2 pathway and early induction of apoptosis. In contrast, the squamous carcinoma KB cells exhibited activation of ATR-Chk1 pathway, a persistent G 2/M-phase arrest, cellular senescence, mitotic catastrophe and delayed apoptosis, suggesting a defective ATM pathway. The cellular response to UV-induced DNA damage, which activates ATR-Chk1 pathway, was similar in the two cell lines exhibiting early apoptosis induction. Inhibition of ATM in A2780 cells, resulting in reduced phosphorylation of Chk2, enhanced ST1968-induced apoptosis, but had no effect in KB cells. The susceptibility to camptothecin-induced apoptosis of A2780 cells was likely p53-dependent but not related to the activation of the ATM pathway. In contrast, the inhibition of Chk1 enhanced apoptosis response in KB cell but not in A2780. Thus, depending on the biological context, the camptothecin activated ATM-Chk2 or ATR-Chk1 pathways, both having a protective role. In conclusion, our results are consistent with the interpretation that the modality of cell death response is not the critical determinant of sensitivity to camptothecins, and support the interest of inhibition of checkpoint kinases to improve the efficacy of camptothecins.

The p53-inducible gene 3 (PIG3) contributes to early cellular response to DNA damage

The p53-inducible gene 3 (PIG3) is originally isolated as a p53 downstream target gene, but its function remains unknown. Here, we report a role of PIG3 in the activation of DNA damage checkpoints, after UV irradiation or radiomimetic drug neocarzinostatin (NCS). We show that depletion of endogenous PIG3 sensitizes cells to DNA damage agents, and impaired DNA repair. PIG3 depletion also allows for UV- and NCS-resistant DNA synthesis and permits cells to progress into mitosis, indicating that PIG3 knockdown can suppress intra-S phase and G2/M checkpoints. PIG3-depleted cells show reduced Chk1 and Chk2 phosphorylation after DNA damage, which may directly contribute to checkpoint bypass. PIG3 exhibited diffuse nuclear staining in the majority of untreated cells and forms discrete nuclear foci in response to DNA damage. PIG3 colocalizes with gamma-H2AX and 53BP1 to sites of DNA damage after DNA damage, and binds to a gamma-H2AX. Notably, PIG3 depletion decreases the efficient induction and maintenance of H2AX phosphorylation after DNA damage. Moreover, PIG3 contributes to the recruitment of 53BP1, Mre11, Rad50 and Nbs1 to the sites of DNA break lesions in response to DNA damage. Our combined results suggest that PIG3 is a critical component of the DNA damage response pathway and has a direct role in the transmission of the DNA damage signal from damaged DNA to the intra-S and G2/M checkpoint machinery in human cells.

Abstract B248: Characterization and preclinical development of LCI-1, a selective and potent Chk1 inhibitor in phase 1 clinical trials

Abstract Interference of DNA damage checkpoints has been demonstrated to be a highly effective means of increasing the cytotoxicity of a wide number of anti-cancer therapies. Ionizing radiation, DNA cross-linkers, topoisomerase inhibitors and anti-metabolites all cause severe cellular DNA damage and activation of multiple DNA damage checkpoints. Cell cycle arrest at these checkpoints protects injured cells from apoptotic cell death until the DNA damage can be repaired. In the absence of functioning DNA damage checkpoints, cells with damaged DNA proceed into premature mitosis followed by rapid apoptotic death. A key protein kinase involved in activating and maintaining the S and G2/M checkpoints is Chk1. Pharmacological inhibition of Chk1 in the absence of p53 leads to abrogation of the DNA damage checkpoints and has been shown to enhance the preclinical activity of many standard of care chemotherapeutic agents. LCI-1 is a novel small molecule inhibitor of Chk1 (IC50 = 7 nM). In cell-based experiments LCI-1 inhibited doxorubicin-dependent autophosphorylation of Chk1 (IC50 = 52 nM). Treatment of cells with LCI-1 alone caused a phenotype identical to that reported for cells depleted of Chk1, demonstrating the mechanism-dependent activity of the compound. Treated with 100 nM doxorubicin, HeLa cells arrested at the G2M checkpoint. When treated 24 hours later with LCI- 1, the arrested cells traversed the G2M checkpoint, allowing cells to proceed into mitosis with unresolved replicated chromosomes. Consistent with abrogation of the Chk1-dependent G2M checkpoint, HT-29 cells (mutant p53) treated with LCI-1 were more sensitive to killing by gemcitabine then were LCI-1 treated HCT116 cells (wild-type p53). In vivo, LCI-1 effectively inhibited Chk1 autophosphorylation, as well as released the S and G2M block induced by gemcitabine treatment. When used preclinically in combination with DNA damaging agents, LCI-1 increased DNA damage and cell death over gemcitabine alone. These results indicate that LCI-1 may prove to be an effective potentiator of DNA damaging therapies in the clinic. LCI-1 is currently undergoing clinical testing in combination with gemcitabine and pemetrexed. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B248.

