Activation Of DNA Repair Pathways Research Articles

Selective Inhibition of p300 HAT Blocks Cell Cycle Progression, Induces Cellular Senescence, and Inhibits the DNA Damage Response in Melanoma Cells

Epigenetic events, including covalent post-translational modifications of histones, have been demonstrated to play critical roles in tumor development and progression. The transcriptional coactivator p300/CBP possesses both histone acetyltransferase (HAT) activity as well as scaffolding properties that directly influence the transcriptional activation of targeted genes. We have used a potent and specific inhibitor of p300/CBP HAT activity, C646, in order to evaluate the functional contributions of p300/CBP HAT to tumor development and progression. Here we report that C646 inhibits the growth of human melanoma and other tumor cells and promotes cellular senescence. Global assessment of the p300 HAT transcriptome in human melanoma identified functional roles in promoting cell cycle progression, chromatin assembly and activation of DNA repair pathways through direct transcriptional regulatory mechanisms. Additionally, C646 is shown to promote sensitivity to DNA damaging agents, leading to the enhanced apoptosis of melanoma cells following combination treatment with cisplatin. Together, our data suggest that p300 HAT activity mediates critical growth regulatory pathways in tumor cells and may serve as a potential therapeutic target for melanoma and other malignancies by promoting cellular responses to DNA damaging agents that are currently ineffective against specific cancers.

Abstract 1589: LKB1 overexpression regulates DNA repair pathway and sensitivity to radiation in LKB1 mutant non-small cell lung cancer cell lines .

Abstract Background: LKB1 is a serine/threonine kinase which is mutated in 20-30% of non-small cell lung cancers (NSCLC).In general, DNA repair pathway activation leads cellular resistant to DNA damaging agents. The loss of LKB1 leads NSCLC to be more invasive and resistant to DNA damage based therapies. We hypothesize that loss of LKB1 may contribute to DNA damage response. We investigated LKB1- mediated signaling pathways by stably overexpressing LKB1 in its mutated NSCLC cell lines, A549 and H460. Methods: Expression of 139 proteins was analyzed by reverse phase protein array (RPPA) in LKB1 overexpressing cell lines along with the appropriate vector control lines. Differences in protein expression at baseline in clones with LKB1 versus clones with vector control were assessed by unpaired T-test with Welch's correction. Ionizing radiation sensitivity (reproductive cell death) was assessed by clonogenic cell survival assay (CSA). Results: LKB1 overexpression in A549 and H460 showed a significant downregulation of genes involved in DNA damage and repair pathway, such as RAD50 (1.4-fold relative expression, p< 0.05 and 1.5-fold relative expression p<0.02 respectively), XRCC1 (1.2-fold relative expression p<0.005 and 1.1-fold relative expression p<0.05, respectively), and DNA-PKCS (1.2-fold relative expression p≈0.067 and 1.2-fold relative expression p<0.01. Also, CSA showed that A549 with LKB1 overexpression led to enhanced sensitization of cells to radiation (DER = 2) which is in consistent with the downregulation of RAD50, XRCC1 and DNA-PKCS. Furthermore, expression of CHK2 and phosphorylation of CHK1 are downregulated, H460 with LKB1 overexpression showed a significant decreasing of CHK1 phosphorylation level (p<0.05). Conclusion: Our results suggest that LKB1 overexpression modulates DNA repair capacity through downregulation of RAD50, XRCC1, DNA-PKCS, CHK1 and CHK2. LKB1 loss may lead to the resistance to DNA damaging induced cell death. Citation Format: Chao Yang, Uma Giri, Maria Angelica Cortez, Jayanthi Gudikote, YouHong Fan, Lixia Diao, Lauren A. Byers, John V. Heymach. LKB1 overexpression regulates DNA repair pathway and sensitivity to radiation in LKB1 mutant non-small cell lung cancer cell lines . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1589. doi:10.1158/1538-7445.AM2013-1589

