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Journal Cover   Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis
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   Hybrid Journal Hybrid journal (It can contain Open Access articles)
   ISSN (Print) 0027-5107
   Published by Elsevier Homepage  [2800 journals]
  • Efficient ligase 3-dependent microhomology-mediated end joining repair of
           DNA double-strand breaks in zebrafish embryos
    • Abstract: Publication date: Available online 20 August 2015
      Source:Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis
      Author(s): Mu-Dan He, Feng-Hua Zhang, Hua-Lin Wang, Hou-Peng Wang, Zuo-Yan Zhu, Yong-Hua Sun
      DNA double-strand break (DSB) repair is of considerable importance for genomic integrity. Homologous recombination (HR) and non-homologous end joining (NHEJ) are considered as two major mechanistically distinct pathways involved in repairing DSBs. In recent years, another DSB repair pathway, namely, microhomology-mediated end joining (MMEJ), has received increasing attention. MMEJ is generally believed to utilize an alternative mechanism to repair DSBs when NHEJ and other mechanisms fail. In this study, we utilized zebrafish as an in vivo model to study DSB repair and demonstrated that efficient MMEJ repair occurred in the zebrafish genome when DSBs were induced using TALEN (transcription activator-like effector nuclease) or CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 technologies. The wide existence of MMEJ repair events in zebrafish embryos was further demonstrated via the injection of several in vitro -designed exogenous MMEJ reporters. Interestingly, the inhibition of endogenous ligase 4 activity significantly increased MMEJ frequency, and the inhibition of ligase 3 activity severely decreased MMEJ activity. These results suggest that MMEJ in zebrafish is dependent on ligase 3 but independent of ligase 4. This study will enhance our understanding of the mechanisms of MMEJ in vivo and facilitate inducing desirable mutations via DSB-induced repair.


      PubDate: 2015-08-21T00:40:32Z
       
  • The key role of miR-21-regulated SOD2 in the medium-mediated bystander
           
    • Abstract: Publication date: Available online 14 August 2015
      Source:Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis
      Author(s): Wenqian Tian, Xiaoming Yin, Longxiao Wang, Jingdong Wang, Wei Zhu, Jianping Cao, Hongying Yang
      Radiation-induced bystander effect (RIBE) is well accepted in the radiation research field by now, but the underlying molecular mechanisms for better understanding this phenomenon caused by intercellular communication and intracellular signal transduction are still incomplete. Although our previous study has demonstrated an important role of miR-21 of unirradiated bystander cells in RIBEs, the direct evidence for the hypothesis that RIBE is epigenetically regulated is still limited and how miR-21 mediates RIBEs is unknown. Reactive oxygen species (ROS) have been demonstrated to be involved in RIBEs, however, the roles of anti-oxidative stress system of cells in RIBEs are unclear. Using transwell insert co-culture system, we investigated medium-mediated bystander responses in WS1 human fibroblasts after co-culture with HaCaT keratinocytes traversed by α-particles. Results showed that the ROS levels in unirradiated bystander WS1 cells were significantly elevated after 30min of co-culture, and 53BP1 foci, a surrogate marker of DNA damage, were obviously induced after 3h of co-culture. This indicates the occurrence of oxidative stress and DNA damage in bystander WS1 cells after co-culture with irradiated keratinocytes. Furthermore, the expression of miR-21 was increased in bystander WS1 cells, downregulation of miR-21 eliminated the bystander responses, overexpression of miR-21 alone could induce bystander-like oxidative stress and DNA damage in WS1 cells. These data indicate an important mediating role of miR-21 in RIBEs. In addition, MnSOD or SOD2 in WS1 cells was involved in the bystander effects, overexpression of SOD2 abolished the bystander oxidative stress and DNA damage, indicating that SOD2 was critical to the induction of RIBEs. Moreover, we found that miR-21 regulated SOD2, suggesting that miR-21 might mediate bystander responses through its regulation on SOD2. In conclusion, this study revealed a profound role of miR-21-regulated SOD2 of unirradiated WS1 cells in bystander effects induced by α-irradiated HaCaT keratinocytes.


