Presence Of Double-strand Breaks Research Articles

Evidence for persistent UV-induced DNA damage and altered DNA damage response in xeroderma pigmentosa patient corneas

Xeroderma pigmentosum (XP) is a rare genetic disorder characterized by injury to the ocular surface due to exposure to ultraviolet (UV) radiation. UV-induced damage in the cells leads to the formation of cyclobutane pyrimidine dimers (CPDs) and 6-4 pyrimidine-pyrimidone photoproducts that are repaired by the NER (Nucleotide Excision Repair) pathway. Mutations in the genes coding for NER proteins, as reported in XP patients, would lead to sub-optimal damage repair resulting in clinical signs varying from photo-keratitis to cancerous lesions on the ocular surface. Here, we aimed to provide evidence for the accumulation of DNA damage and activation of DNA repair pathway proteins in the corneal cells of patients with XP. Corneal buttons of patients who underwent penetrating keratoplasty were stained to quantify DNA damage and the presence of activated DNA damage response proteins (DDR) using specific antibodies. Positive staining for pH2A.X and thymidine dimers confirmed the presence of DNA damage in the corneal cells. Positive cells were found in both control corneas and XP samples however, unlike normal tissues, positive cells were found in all cell layers of XP samples indicating that these cells were sensitive to very low levels of UV. pH2A.X-positive cells were significantly more in XP corneas (p < 0.05) indicating the presence of double strand breaks in these tissues. A positive expression of phosphorylated-forms of DDR proteins was noted in XP corneas (unlike controls) such as ataxia telangiectasia mutated/Rad-3 related proteins (ATM/ATR), breast cancer-1 and checkpoint kinases-1 and -2. Nuclear localization of XPA was noted in XP samples which co-localized (calculated using Pearson's correlation) with pATM (0.9 ± 0.007) and pATR (0.6 ± 0.053). The increased presence of these in the nucleus confirms that unresolved DNA damage was accumulating in these cells thereby leading to prolonged activation of the damage response proteins. An increase in pp53 and TUNEL positive cells in the XP corneas indicated cell death likely driven by the p53 pathway. For comparison, cultured normal corneal epithelial cells were exposed to UV-radiation and stained for DDR proteins at 3, 6 and 24 h after irradiation to quantify the time taken by cells with intact DDR pathway to repair damage. These cells, when exposed to UV showed nuclear translocation of DDR proteins at 3 and 6 h which reduced significantly by 24 h confirming that the damaged DNA was being actively repaired leading to cell survival. The persistent presence of the DDR proteins in XP corneas indicates that damage is being actively recognized and DNA replication is stalled, thereby causing accumulation of damaged DNA leading to cell death, which would explain the cancer incidence and cell loss reported in these patients.

The plant-specific DDR factor SOG1 increases chromatin mobility in response to DNA damage.

Homologous recombination (HR) is a conservative DNA repair pathway in which intact homologous sequences are used as a template for repair. How the homology search happens in the crowded space of the cell nucleus is, however, still poorly understood. Here, we measure chromosome and double-strand break (DSB) site mobility in Arabidopsis thaliana, using lacO/LacI lines and two GFP-tagged HR reporters. We observe an increase in chromatin mobility upon the induction of DNA damage, specifically at the S/G2 phases of the cell cycle. This increase in mobility is lost in the sog1-1 mutant, a central transcription factor of the DNA damage response in plants. Also, DSB sites show particularly high mobility levels and their enhanced mobility requires the HR factor RAD54. Our data suggest that repair mechanisms promote chromatin mobility upon DNA damage, implying a role of this process in the early steps of the DNA damage response.

TMT-Based Proteomic Analysis of Human Spermatozoa from Unexplained Recurrent Miscarriage Patients before and after Oral Antioxidant Treatment.

Recently, sperm quality and the presence of double-stranded breaks (DSB) has been pointed out as a possible cause of recurrent miscarriage, and the use of antioxidants has expanded as a treatment for male infertility. The aim of the present study was to analyze the proteomic effects of antioxidants on sperm from RM patients with high incidence of DSB. Proteomic analysis was performed using a tandem mass tag labeling technique, and subsequently compared with the PANTHER database for DEPs, and the STRING database for protein–protein interactions (PPI). Differentially expressed proteins (DEPs) both before and after antioxidant oral treatment were identified. PPI involving DEPs clustered into networks related to cell metabolism, cytoskeleton, and DNA damage. Results show that the sperm proteomic profiles before and after antioxidant treatment do not significantly differ from each other. However, some DEPs found after the antioxidant treatment shifted towards a DEPs profile typical of fertile donors. This indirect measurement suggests an improvement caused by antioxidants on the expression of several proteins. Among them were proteins involved in sperm DNA remodeling (LMO7, MMP28, BNC2, H2B, and PRDM2). The results presented here represent the first approach in the analysis and repair of the proteomic change caused by antioxidants in recurrent miscarriage patients, elucidating biomarkers that may be useful for the diagnosis and further sperm selection in this type of patient. Further studies should be conducted to validate the usefulness of these biomarkers in larger study groups.