A STUbL wards off telomere fusions

Protection of chromosomal ends from erroneous recognition as double‐strand breaks (DSBs) is critical for maintaining genome integrity. Unprotected ends can activate DNA repair pathways leading to catastrophic fusions or rearrangements of chromosomes. A study in The EMBO Journal identifies Uls1 as a new player in this end‐protection process, utilizing SUMO and ubiquitin modifications to prevent chromosomal fusions by clearing a polysumoylated form of Rap1. Telomeres, the ends of linear chromosomes, pose a tremendous challenge for eukaryotic cells, because their resemblance to DNA DSBs puts them at constant risk of being targeted by repair pathways. Thus, these ends must be kept off the radar of the two major DSB repair pathways, non‐homologous end joining (NHEJ) and homologous recombination (HR). A number of studies in the model system budding yeast previously identified a central role for the Rap1 protein in preventing NHEJ at telomeres. Rap1 is an evolutionarily conserved protein that binds to the double‐stranded DNA region of telomeres. It inhibits NHEJ via at least three different pathways (Marcand et al , 2008). Two of these are mediated by its binding partners Rif2 and Sir4, respectively, with a less well‐characterized third pathway independent of either interactor. How these pathways enable NHEJ evasion is not entirely clear, but recent findings of posttranslational modifications of telomere‐binding proteins, particularly by SUMO, hinted at a possible role for dynamic protein modifications in regulating telomeric functions. Attachment of the SUMO protein to a lysine residue in target proteins can change both their interaction profiles, by allowing other proteins to bind or impairing interactions, and their biochemical functions. Additionally, since the SUMO polypeptide itself contains lysines, poly‐SUMO chains can be generated, which …

Abstract 2751: Fanconi anemia pathway and DNA repair-based prognostic signatures in ovarian cancer

Abstract Introduction: Of the 21,000 patients diagnosed annually in the United States with epithelial ovarian cancer, approximately 75% present with Stage III-IV disease. The vast majority of these patients are treated with cytotoxic chemotherapy regimens, where clinical response may be associated with activity of DNA repair pathways in tumors. Characterizing these repair pathways should help predict clinical response to specific chemotherapeutic agents. Methods: We devised a set of 150 DNA repair genes with documented functional importance in key DNA repair pathways including base excision repair (BER), Fanconi Anemia (FA), homologous recombination (HR), mismatch repair (MMR), non-homologous end joining (NHEJ), nucleotide excision repair (NER) and translesion synthesis (TLS). We extracted microarray expression data for these genes for 511 tumors and 8 normal specimens using data from Agilent Custom 244K Gene Expression and Affymetrix Human Genome U133A arrays. We performed Significance Analysis of Microarrays (SAM) to determine which of the 150 DNA repair genes were significantly more expressed in tumor vs. normal specimens. We then generated Kaplan-Meier survival curves using overall survival data. Results: Using the Affymetrix data, we identified 57 genes that were significantly up-regulated with a median fold change of 3.15 (range, 2.03-6.07) and 6 genes that were significantly down-regulated with a median fold change of 2.67 (range, 2.11-4.74) among tumor vs. normal tissue samples by SAM (delta 1.013, false discovery rate 0%). SAM using the Agilent data showed similar results; a total of 46 genes were significant hits in both datasets. A DNA repair signature consisting of genes significant in both platforms was defined and revealed a correlation between high expression values and decreased survival (median overall survival 4.5 vs. 5.2 years, log-rank p = 0.0086). DNA repair signatures specific to individual repair pathways were defined and demonstrated that high FA pathway (BRCA2, FAAP24, FANCA, FANCE, FANCG, FANCI and PALB2) expression in particular was inversely correlated with survival (median overall survival 4.1 vs. 5.2 years, log-rank p = 0.029 and 0.019 for the Affymetrix and Agilent datasets, respectively). Conclusion: Evidence from literature supports the critical role of the Fanconi Anemia (FA) pathway in repairing DNA damage caused by platinum-based agents such as cisplatin and carboplatin, commonly utilized to treat ovarian cancer patients. We show that a DNA repair signature specific to FA can be utilized to identify patients with high FA expression, whom we hypothesize may be more resistant to traditional platinum-based therapy. 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 2751. doi:10.1158/1538-7445.AM2011-2751