      PubDate: 2015-08-17T06:11:09Z
       
  • DNA sequence context greatly affects the accuracy of bypass across an
           ultraviolet light 6-4 photoproduct in mammalian cells
    • Abstract: Publication date: Available online 13 August 2015
      Source:Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis
      Author(s): Pola Shriber, Yael Leitner-Dagan, Nicholas Geacintov, Tamar Paz-Elizur, Zvi Livneh
      Translesion DNA synthesis (TLS) is a DNA damage tolerance mechanism carried out by low-fidelity DNA polymerases that bypass DNA lesions, which overcomes replication stalling. Despite the miscoding nature of most common DNA lesions, several of them are bypassed in mammalian cells in a relatively accurate manner, which plays a key role maintaining a low mutation load. Whereas it is generally agreed that TLS across the major UV and sunlight induced DNA lesion, the cyclobutane pyrimidine dimer (CPD), is accurate, there were conflicting reports on whether the same is true for the thymine-thymine pyrimidine-pyrimidone(6-4) ultraviolet light photoproduct (TT6-4PP), which represents the second most common class of UV lesions. Using a TLS assay system based on gapped plasmids carrying site-specific TT6-4PP lesions in defined sequence contexts we show that the DNA sequence context markedly affected both the extent and accuracy of TLS. The sequence exhibiting higher TLS exhibited also higher error-frequency, caused primarily by semi-targeted mutations, at the nearest nucleotides flanking the lesion. Our results resolve the discrepancy reported on TLS across TT6-4PP, and suggest that TLS is more accurate in human cells than in mouse cells.


      PubDate: 2015-08-17T06:11:09Z
       
  • Reduced gene expression levels after chronic exposure to high
           concentrations of air pollutants
    • Abstract: Publication date: Available online 11 August 2015
      Source:Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis
      Author(s): Pavel Rossner, Elena Tulupova, Andrea Rossnerova, Helena Libalova, Katerina Honkova, Hans Gmuender, Anna Pastorkova, Vlasta Svecova, Jan Topinka, Radim J. Sram
      We analyzed the ability of particulate matter (PM) and chemicals adsorbed onto it to induce diverse gene expression profiles in subjects living in two regions of the Czech Republic differing in levels and sources of the air pollution. A total of 312 samples from polluted Ostrava region and 154 control samples from Prague were collected in winter 2009, summer 2009 and winter 2010. The highest concentrations of air pollutants were detected in winter 2010 when the subjects were exposed to: PM of aerodynamic diameter < 2.5μm (PM2.5) (70 vs. 44.9μg/m3); benzo[a]pyrene (9.02 vs. 2.56 ng/m3) and benzene (10.2 vs. 5.5μg/m3) in Ostrava and Prague, respectively. Global gene expression analysis of total RNA extracted from leukocytes was performed using Illumina Expression BeadChips microarrays. The expression of selected genes was verified by quantitative real-time PCR (qRT-PCR). Gene expression profiles differed by locations and seasons. Despite lower concentrations of air pollutants a higher number of differentially expressed genes and affected KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways was found in subjects from Prague. In both locations immune response pathways were affected, in Prague also neurodegenerative diseases-related pathways. Over-representation of the latter pathways was associated with the exposure to PM2.5. The qRT-PCR analysis showed a significant decrease in expression of APEX, ATM, FAS, GSTM1, IL1B and RAD21 in subjects from Ostrava, in a comparison of winter 2010 and summer 2009. In Prague, an increase in gene expression was observed for GADD45A and PTGS2. In conclusion, high concentrations of pollutants in Ostrava were not associated with higher number of differentially expressed genes, affected KEGG pathways and expression levels of selected genes. This observation suggests that chronic exposure to air pollution may result in reduced gene expression response with possible negative health consequences.


      PubDate: 2015-08-12T04:34:38Z
       
  • TITLE PAGE (EDI BOARD)
    • Abstract: Publication date: June 2015
      Source:Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, Volume 776




      PubDate: 2015-08-08T04:18:33Z
       
  • Editorial
    • Abstract: Publication date: June 2015
      Source:Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, Volume 776
      Author(s): Elza Tiemi Sakamoto Hojo, Carlos Frederico Martins Menck, Daisy Maria Favero Salvadori



      PubDate: 2015-08-08T04:18:33Z
       
  • TITLE PAGE (EDI BOARD)
    • Abstract: Publication date: August 2015
      Source:Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, Volume 778