Oncogenic RAS Enables DNA Damage- and p53-Dependent Differentiation of Acute Myeloid Leukemia Cells in Response to Chemotherapy.

Abstract 5040 IntroductionAcute myeloid leukemia (AML) is a clonal disease originating from myeloid progenitor cells with a heterogeneous genetic background. High-dose cytarabine is used as the standard consolidation chemotherapy. Oncogenic RAS mutations are frequently observed in AML, and patients with RAS mutations benefit most from high-dose cytarabine as postremission therapy (Neubauer et al., J Clin Oncol 2008). The molecular reason for this phenomenon is not well understood. MethodsWe used bone marrow cells expressing a conditional MLL-ENL-ER oncogene to investigate the interaction of oncogenic RAS and chemotherapeutic agents. In addition, we used primary human inversion 16 positive AML samples with or without oncogenic RAS mutations to corroborate our findings. ResultsThere was no difference in cell cycle kinetics, apoptosis or cellular senescence in MLL-ENL cell expressing control vector vs. MLL-ENL cells expressing oncogenic RAS in response to cytarabine treatment. However, we observed an increased activation of DNA damage checkpoints in MLL-ENL-cells expressing oncogenic RAS after incubation with chemotherapeutic agents. This resulted in an Atm/r as well as p53-dependent genetic program causing dramatically reduction of clonogenicity in MLL-ENL cells expressing oncogenic RAS due to induction of myeloid differentiation. Co-expression of dominant – negative p53 completely abolished this differentiation resulting in the rescue of the clonogic potential. Activation of p53 as a result of inhibition of Mdm2-mediated degradation of p53 further enhances this myeloid differentiation. Of note, in primary AML cases, oncogenic RAS also was associated with a more differentiated phenotype. ConclusionsThe data can explain the beneficial effects observed in AML patients with oncogenic RAS mutations treated with high dosages of cytarabine and suggest that induction of p53-dependent differentiation, e.g. by interfering with Mdm2-mediated degradation, may be a rational approach to increase cure rate in response to chemotherapy since stronger acitvation of p53 by oncogenic RAS and chemotherapeutic agents cause the differentiation of hematopoietic stem cells to more differentiated progenitors. The data also support the notion that the therapeutic success of cytotoxic drugs may depend on their ability to promote the differentiation of tumor-initiating cells. DisclosuresNo relevant conflicts of interest to declare.

Oncogenic RAS Enables DNA Damage- and p53-Dependent Differentiation of Acute Myeloid Leukemia Cells in Response to Chemotherapy

Acute myeloid leukemia (AML) is a clonal disease originating from myeloid progenitor cells with a heterogeneous genetic background. High-dose cytarabine is used as the standard consolidation chemotherapy. Oncogenic RAS mutations are frequently observed in AML, and are associated with beneficial response to cytarabine. Why AML-patients with oncogenic RAS benefit most from high-dose cytarabine post-remission therapy is not well understood. Here we used bone marrow cells expressing a conditional MLL-ENL-ER oncogene to investigate the interaction of oncogenic RAS and chemotherapeutic agents. We show that oncogenic RAS synergizes with cytotoxic agents such as cytarabine in activation of DNA damage checkpoints, resulting in a p53-dependent genetic program that reduces clonogenicity and increases myeloid differentiation. Our data can explain the beneficial effects observed for AML patients with oncogenic RAS treated with higher dosages of cytarabine and suggest that induction of p53-dependent differentiation, e.g. by interfering with Mdm2-mediated degradation, may be a rational approach to increase cure rate in response to chemotherapy. The data also support the notion that the therapeutic success of cytotoxic drugs may depend on their ability to promote the differentiation of tumor-initiating cells.