Challenges of drug resistance in the management of pancreatic cancer

The current treatment of choice for metastatic pancreatic cancer involves single-agent gemcitabine or a combination of gemcitabine with capecitabine or erlotinib (a tyrosine kinase inhibitor). Only 25–30% of patients respond to this treatment and patients who do respond initially ultimately exhibit disease progression. Median survival for pancreatic cancer patients has reached a plateau due to inherent and acquired resistance to these agents. Key molecular factors implicated in this resistance include: deficiencies in drug uptake, alteration of drug targets, activation of DNA repair pathways, resistance to apoptosis and the contribution of the tumor microenvironment. Moreover, for newer agents including tyrosine kinase inhibitors, overexpression of signaling proteins, mutations in kinase domains, activation of alternative pathways, mutations of genes downstream of the target and/or amplification of the target represent key challenges for treatment efficacy. Here we will review the contribution of known mechanisms and markers of resistance to key pancreatic cancer drug treatments.

Mammalian Rap1 widens its impact

Three recent studies reveal unexpected functions of Rap1, a member of the shelterin complex that protects chromosome ends from the activity of DNA repair pathways. Rap1 not only protects telomeres from sister chromatid exchange, but also functions in genome-wide transcriptional regulation and NF-κB-dependent signalling, revealing new perspectives for the telomere field.

Evidence Linking Genetics, Environment, and Epigenetics to Impaired DNA Repair in Alzheimer's Disease

Increasing evidence suggests that the repair of DNA lesions, particularly oxidative DNA lesions, might be compromised in Alzheimer's disease (AD). Studies performed in brains and peripheral tissues of both AD patients and individuals affected by mild cognitive impairment (MCI) revealed that oxidative DNA damage is one of the earliest detectable events during the progression from healthy aging to dementia. Moreover, the increase in DNA damage is paralleled by a decrease in DNA repair activities. Several hypotheses are currently tested in order to explain the decreased DNA repair activity observed in MCI and AD subjects. Some authors have suggested that mutations or polymorphisms in DNA repair genes might impair DNA repair. However, this hypothesis does not seem to be confirmed by recent genetic association studies. Others suggest that DNA repair proteins might be inactivated by oxidative induced post-translational modifications or degradation. There is also indication that different isoforms of the same repair protein might be involved in the progression from early to late stages AD. Moreover, a widespread activation of DNA repair pathways might generate death signals ending with neuronal apoptosis. A link between environmental induced epigenetic modification, oxidation, and repair of AD related genes has been also proposed. Most of these studies have been performed during the last few years, and we are still at the beginning of understanding the complex interplay between oxidative DNA damage, DNA repair, and neuronal death in the brain leading to Alzheimer's dementia, making this topic an exciting and promising field for future investigation.

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.

Loss of Rap1 Induces Telomere Recombination in the Absence of NHEJ or a DNA Damage Signal

Shelterin is an essential telomeric protein complex that prevents DNA damage signaling and DNA repair at mammalian chromosome ends. Here we report on the role of the TRF2-interacting factor Rap1, a conserved shelterin subunit of unknown function. We removed Rap1 from mouse telomeres either through gene deletion or by replacing TRF2 with a mutant that does not bind Rap1. Rap1 was dispensable for the essential functions of TRF2--repression of ATM kinase signaling and nonhomologous end joining (NHEJ)--and mice lacking telomeric Rap1 were viable and fertile. However, Rap1 was critical for the repression of homology-directed repair (HDR), which can alter telomere length. The data reveal that HDR at telomeres can take place in the absence of DNA damage foci and underscore the functional compartmentalization within shelterin.