SOG1 transcription factor promotes the onset of endoreduplication under salinity stress in Arabidopsis

As like in mammalian system, the DNA damage responsive cell cycle checkpoint functions play crucial role for maintenance of genome stability in plants through repairing of damages in DNA and induction of programmed cell death or endoreduplication by extensive regulation of progression of cell cycle. ATM and ATR (ATAXIA-TELANGIECTASIA-MUTATED and -RAD3-RELATED) function as sensor kinases and play key role in the transmission of DNA damage signals to the downstream components of cell cycle regulatory network. The plant-specific NAC domain family transcription factor SOG1 (SUPPRESSOR OF GAMMA RESPONSE 1) plays crucial role in transducing signals from both ATM and ATR in presence of double strand breaks (DSBs) in the genome and found to play crucial role in the regulation of key genes involved in cell cycle progression, DNA damage repair, endoreduplication and programmed cell death. Here we report that Arabidopsis exposed to high salinity shows generation of oxidative stress induced DSBs along with the concomitant induction of endoreduplication, displaying increased cell size and DNA ploidy level without any change in chromosome number. These responses were significantly prominent in SOG1 overexpression line than wild-type Arabidopsis, while sog1 mutant lines showed much compromised induction of endoreduplication under salinity stress. We have found that both ATM-SOG1 and ATR-SOG1 pathways are involved in the salinity mediated induction of endoreduplication. SOG1was found to promote G2-M phase arrest in Arabidopsis under salinity stress by downregulating the expression of the key cell cycle regulators, including CDKB1;1, CDKB2;1, and CYCB1;1, while upregulating the expression of WEE1 kinase, CCS52A and E2Fa, which act as important regulators for induction of endoreduplication. Our results suggest that Arabidopsis undergoes endoreduplicative cycle in response to salinity induced DSBs, showcasing an adaptive response in plants under salinity stress.

Targeted mutagenesis of Mycoplasma gallisepticum using its endogenous CRISPR/Cas system

New, more efficient methods are needed to facilitate studies of gene function in the mycoplasmas. CRISPR/Cas systems, which provide bacteria with acquired immunity against invading nucleic acids, have been developed as tools for genomic editing in a wide range of organisms. We explored the potential for using the endogenous Mycoplasma gallisepticum CRISPR/Cas system to introduce targeted mutations into the chromosome of this important animal pathogen. Three constructs carrying different CRISPR arrays targeting regions in the ksgA gene (pK1-CRISPR, pK-CRISPR-1 and pK-CRISPR-2) were assembled and introduced into M. gallisepticum on an oriC plasmid. The loss of KsgA prevents ribosomal methylation, which in turn confers resistance to the aminoglycoside antimicrobial kasugamycin, enabling selection for ksgA mutants. Analyses of the complete sequence of the ksgA gene in 78 resistant transformants revealed various modifications of the target region, presumably caused by the directed CRISPR/Cas activity of M. gallisepticum. The analyses suggested that M. gallisepticum may utilize a non-homologous end joining (NHEJ) repair system, which can result in deletion or duplication of a short DNA segment in the presence of double-stranded breaks. This study has generated an improved understanding of the M. gallisepticum CRISPR/Cas system, and may also facilitate further development of tools to genetically modify this important pathogen.

Human Embryonic Stem Cells Acquire Responsiveness to TRAIL upon Exposure to Cisplatin.