      PubDate: 2015-08-04T03:53:36Z
       
  • Positive selection of AS3MT to arsenic water in Andean populations
    • Abstract: Publication date: Available online 29 July 2015
      Source:Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis
      Author(s): Christina A. Eichstaedt, Tiago Antao, Alexia Cardona, Luca Pagani, Toomas Kivisild, Maru Mormina
      Arsenic is a carcinogen associated with skin lesions and cardiovascular diseases. The Colla population from the Puna region in Northwest Argentinean is exposed to levels of arsenic in drinking water exceeding the recommended maximum by a factor of 20. Yet, they thrive in this challenging environment since thousands of years and therefore we hypothesise strong selection signatures in genes involved in arsenic metabolism. We analysed genome-wide genotype data for 730,000 loci in 25Collas, considering 24 individuals of the neighbouring Calchaquíes and 24 Wichí from the Gran Chaco region in the Argentine province of Salta as control groups. We identified a strong signal of positive selection in the main arsenic methyltransferase AS3MT gene, which has been previously associated with lower concentrations of the most toxic product of arsenic metabolism monomethylarsonic acid. This study confirms recent studies reporting selection signals in the AS3MT gene albeit using different samples, tests and control populations.


      PubDate: 2015-07-30T21:36:34Z
       
  • Studies of adaptive response and mutation induction in the chronically and
           acutely exposed MCF-10A cells to ionising radiation
    • Abstract: Publication date: Available online 29 July 2015
      Source:Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis
      Author(s): Sara Shakeri Manes, Traimate Sangsuwan, Andrzej Wojcik, Siamak Haghdoost
      A phenomenon in which exposure to a low adapting dose of radiation makes cells more resistant to the effects of a subsequent high dose exposure is termed radio-adaptive response. Adaptive response could hypothetically reduce the risk of late adverse effects of chronic or acute radiation exposures in humans. Understanding the underlying mechanisms of such responses are of relevance for radiation protection as well as for the clinical applications of radiation in medicine. However, due to the variability of responses depending on the model system and radiation condition, there is a need to further study under what conditions adaptive response can be induced. In this study, we analyzed if there is a dose rate dependence for the adapting dose, assuming that the adapting dose induces DNA response/repair pathways that are dose rate dependent. MCF-10A cells were exposed to a 50 mGy adapting dose administered acutely (0.40 Gy/min) or chronically (1.4 mGy/h or 4.1 mGy/h) and then irradiated by high acute challenging doses. The endpoints of study include clonogenic cell survival and mutation frequency at X-linked hprt locus. In another series of experiment, cells were exposed to 100 mGy and 1 Gy at different dose rates (acutely and chronically) and then the mutation frequencies were studied. Adaptive response was absent at the level of clonogenic survival and present at the level of mutations only at 1.4 mGy/h administration of adapting dose. Overall, no dose rate effect of the adapting dose was observed at the level of clonogenic survival or mutant frequency. Importantly, no dose rate effect was found comparing acute and chronic exposures.


      PubDate: 2015-07-30T21:36:34Z
       
  • Micronucleus frequency in peripheral blood lymphocytes and frailty status
           in elderly. A lack of association with clinical features
    • Abstract: Publication date: Available online 29 July 2015
      Source:Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis
      Author(s): Valdiglesias Vanessa, Bonassi Stefano, Dell'Armi Valentina, Silvana Settanni, Michela Celi, Simona Mastropaolo, Manuela Antocicco, Fini Massimo, Graziano Onder
      Frailty is a condition of vulnerability that carries an increased risk of poor outcome in elder adults. Frail individuals show fatigue, weight loss, muscle weakness, and a reduced physical function, and are known to frequently experience disability, social isolation, and institutionalization. Identifying frail people is a critical step for geriatricians to provide timely geriatric care and, eventually, to improve the quality of life in elderly. The aim of the present study is to investigate the association between frailty status and micronucleus (MN) frequency, a known marker of genomic instability, in a sample of elder adults. Several clinical features were evaluated and their possible association with MN frequency was tested. Criteria proposed by Fried were used to identify frail subjects. Overall, 180 elder adults entered the study, 93 of them (51.7%) frail. No association between MN frequency and frailty status was found under the specific conditions tested in this study(Mean Ratio = 1.06; 95% CI 0.96 - 1.18). The inclusion of MN frequency in the Fried's frailty scale minimally improved the classification of study subjects according to the Multidimensional Prognostic Index (MPI). The presence of genomic instability in the ageing process and in most chronic diseases, demands further investigation on this issue.