Promyelocytic Leukemia Nuclear Bodies Link the DNA Damage Repair Pathway with Hepatitis B Virus Replication: Implications for Hepatitis B Virus Exacerbation during Chemotherapy and Radiotherapy

The mechanism responsible for hepatitis B virus (HBV) exacerbation during chemotherapy and radiotherapy remains unknown. We investigated whether the activation of DNA repair pathways influences HBV replication. The upregulation of the promyelocytic leukemia (PML) protein and its associated PML nuclear body (PML-NB) by chemotherapy and irradiation-induced DNA repair signaling correlated with the upregulation of HBV pregenomic transcription, HBV-core expression, and HBV DNA replication. The HBV-core protein and HBV DNA localized to PML-NBs, where they associated with PML and histone deacetylase 1 (HDAC1). Chemotherapy and radiotherapy affected the interactions between PML, HBV-core, and HDAC1. The enhanced protein-protein interaction between PML and HBV-core inhibited PML-mediated apoptosis and decreased PML-associated HDAC activity. The reversal of HDAC-mediated repression on the HBV covalently closed circular DNA basal core promoter resulted in the amplification of HBV-core and pregenomic expression. These results suggest that PML in PML-NBs links the DNA damage response with HBV replication and may cooperate with HBV-core and HDAC1 on the HBV covalently closed circular DNA basal core promoter to form a positive feedback loop for HBV exacerbation during chemotherapy and radiotherapy.

MiRNAs modulate the drug response of tumor cells

Chemotherapy is one of the major treatments of malignant carcinomas. However, its efficiency is affected by both intrinsic and acquired resistance to anticancer drugs. The cellular mechanisms of drug resistance include the overexpression of energy-dependent transporters that eject anticancer drugs from cells such as p-glycoprotein and multidrug resistance related protein (MRP), the mutation of drug targets, the activation of DNA repair pathways, the defects in cellular death pathways and so on. The genetic and epigenetic changes of these genes can lead to cancer drug resistance. Among these mechanisms, microRNAs (miRNAs) which are critical and essential for many important processes such as development, differentiation, and even carcinogenesis have been reported to regulate the chemo-sensitivity of tumor cells. In this paper we briefly review the relationship between miRNA and cancer drug resistance.

ATM Gene Alterations in CLL Patients Induce Gene Profile Clusters and Predict Disease Progression.