Tumor necrosis factor-related apoptosis-inducing ligand—TRAIL—is a protein operating as a ligand capable of inducing apoptosis particularly in cancerously transformed cells, while normal healthy cells are typically nonresponsive. We have previously demonstrated that pluripotent human embryonic stem cells (hESC) are also refractory to TRAIL, even though they express all canonical components of the death receptor-induced apoptosis pathway. In this study, we have examined a capacity of DNA damage to provoke sensitivity of hESC to TRAIL. The extent of DNA damage, behavior of molecules involved in apoptosis, and response of hESC to TRAIL were investigated. The exposure of hESC to 1 μM and 2 μM concentrations of cisplatin have led to the formation of 53BP1 and γH2AX foci, indicating the presence of double-strand breaks in DNA, without affecting the expression of proteins contributing to mitochondrial membrane integrity. Interestingly, cisplatin upregulated critical components of the extrinsic apoptotic pathway—initiator caspase 8, effector caspase 3, and the cell death receptors. The observed increase of expression of the extrinsic apoptotic pathway components was sufficient to sensitize hESC to TRAIL-induced apoptosis; immense cell dying accompanied by enhanced PARP cleavage, processing of caspase 8, and full activation of caspase 3 were all observed after the treatment combining cisplatin and TRAIL. Finally, we have demonstrated the central role of caspase 8 in this process, since its downregulation abrogated the sensitizing effect of cisplatin.

Mek1 coordinates meiotic progression with DNA break repair by directly phosphorylating and inhibiting the yeast pachytene exit regulator Ndt80.

Meiotic recombination plays a critical role in sexual reproduction by creating crossovers between homologous chromosomes. These crossovers, along with sister chromatid cohesion, connect homologs to enable proper segregation at Meiosis I. Recombination is initiated by programmed double strand breaks (DSBs) at particular regions of the genome. The meiotic recombination checkpoint uses meiosis-specific modifications to the DSB-induced DNA damage response to provide time to convert these breaks into interhomolog crossovers by delaying entry into Meiosis I until the DSBs have been repaired. The meiosis-specific kinase, Mek1, is a key regulator of meiotic recombination pathway choice, as well as being required for the meiotic recombination checkpoint. The major target of this checkpoint is the meiosis-specific transcription factor, Ndt80, which is essential to express genes necessary for completion of recombination and meiotic progression. The molecular mechanism by which cells monitor meiotic DSB repair to allow entry into Meiosis I with unbroken chromosomes was unknown. Using genetic and biochemical approaches, this work demonstrates that in the presence of DSBs, activated Mek1 binds to Ndt80 and phosphorylates the transcription factor, thus inhibiting DNA binding and preventing Ndt80’s function as a transcriptional activator. Repair of DSBs by recombination reduces Mek1 activity, resulting in removal of the inhibitory Mek1 phosphates. Phosphorylation of Ndt80 by the meiosis-specific kinase, Ime2, then results in fully activated Ndt80. Ndt80 upregulates transcription of its own gene, as well as target genes, resulting in prophase exit and progression through meiosis.

Laminarin based AgNPs using brown seaweed Turbinaria ornata and its induction of apoptosis in human retinoblastoma Y79 cancer cell lines

Biosynthesis of nanoparticles using isolated compounds from various sources is accepting interest due to their broad array of biological activities and biocompatibility. This paper presents a simple; cost effective and green synthesis of silver nanoparticles (AgNPs) using the polysaccharide, laminarin a storage compound obtained from the brown algae Turbinaria ornata (T. ornata). Initially, the water soluble polysaccharide, laminarin was extracted, purified and analyzed using Matrix Assisted Laser Desorption Ionization Time-of-Flight Mass Spectroscopy (MALDI-TOF MS) and Proton Nuclear Magnetic Resonance (1H NMR). Further, the silver nanoparticles (AgNPs) were synthesized using the isolated laminarin and were characterized by Ultraviolet - visible (UV–vis) spectrophotometer, colour value analysis, Fourier Transform Infrared Spectroscopy (FTIR), X-ray Diffraction (XRD) and High Resolution Transmission Electron Microscopy (HR-TEM). The free radical scavenging activities were performed and the effect of cytotoxicity against retinoblastoma Y79 cell lines was also evaluated by in vitro studies. Induction of apoptosis was evident by the percentage of cells arrested in G2/M phase using flow cytometry analysis and was further confirmed by DNA fragmentation study which identified the presence of double strand break.