      PubDate: 2015-07-30T21:36:34Z
       
  • Defective DNA repair and increased chromatin binding of DNA repair factors
           in Down syndrome fibroblasts
    • Abstract: Publication date: Available online 26 July 2015
      Source:Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis
      Author(s): Daniela Necchi, Antonella Pinto, Micol Tillhon, Ilaria Dutto, Melania Maria Serafini, Cristina Lanni, Stefano Govoni, Marco Racchi, Ennio Prosperi
      Down syndrome (DS) is characterized by genetic instability,neurodegeneration, and premature aging. However, the molecular mechanisms leading to thisphenotype are not yet well understood. Here, we report that DS fibroblasts from both fetal and adult donorsshow the presence of oxidative DNA base damage, such asdihydro-8-oxoguanine (8-oxodG), and activation of a DNA damage response (DDR), already during unperturbed growth conditions. DDR with checkpoint activation was indicated by histone H2AX and Chk2 protein phosphorylation, and by increased p53 protein levels. In addition, both fetal and adult DS fibroblasts were more sensitive to oxidative DNA damage induced by potassium bromate, and were defective in the removal of 8-oxodG, as compared with age-matched cells from controlhealthy donors. The analysis of core proteins participating in base excision repair (BER), such as XRCC1 and DNA polymerase, showed that higher amounts of these factors were bound to chromatin in DS than in control cells, even in the absence of DNA damage. These findings occurred in concomitance with increased levels of phosphorylated XRCC1 detected in DS cells. These results indicate thatDS cells exhibit a BERdeficiency, whichis associated with prolonged chromatin association of core BER factors.


      PubDate: 2015-07-27T07:38:17Z
       
  • Effect of Proton and Gamma Irradiation on Human Lung Carcinoma Cells: Gene
           Expression, Cell Cycle, Cell Death, Epithelial-Mesenchymal Transition and
           Cancer-Stem Cell trait as Biological End Points
    • Abstract: Publication date: Available online 26 July 2015
      Source:Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis
      Author(s): Himanshi Narang, Amit Kumar, Nagesh Bhat, Badri N. Pandey, Anu Ghosh
      Proton beam therapy is a cutting edge modality over conventional gamma radiotherapy because of its physical dose deposition advantage. However, not much is known about its biological effects vis-a-vis gamma irradiation. Here we investigated the effect of proton- and gamma- irradiation on cell cycle, death, epithelial-mesenchymal transition (EMT) and “stemness” in human non-small cell lung carcinoma cells (A549). Proton beam (3 MeV) was two times more cytotoxic than gamma radiation and induced higher and longer cell cycle arrest. At equivalent doses, numbers of genes responsive to proton irradiation were ten times higher than those responsive to gamma irradiation. At equitoxic doses, the proton-irradiated cells had reduced cell adhesion and migration ability as compared to the gamma-irradiated cells. It was also more effective in reducing population of Cancer Stem Cell (CSC) like cells as revealed by aldehyde dehydrogenase activity and surface phenotyping by CD44+, a CSC marker. These results can have significant implications for proton therapy in the context of suppression of molecular and cellular processes that are fundamental to tumor expansion.


      PubDate: 2015-07-27T07:38:17Z
       
  • Discrimination of Driver and Passenger Mutations in Epidermal Growth
           Factor Receptor in Cancer
    • Abstract: Publication date: Available online 20 July 2015
      Source:Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis
      Author(s): P. Anoosha, Liang-Tsung Huang, R.Sakthivel, D. Karunagaran, M. Michael Gromiha
      Cancer is one of the most life-threatening diseases and mutations in several genes are the vital cause in tumorigenesis. Protein kinases play essential roles in cancer progression and specifically, Epidermal Growth Factor Receptor (EGFR) is an important target for cancer therapy. In this work, we have developed a method to classify Single Amino acid Polymorphisms (SAPs) in EGFR into disease-causing (driver) and neutral (passenger) mutations using both sequence and structure based features of the mutation site by machine learning approaches. We compiled a set of 222 features and selected a set of 21 properties utilizing feature selection methods, for maximizing the prediction performance. In a set of 540 mutants, we obtained an overall classification accuracy of 67.8% with 10 fold cross validation using Support Vector Machines. Further, the mutations have been grouped into four sets based on secondary structure and accessible surface area, which enhanced the overall classification accuracy to 80.2%, 81.9%, 77.9% and 75.1% for helix, strand, coil-buried and coil-exposed mutants, respectively. The method was tested with a blind dataset of 60 mutations, which showed an average accuracy of 85.4%. These accuracy levels are superior to other methods available in the literature for EGFR mutants, with an increase of more than 30%. Moreover, we have screened all possible single amino acid polymorphisms (SAPs) in EGFR and suggested the probable driver and passenger mutations, which would help in the development of mutation specific drugs for cancer treatment.