Chronic lymphocytic leukemia (CLL) presents a highly variable clinical course, with some patients surviving for many years without requiring treatment, while others have rapidly progressing disease, require aggressive treatment and show a short life expectancy. Several biological features of the leukemic cells - the mutational status of the immunoglobulin (Ig) heavy (H) chain variable (V) genes, cytogenetic alterations, ZAP-70 and CD38 antigen expression, p53 dysfunctions – bear prognostic value and allow to stratify patients into risk categories. The deletion of the chromosome region 11q22–q23 that occurs in 10–20% of cases represents the second most common genetic abnormality in CLL and defines a subgroup of patients characterized by advanced progressive disease and poor survival. The ATM (ataxia telangiectasia-mutated) gene is located in this region and several studies suggest that the 11q deletion results in ATM gene inactivation. The ATM gene consists of 66 exons, 62 (5–66) of which are coding exons. The ATM protein is a nuclear serine/threonine kinase of 350 kDa induced by chromosomal double-strand breaks that arise endogenously or after exposure to DNA-damaging agents, including ionizing radiations and drugs. It protects the integrity of the genome in several ways: it regulates the arrest of the cell cycle at the G1/S and G2/M phases to prevent the processing of damaged DNA; it activates DNA repair pathways and induces apoptosis if the DNA damage cannot be repaired. In the present study we examined the status of the ATM gene by DHPLC (Denaturing High Performance Liquid Cromatography) in a series of untreated CLL patients studied at the Division of Hematology, “Sapienza” University of Rome. In addition, a multiplex gene dosage analysis of the ATM gene was performed by MLPA (Multiplex Ligation Probe Amplification) in all patients. The results obtained have been correlated with the molecular status of the IgVH genes, the presence of cytogenetic abnormalities, ZAP-70 and CD38 expression, p53 mutations, and with the gene expression profile. Mutational screening of the ATM gene identified mutations in 8 of the 57 CLL patients studied (14%): 6 missense, 8095C>T (Pro2699Ser), 8071C>T (Arg2691Cys), 2476A>C (Ile826Leu) and, in 3 patients, 1435G>T (Asp479Tyr); 1 frameshift: 2502insA; 1 splicing mutation: IVS29+5G>A. In addition, 14 different variants and polymorphisms of unknown functional significance were found. The 8 ATM mutated cases showed the following features: 6 were IgVH unmutated, 4 were ZAP70+, 5 were CD38+, 4 harbored a 13q14 deletion, only 1 patient showed a 17p13 deletion and 2 a 14q32 deletion; furthermore, 2/8 patients showed a p53 gene mutation; none showed the 11q deletion. MLPA analysis identified 6 patients with 11q deletion and all of them showed at least 20% of cells positive for the 11q23 deletion evaluated by FISH; only 1 case showed a 17p13 deletion. In these cases, the IgVH gene was always unmutated and ZAP70 was positive; 4 cases were also CD38+. ATM gene mutated and deleted CLL cases showed a peculiar gene profile characterized by the up-regulation and down-regulation of genes involved in apoptosis and DNA repair function. On the contrary, no gene profile difference was found in patients carrying ATM gene polymorphisms. Patients with ATM gene mutated and deleted showed a short time to treatment, 23 and 19 months respectively. The results of this study indicate that ATM gene alterations - mutations or deletions - account for 24% of untreated CLL patients, are associated with a defined gene profile and are characterized by a poor prognosis. It is important to underline that these patients have been evaluated before any treatment and that ATM mutations have been found in the absence of 11q23 deletions. ATM alterations appear to represent the most frequent genetic abnormality with unfavorable prognostic implications in CLL and should be investigated prior to initiating therapy in order to plan an appropriate treatment algorithm.

Statins Use a Novel Nijmegen Breakage Syndrome-1–Dependent Pathway to Accelerate DNA Repair in Vascular Smooth Muscle Cells

Although the hydroxymethylglutaryl-coenzyme A reductase inhibitors (statins) are widely used in atherosclerosis to reduce serum cholesterol, statins have multiple other effects, including direct effects on cells of the vessel wall. Recently, DNA damage, including telomere shortening, has been identified in vascular smooth muscle cells (VSMCs) in human atherosclerosis. Although statins reduce DNA damage in vitro, the mechanisms by which they might protect DNA integrity in VSMCs are unknown. We show that human atherosclerotic plaque VSMCs exhibit increased levels of double-stranded DNA breaks and basal activation of DNA repair pathways involving ataxia telangiectasia-mutated (ATM) and the histone H2AX in vivo and in vitro. Oxidant stress induced DNA damage and activated DNA repair pathways in VSMCs. Statin treatment did not reduce oxidant stress or DNA damage but markedly accelerated DNA repair. Accelerated DNA repair required both the Nijmegen breakage syndrome (NBS)-1 protein and the human double minute protein Hdm2, accompanied by phosphorylation of Hdm2, dissociation of NBS-1 and Hdm2, inhibition of NBS-1 degradation, and accelerated phosphorylation of ATM. Statin treatment reduced VSMC senescence and telomere attrition in culture, accelerated DNA repair and reduced apoptosis in vivo after irradiation, and reduced ATM/ATR (ATM and Rad3-related) activity in atherosclerosis. We conclude that statins activate a novel mechanism of accelerating DNA repair, dependent on NBS-1 stabilization and Hdm2. Statin treatment may delay cell senescence and promote DNA repair in atherosclerosis.