139. Development of a Therapy for Duchenne Muscular Dystrophy Using Either TALEN of Cas9 Proteins

Duchenne muscular dystrophy (DMD) is a hereditary disease due to a mutation of the DMD gene located in the X chromosome. It is mainly caused by the deletion of one or more exons of the DMD gene, which changes the reading frame and results in a premature stop codon and a truncated and inactive dystrophin protein. Nowadays, genome editing by programmable endonucleases like TALENs or the CRISPR/Cas9 systems is a powerful method for developing a treatment for this type of disease. However, the use of DNA encoding these systems leads to a prolonged expression, which may increase the off-target activity of these nucleases. Despite the risk of integration into the genome hence increased probability of side effects, viral vectors remain the most effective delivery system for the nucleases and the sgRNA. The objective of this work was to develop a novel approach using purified TALEN proteins or Cas9 complex (Cas9 protein with crRNA and tracrRNA) for genome editing as potential treatment for DMD. TALEN proteins or Cas9 complex were transduced to generate double strand breaks (DSBs) in the dystrophin gene. Repair of this DSB by non-homologous end joining (NHEJ) could restore the normal reading frame of the DMD gene producing a functional dystrophin protein as already described for Becker patients (Koenig et al. 1989). To develop this approach, DNA coding for a pair of TALEN proteins targeting exon 54 of the DMD gene were engineered and cloned in the pet16b expression vector. The resulting TALEN proteins contained a His-Tag for purification purposes. The proteins were produced in bacteria and the TALEN-His-Tag proteins were purified using a Nickel column. The purity was analyzed by SDS-PAGE electrophoresis. The Cas9 protein, the crRNA and the tracrRNA were obtained commercially. To verify the cleavage activity of the programmable nuclease-complexes, the target genomic region including exon 54 was PCR amplified and the resulting amplicon was incubated with either the TALEN proteins or the Cas9 complex. Cleavage products were analysed by gel electrophoresis. Both types of proteins were able to cut 100% of the amplicons in vitro. The TALEN proteins or the Cas9 complex were tested in Hela IR8 and in human myoblast cell lines. The TALEN proteins were transduced with Cys-(Npys)-(d-Arg)9, Bioporter or the iTop delivery system (propanebetaine). The Cas9 complex was transduced with RNAiMax. The presence of DSBs was evaluated using the Surveyor assay. The Cas9 complex generated the expected cleavage products. These results are similar to those obtained when the plasmids coding for both systems were transfected. This indicates that the Cas9 complex could be used effectively to target a specific gene. In contrary, the TALEN proteins did not induce INDELs detectable by the surveyor assay. This could be due to an insufficient protein transduction. In conclusion, protein therapy using the CRISPR/Cas9 complex could be a promising approach to develop alternative treatments for genetic diseases. We are currently validating this approach in vivo using the hDMD mouse model containing the complete human DMD gene. (‘t Hoen et al. 2008).

Biochemical and proteomic analysis of a potential anticancer agent: Palladium(II) Saccharinate complex of terpyridine acting through double strand break formation.

Metal based chemotherapeutic drugs are widely used as an effective method to defeat various cancers. In this study, the mechanism of action of a novel therapeutic agent, [Pd(sac)(terpy)](sac)·4H2O (sac = saccharinate, and terpy = 2,2':6',2″-terpyridine) was studied. To better understand the proteomic changes in response to this agent, we performed nano LC-MS/MS analyses in human breast cancer cells (MDA-MB-231). Thirty proteins were identified to be differentially expressed more than 40% after drug treatment. Many cellular pathways were affected, including proteins involved in DNA repair, apoptosis, energy metabolism, protein folding, cytoskeleton, pre-mRNA maturation, or protein translation. The changes in protein expression were further verified for XRCC5, which plays a role in double strand break (DSB) repair, and ubiquitin, which is involved in protein degradation and apoptosis. The elevated XRCC5 levels were suggestive of increased DSBs. The presence of DSBs was confirmed by smearing of plasmid DNA in vitro and induction of γH2AX foci in vivo. There was also increased intracellular reactive oxygen species (ROS) formation, as detected by 2',7'-dichlorofluorescein diacetate (DCFDA) staining. Scavenging ROS by N-acetylcysteine rescued cell death in response to Pd(II) treatment, potentially explaining how the Pd(II) complex damaged the DNA. The details of this analysis and the significance will be discussed during the scope of this work.