Coupling of Mutated Met Variants to DNA Repair via Abl and Rad51

Abnormal activation of DNA repair pathways by deregulated signaling of receptor tyrosine kinase systems is a compelling likelihood with significant implications in both cancer biology and treatment. Here, we show that due to a potential substrate switch, mutated variants of the receptor for hepatocyte growth factor Met, but not the wild-type form of the receptor, directly couple to the Abl tyrosine kinase and the Rad51 recombinase, two key signaling elements of homologous recombination-based DNA repair. Treatment of cells that express the mutated receptor variants with the Met inhibitor SU11274 leads, in a mutant-dependent manner, to a reduction of tyrosine phosphorylated levels of Abl and Rad51, impairs radiation-induced nuclear translocation of Rad51, and acts as a radiosensitizer together with the p53 inhibitor pifithrin-alpha by increasing cellular double-strand DNA break levels following exposure to ionizing radiation. Finally, we propose that in order to overcome a mutation-dependent resistance to SU11274, this aberrant molecular axis may alternatively be targeted with the Abl inhibitor, nilotinib.

Telomeres and Aging

Telomeres play a central role in cell fate and aging by adjusting the cellular response to stress and growth stimulation on the basis of previous cell divisions and DNA damage. At least a few hundred nucleotides of telomere repeats must "cap" each chromosome end to avoid activation of DNA repair pathways. Repair of critically short or "uncapped" telomeres by telomerase or recombination is limited in most somatic cells and apoptosis or cellular senescence is triggered when too many "uncapped" telomeres accumulate. The chance of the latter increases as the average telomere length decreases. The average telomere length is set and maintained in cells of the germline which typically express high levels of telomerase. In somatic cells, telomere length is very heterogeneous but typically declines with age, posing a barrier to tumor growth but also contributing to loss of cells with age. Loss of (stem) cells via telomere attrition provides strong selection for abnormal and malignant cells, a process facilitated by the genome instability and aneuploidy triggered by dysfunctional telomeres. The crucial role of telomeres in cell turnover and aging is highlighted by patients with 50% of normal telomerase levels resulting from a mutation in one of the telomerase genes. Short telomeres in such patients are implicated in a variety of disorders including dyskeratosis congenita, aplastic anemia, pulmonary fibrosis, and cancer. Here the role of telomeres and telomerase in human aging and aging-associated diseases is reviewed.

ATR Pathway Is the Primary Pathway for Activating G2/M Checkpoint Induction After Re-replication

DNA replication is tightly controlled to ensure accurate chromosome duplication and segregation in each cell cycle. Inactivation of Geminin, an inhibitor of origin licensing, leads to re-replication in human tumor cells within the same cell cycle and triggers a G(2)/M checkpoint. We find that the primary pathway to signal that re-replication has been detected is the ATR kinase and the Rad9-Rad1-Hus1 (9-1-1) clamp complex together with Rad17-RFC clamp loader. ATM kinase and the Mre11-Rad50-Nbs1 complex do not appear to play significant roles in the checkpoint. Chk1 activation occurs at early stages, whereas Chk2 activation occurs much later. Overall we conclude that ATR/Chk1 pathway is activated at an early time point after the loss of Geminin and contributes to checkpoint arrest essential for the accumulation of re-replicated cells, whereas activation of the ATM/Chk2 pathway is a by-product of DNA re-replication at a later period.

A Novel Poly(ADP-ribose) Polymerase Inhibitor, ABT-888, Sensitizes Malignant Human Cell Lines to Ionizing Radiation Under Oxia and Hypoxia

Poly(ADP-ribose) polymerase (PARP) is a nuclear enzyme that recognizes and binds to DNA breaks, and facilitates the subsequent recruitment of DNA repair proteins, thereby coupling the sensing of DNA damage to the activation of DNA repair pathways. There has been much interest in the development of specific PARP inhibitors to potentiate radiotherapy and chemotherapy. Phase I clinical trials are ongoing, but it is unclear as to whether this agent is effective against hypoxic cells. We therefore tested the role for a novel PARP inhibitor, ABT-888, as a radiosensitizing agent in a preclinical context under oxia and hypoxia.