High ultraviolet C resistance of marine Planctomycetes

Planctomycetes are bacteria with particular characteristics such as internal membrane systems encompassing intracellular compartments, proteinaceous cell walls, cell division by yeast-like budding and large genomes. These bacteria inhabit a wide range of habitats, including marine ecosystems, in which ultra-violet radiation has a potential harmful impact in living organisms. To evaluate the effect of ultra-violet C on the genome of several marine strains of Planctomycetes, we developed an easy and fast DNA diffusion assay in which the cell wall was degraded with papain, the wall-free cells were embedded in an agarose microgel and lysed. The presence of double strand breaks and unwinding by single strand breaks allow DNA diffusion, which is visible as a halo upon DNA staining. The number of cells presenting DNA diffusion correlated with the dose of ultra-violet C or hydrogen peroxide. From DNA damage and viability experiments, we found evidence indicating that some strains of Planctomycetes are significantly resistant to ultra-violet C radiation, showing lower sensitivity than the known resistant Arthrobacter sp. The more resistant strains were those phylogenetically closer to Rhodopirellula baltica, suggesting that these species are adapted to habitats under the influence of ultra-violet radiation. Our results provide evidence indicating that the mechanism of resistance involves DNA damage repair and/or other DNA ultra-violet C-protective mechanism.

DNA in motion during double-strand break repair

DNA organization and dynamics profoundly affect many biological processes such as gene regulation and DNA repair. In this review, we present the latest studies on DNA mobility in the context of DNA damage. Recent studies demonstrate that DNA mobility is dramatically increased in the presence of double-strand breaks (DSBs) in the yeast Saccharomyces cerevisiae. As a consequence, chromosomes explore a larger nuclear volume, facilitating homologous pairing but also increasing the rate of ectopic recombination. Increased DNA dynamics is dependent on several homologous recombination (HR) proteins and we are just beginning to understand how chromosome dynamics is regulated after DNA damage.

Smad7 foci are present in micronuclei induced by heavy particle radiation

DNA damage and reactive oxygen species (ROS) generated by ionizing radiation (IR) activate DNA damage response (DDR) and cytokine signaling pathways, including double strand break (DSB) repair and TGFβ/Smad signaling pathway. Proteins assembled at IR-induced DSB sites can be visualized as foci, including γH2AX, 53BP1, ATM and ATF2. Unrepaired DSBs are thought to be one origin of micronuclei (MN), an indicator of genotoxic stress and chromosomal instability. Studies have detected γH2AX in IR-induced MN, indicating the presence of DSB in MN. Previously we reported that TGFβ downstream proteins Smad7 and phospho-Smad2 (pSmad2) co-localized with DDR proteins following radiation. Here we studied the status of Smad7 and pSmad2 in MN post high linear energy transfer (LET) radiation in human normal and cancerous cells. We observed γH2AX foci in IR-induced MN, whereas 53BP1 and ATF2 were absent. Interestingly, Smad7 foci, but not pSmad2, were detectable in both spontaneous and IR-induced MN. We compared the effect of particle track structures on the yield of MN using 5.6MeV/u boron (B) and 600MeV/u iron (Fe) particles with similar LET (200 and 180keV/μm, respectively) in human fibroblasts. The frequency of MN induced by B was lower than that by Fe particles, albeit the proportion of Smad7-positive to Smad7-negative MN remained constant. An increased frequency of spontaneous MN, with slightly higher ratio of Smad7 or γH2AX positive, was found in human prostate cancer cells (PC3) compared to normal cells. 24h after 1Gy of Fe particles exposure, the yield of MN increased, and the majority (∼70%) carried γH2AX and Smad7. Phospho-ATM (Ser1981) foci were found in both spontaneous and IR-induced MN in PC3 cells, displaying a much lower frequency compared to γH2AX and Smad7. Our data suggest a unique role of Smad7 in IR-induced MN formation, which may associate with DNA repair, apoptosis and genomic instability.

Abstract 2028: Krüppel-like factor 4 null mouse embryonic fibroblasts exhibit DNA repair defects post exposure to gamma-irradiation

Abstract BACKGROUND: Krüppel-like factor 4 (KLF4) is a zinc finger transcription factor that exerts a potent inhibitory effect on cell cycle progression. KLF4 functions as a tumor suppressor in colorectal cancer and is involved in regulating centrosome duplication. Abnormal amplification of centrosomes has been shown to contribute to genetic instability by increasing the frequency of mitotic defects. AIM: To determine whether KLF4 is involved in maintaining genetic stability during cell division. METHODS: Mice heterozygous for the Klf4 alleles (Klf4+/−) on a C57BL/6 background were crossbred. Mouse embryonic fibroblasts (MEFs) that are wild type (Klf4+/+), heterozygous (Klf4+/−) or null (Klf4−/−) for the Klf4 alleles were derived from day-13.5 embryos. Post-senescence MEFs were derived by passing cells to senescence and maintaining them in culture until they spontaneously regained proliferative. Immuno-detection of histone variant γ-H2AX was performed to assess chromosome integrity. The levels of cell cycle regulators such as Cdk2, Cyclin E, and Cyclin D were determined by Western blotting. The presence of double strand breaks (DSB) was determined by western blot and immunofluorescence for histone variant -H2AX contains nuclear foci. Flow cytometry has been used to determine the level of DNA damage in both wildtype and klf4 null cells following gamma-irradiation. Furthermore, Expression profiles of fibroblasts isolated from mouse embryos wild type or null for the Klf4 alleles were examined by DNA microarrays. RESULTS: Cells that exhibits DSBs in their DNA can develop genomic instability, which can result in cancer formation. Compared to Klf4+/+ and Klf4−/− MEFs exposed to α-irradiation showed time-dependent increase in levels of histone variant -H2AX that persisted for up to 48 hours after the α-irradiation. On the other hand, Klf4+/+ halt cell cycle progression to allow repair of DNA damage. Western blot analysis showed that Cdk2, Cyclin E, Cyclin D and histone variant -H2AX expression were high in Klf4−/− MEFs. Furthermore, we found many of the down-regulated genes in Klf4-null MEFs encode DNA repair, and antioxidant genes. CONCLUSIONS: Results of this study demonstrate that Klf4 null mouse embryonic fibroblasts exhibit an impaired DNA damage response post gamma irradiation, when compared to wild-type cells. This data strongly suggest that the role for Klf4 in DNA damage repair in mouse embryonic fibroblast cells. These results indicate that KLF4 plays a crucial role in maintaining genetic stability and support the previous findings that KLF4 is a tumor suppressor. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2028. doi:1538-7445.AM2012-2028

Valproic acid, a histone deacetylase inhibitor, enhances radiosensitivity in esophageal squamous cell carcinoma

Histone deacetylase (HDAC) inhibitors have been shown to enhance radiation response in various cancer cell lines. Valproic acid (VPA) has been used in clinical practice for the treatment of epilepsy and other seizure disorders and is also one of the most represented HDAC inhibitors. The aim of this study was to evaluate the radiosensitizing ability of VPA and its mechanisms in four esophageal squamous cell carcinoma (ESCC) cell lines (TE9, TE10, TE11 and TE14). VPA inhibited the viability of all ESCC cells in a dose-dependent manner. The 50% inhibitory concentration (IC50) value of VPA in each cell line was between 1.02-2.15 mM, which is higher than clinically used safe concentrations. VPA induced the hyperacetylation of histones H3 and H4, as well as apoptosis and had a radiosensitizing effect on all four ESCC cell lines at a concentration of 0.5 mM which is equivalent to the therapeutic plasma concentration of anti-epilepsy therapy in humans. The radiosensitization was accompanied by an increase in γH2AX levels, indicating the presence of double-strand breaks (DSBs), and decrease in Rad51 expression, a DSB repair protein. These results suggest that a clinically safe dose of VPA can enhance radiation-induced cytotoxicity in human ESCC cells by chromatin decondensation with histone hyperacetylation and downregulation of Rad51. In conclusion, VPA appears to be a safe and promising radiosensitizer for esophageal cancer radiotherapy.

Sublethal Doses of β-Amyloid Peptide Abrogate DNA-dependent Protein Kinase Activity

Accumulation of DNA damage and deficiency in DNA repair potentially contribute to the progressive neuronal loss in neurodegenerative disorders, including Alzheimer disease (AD). In multicellular eukaryotes, double strand breaks (DSBs), the most lethal form of DNA damage, are mainly repaired by the nonhomologous end joining pathway, which relies on DNA-PK complex activity. Both the presence of DSBs and a decreased end joining activity have been reported in AD brains, but the molecular player causing DNA repair dysfunction is still undetermined. β-Amyloid (Aβ), a potential proximate effector of neurotoxicity in AD, might exert cytotoxic effects by reactive oxygen species generation and oxidative stress induction, which may then cause DNA damage. Here, we show that in PC12 cells sublethal concentrations of aggregated Aβ(25-35) inhibit DNA-PK kinase activity, compromising DSB repair and sensitizing cells to nonlethal oxidative injury. The inhibition of DNA-PK activity is associated with down-regulation of the catalytic subunit DNA-PK (DNA-PKcs) protein levels, caused by oxidative stress and reversed by antioxidant treatment. Moreover, we show that sublethal doses of Aβ(1-42) oligomers enter the nucleus of PC12 cells, accumulate as insoluble oligomeric species, and reduce DNA-PK kinase activity, although in the absence of oxidative stress. Overall, these findings suggest that Aβ mediates inhibition of the DNA-PK-dependent nonhomologous end joining pathway contributing to the accumulation of DSBs that, if not efficiently repaired, may lead to the neuronal loss observed in AD.