A systems biology analysis of protein–protein interactions between yeast superoxide dismutases and DNA repair pathways

Superoxide dismutases (SODs) are widely distributed in eukaryotic and prokaryotic species and are responsible for O 2 − scavenging and dismutation to H 2O 2 and O 2. Mutations in the cytoplasmic (Sod1p) or mitochondrial (Sod2p) form of SODs result in aging, neurodegenerative diseases, and carcinogenesis. Diminished activity of SODs leads to reduced activity of DNA repair pathways, and overexpression of SODs in cells defective for DNA repair increases their level of chromatin damage. Unfortunately, little is understood regarding the interplay between SODs and DNA repair proteins and their role in protecting the genome from oxidative damage. To elucidate the association between yeast SODs and DNA repair mechanisms, a systems biology study was performed employing algorithms of literature data mining and the construction of physical protein–protein interactions from large yeast protein databases. The results obtained in this work allow us to draw two models suggesting that yeast SODs act as O 2 − sensors under conditions of redox imbalance, activating and controlling specific DNA repair mechanisms (e.g., recombinational and excision repair pathways), chromatin remodeling, and synthesis of dNTPs.

DYRK2 Is Targeted to the Nucleus and Controls p53 via Ser46 Phosphorylation in the Apoptotic Response to DNA Damage

Genotoxic stress exerts biological activity by activating downstream effectors, including the p53 tumor suppressor. p53 regulates cell-cycle checkpoint and induction of apoptosis in response to DNA damage; however, molecular mechanisms responsible for committing to these distinct functions remain to be elucidated. Recent studies demonstrated that phosphorylation of p53 at Ser46 is associated with induction of p53AIP1 expression, resulting in commitment to apoptotic cell death. In this regard, the role for Ser46 kinases in p53-dependent apoptosis has been established; however, the kinases responsible for Ser46 phosphorylation have yet to be identified. Here, we demonstrate that the dual-specificity tyrosine-phosphorylation-regulated kinase 2 (DYRK2) directly phosphorylates p53 at Ser46. Upon exposure to genotoxic stress, DYRK2 translocates into the nucleus for Ser46 phosphorylation. Consistent with these results, DYRK2 induces p53AIP1 expression and apoptosis in a Ser46 phosphorylation-dependent manner. These findings indicate that DYRK2 regulates p53 to induce apoptosis in response to DNA damage.

KLF4 and KLF5 regulate proliferation, Apoptosis and invasion in esophageal cancer cells

KLF4 and KLF5, members of the KLF family of transcription factors, play key roles in proliferation, differentiation, and carcinogenesis in a number of gastrointestinal tissues. While KLF4 is expressed in differentiating epithelial cells, KLF5 is found in proliferating cells of the gastrointestinal tract, including the esophagus. KLF4 regulates a number of genes vital for esophageal epithelial differentiation, and decreased expression of KLF4 is seen in esophageal squamous cancers. Nonetheless, the roles of KLF4 and KLF5 in esophageal tumor progression are not known. Here, using TE2 cells stably infected with retroviral vectors to express KLF4 or KLF5, we demonstrate that KLF4 and KLF5 are key players in a number of cellular processes critical for esophageal carcinogenesis. TE2 cells, derived from a patient with poorly differentiated esophageal squamous cancer, normally lack KLF4 and KLF5. Expression of KLF5 in TE2 cells inhibits proliferation, and both KLF4 and KLF5 decrease viability after treatment with hydrogen peroxide and increase anoikis. In response to DNA damage from UV irradiation, viability is decreased in KLF5 but not KLF4 infected cells. Both KLF4 and KLF5 upregulate the cdk inhibitor p21waf1/cip1 following UV irradiation, but the pro-apoptotic protein BAX is markedly induced only by KLF5. Thus KLF4 may preferentially activate DNA repair pathways while KLF5 induces both DNA repair and apoptosis after UV irradiation. Expression of KLF4 or KLF5 in TE2 cells also inhibits invasion, consistent with a role for each in preventing tumor metastasis. In summary, KLF4 and KLF5 regulate esophageal carcinogenesis by affecting proliferation, apoptosis, and invasion.