Gain‐of‐function mutant p53 but not p53 deletion promotes head and neck cancer progression in response to oncogenic K‐ras

Mutations in p53 occur in over 50% of the human head and neck squamous cell carcinomas (SCCHN). The majority of these mutations result in the expression of mutant forms of p53, rather than deletions in the p53 gene. Some p53 mutants are associated with poor prognosis in SCCHN patients. However, the molecular mechanisms that determine the poor outcome of cancers carrying p53 mutations are unknown. Here, we generated a mouse model for SCCHN and found that activation of the endogenous p53 gain-of-function mutation p53$^{\rm{R172H}}$, but not deletion of p53, cooperates with oncogenic K-ras during SCCHN initiation, accelerates oral tumour growth, and promotes progression to carcinoma. Mechanistically, expression profiling of the tumours that developed in these mice and studies using cell lines derived from these tumours determined that mutant p53 induces the expression of genes involved in mitosis, including cyclin B1 and cyclin A, and accelerates entry in mitosis. Additionally, we discovered that this oncogenic function of mutant p53 was dependent on K-ras because the expression of cyclin B1 and cyclin A decreased, and entry in mitosis was delayed, after suppressing K-ras expression in oral tumour cells that express p53$^{\rm{R172H}}$. The presence of double-strand breaks in the tumours suggests that oncogene-dependent DNA damage resulting from K-ras activation promotes the oncogenic function of mutant p53. Accordingly, DNA damage induced by doxorubicin also induced increased expression of cyclin B1 and cyclin A in cells that express p53$^{\rm{R172H}}$. These findings represent strong in vivo evidence for an oncogenic function of endogenous p53 gain-of-function mutations in SCCHN and provide a mechanistic explanation for the genetic interaction between oncogenic K-ras and mutant p53.

Abstract 3917: EMSY amplification and overexpression is not associated with defective homologous recombination and does not predict sensitivity to cisplatin or PARP inhibitors

Abstract EMSY is a gene amplified in 13-18% of breast and ovarian cancers, and encodes a protein reported to be a binding partner for BRCA2, that when overexpressed causes impairment of BRCA2 functions and so might constitute a mechanism for BRCA2 inactivation in non-hereditary breast and ovarian cancers. We hypothesised that if EMSY amplification abrogates BRCA2 function, cells harbouring EMSY gene amplification could have an impaired ability to elicit competent homologous recombination (HR) DNA repair in the presence of DNA double strand breaks (DSBs) and thus an increased sensitivity to DNA cross-linking agents and PARP inhibitors. EMSY amplification may therefore constitute a biomarker for response to these therapies. 59 cell lines were subjected to microarray based comparative genomic hybridisation (aCGH) with a resolution of 50Kb. Ten cell lines harbouring EMSY amplification were identified. These cell lines were matched by anatomical site and biomarker expression with cell lines lacking EMSY gene amplification. In addition, CAPAN1 (BRCA2 mutant), MDAMB436 and SUM149 (BRCA1 mutant) cells were included as controls. The associations between EMSY copy number, mRNA and protein expression were determined by aCGH and fluorescence in situ hybridisation, quantitative real-time PCR and western blotting respectively. Cell viability was assessed following transfection with validated short interfering RNAi (siRNA) against EMSY. Formation of foci of phosphorylated H2AX (γH2AX), a surrogate marker of the presence of double strand breaks, and RAD51, a surrogate marker of competent HR DNA repair, were assessed in cell lines with and without EMSY gene amplification following 10Gy of ionising radiation and treatment with cisplatin or the PARP inhibitor olaparib. Cell viability following treatment with cisplatin or olaparib was assessed to determine sensitivity of cell lines with and without EMSY amplification. EMSY is not consistently overexpressed at the mRNA and protein levels in cancer cells harbouring EMSY gene amplification. No significant difference in viability was seen in cells with or without EMSY amplification following silencing of EMSY using siRNA. Cell lines with EMSY amplification were able to elicit RAD51 foci formation in the presence of DNA double strand breaks, and did not differ from cancer cells devoid of EMSY amplification in their sensitivity to cisplatin and olaparib. These cells also showed a lower sensitivity to these drugs than CAPAN1, MDA-MB436 and SUM149 cells. EMSY amplification is not associated with an impairment of cancer cells to elicit RAD51 foci formation in the presence of DNA double strand breaks and is not associated with increased sensitivity to cisplatin or olaparib. Its potential use as a biomarker for response to cisplatin and PARP inhibitors should therefore be viewed with caution. 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 3917. doi:10.1158/1538-7445.AM2011-3917

Double-Strand Breaks in Heterochromatin Move Outside of a Dynamic HP1a Domain to Complete Recombinational Repair

Double-strand breaks (DSBs) in heterochromatic repetitive DNAs pose significant threats to genome integrity, but information about how such lesions are processed and repaired is sparse. We observe dramatic expansion and dynamic protrusions of the heterochromatin domain in response to ionizing radiation (IR) in Drosophila cells. We also find that heterochromatic DSBs are repaired by homologous recombination (HR) but with striking differences from euchromatin. Proteins involved in early HR events (resection) are rapidly recruited to DSBs within heterochromatin. In contrast, Rad51, which mediates strand invasion, only associates with DSBs that relocalize outside of the domain. Heterochromatin expansion and relocalization of foci require checkpoint and resection proteins. Finally, the Smc5/6 complex is enriched in heterochromatin and is required to exclude Rad51 from the domain and prevent abnormal recombination. We propose that the spatial and temporal control of DSB repair in heterochromatin safeguards genome stability by preventing aberrant exchanges between repeats.

Nucleosome resection at a double-strand break during Non-Homologous Ends Joining in mammalian cells - implications from repressive chromatin organization and the role of ARTEMIS

BackgroundThe S. cerevisiae mating type switch model of double-strand break (DSB) repair, utilizing the HO endonuclease, is one of the best studied systems for both Homologous Recombination Repair (HRR) and direct ends-joining repair (Non-Homologous Ends Joining - NHEJ). We have recently transposed that system to a mammalian cell culture model taking advantage of an adenovirus expressing HO and an integrated genomic target. This made it possible to compare directly the mechanism of repair between yeast and mammalian cells for the same type of induced DSB. Studies of DSB repair have emphasized commonality of features, proteins and machineries between organisms, and differences when conservation is not found. Two proteins that stand out that differ between yeast and mammalian cells are DNA-PK, a protein kinase that is activated by the presence of DSBs, and Artemis, a nuclease whose activity is modulated by DNA-PK and ATM. In this report we describe how these two proteins may be involved in a specific pattern of ends-processing at the DSB, particularly in the context of heterochromatin.FindingsWe previously published that the repair of the HO-induced DSB was generally accurate and occurred by simple rejoining of the cohesive 3'-overhangs generated by HO. During continuous passage of those cells in the absence of puromycin selection, the locus appears to have become more heterochromatic and silenced by displaying several features. 1) The site had become less accessible to cleavage by the HO endonuclease; 2) the expression of the puro mRNA, which confers resistance to puromycin, had become reduced; 3) occupancy of nucleosomes at the site (ChIP for histone H3) was increased, an indicator for more condensed chromatin. After reselection of these cells by addition of puromycin, many of these features were reversed. However, even the reselected cells were not identical in the pattern of cleavage and repair as the cells when originally created. Specifically, the pattern of repair revealed discrete deletions at the DSB that indicated unit losses of nucleosomes (or other protein complexes) before religation, represented by a ladder of PCR products reminiscent of an internucleosomal cleavage that is typically observed during apoptosis. This pattern of cleavage suggested to us that perhaps, Artemis, a protein that is believed to generate the internucleosomal fragments during apoptosis and in DSB repair, was involved in that specific pattern of ends-processing. Preliminary evidence indicates that this may be the case, since knock-down of Artemis with siRNA eliminated the laddering pattern and revealed instead an extensive exonucleolytic processing of the ends before religation.Conclusionse have generated a system in mammalian cells where the absence of positive selection resulted in chromatin remodeling at the target locus that recapitulates many of the features of the mating-type switching system in yeast. Specifically, just as for yeast HML and HMR, the locus had become transcriptionally repressed; accessibility to cleavage by the HO endonuclease was reduced; and processing of the ends was drastically changed. The switch was from high-fidelity religation of the cohesive ends, to a pattern of release of internucleosomal fragments, perhaps in search of micro-homology stretches for ligation. This is consistent with reports that the involvement of ATM, DNA-PK and Artemis in DSB repair is largely focused to heterochromatic regions, and not required for the majority of IR-induced DSB repair foci in euchromatin.