      PubDate: 2015-07-23T00:29:48Z
       
  • Point mutations in Escherichia coli DNA pol V that confer resistance to
           non-cognate DNA damage also alter protein-protein interactions
    • Abstract: Publication date: Available online 13 July 2015
      Source:Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis
      Author(s): Lisa A. Hawver , Mohammad Tehrani , Nicole M. Antczak , Danielle Kania , Stephanie Muser , Jana Sefcikova , Penny J. Beuning
      Y-family DNA polymerases are important for conferring cellular resistance to DNA damaging agents in part due to their specialized ability to copy damaged DNA. The Escherichia coli Y-family DNA polymerases are encoded by the umuDC and dinB genes. UmuC and the cleaved form of UmuD, UmuD′, form UmuD′2C (pol V), which is able to bypass UV photoproducts such as cyclobutane pyrimidine dimers and 6-4 thymine-thymine dimers, whereas DinB is specialized to copy N2-dG adducts, such as N2-furfuryl-dG. To better understand this inherent specificity, we used hydroxylamine to generate a random library of UmuC variants from which we then selected those with the ability to confer survival to nitrofurazone (NFZ), which is believed to cause N2-furfuryl-dG lesions. We tested the ability of three of the selected UmuC variants, A9 V, H282P, and T412I, to bypass N2-furfuryl-dG in vitro, and discovered that pol V containing UmuC A9 V has overall modestly better primer extension activity than WT pol V, whereas the UmuC T412I and UmuC H282P mutations result in much lower primer extension efficiency. Upon further characterization, we found that the ability of the UmuC variant A9 V to render cells UV-mutable is dependent on the proper length of the arm of UmuD′. Cells harboring UmuC variants T412I and H282P show enhanced cleavage of UmuD to form UmuD′, which, together with our other observations, suggests that this may be due to a disruption of a direct interaction between UmuC and UmuD. Thus, we find that protein interactions as well as protein conformation appear to be crucial for resistance to specific types of DNA damage.
      Graphical abstract image

      PubDate: 2015-07-14T23:58:23Z
       
  • Reduced cellular DNA repair capacity after environmentally relevant
           arsenic exposure. Influence of Ogg1 deficiency
    • Abstract: Publication date: Available online 13 July 2015
      Source:Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis
      Author(s): Jordi Bach , Jana Peremartí , Balasubramnayam Annangi , Ricard Marcos , Alba Hernández
      Inorganic arsenic (i-As) is a genotoxic and carcinogenic environmental contaminant known to affect millions of people worldwide. Our previous work demonstrated that chronic sub-toxic i-As concentrations were able to induce biologically significant levels of genotoxic and oxidative DNA damage that were strongly influenced by the Ogg1 genotype. In order to study the nature of the observed levels of damage and the observed differences between MEF Ogg1+/+ and Ogg1−/− genetic backgrounds, the genotoxic and oxidative DNA repair kinetics of 18-weeks exposed MEF cells were evaluated by the comet assay. Results indicate that MEF Ogg1+/+ and Ogg1−/− cells chronically exposed to i-As repair the DNA damage induced by arsenite, potassium bromide and UVC radiation less efficiently than control cells, being that observation clearly more pronounced in MEF Ogg1−/− cells. Consequently, exposed cells accumulate a higher percentage of unrepaired DNA damage at the end of the repair period. As an attempt to eliminate i-As associated toxicity, chronically exposed MEF Ogg1−/− cells overexpress the arsenic metabolizing enzyme As3mt. This adaptive response confers cells a significant resistance to i-As–induced cell death, but at expenses of accumulating high levels of DNA damage due to their repair impairment. Overall, the work presented here evidences that i-As chronic exposure disrupts the normal cellular repair function, and that oxidative DNA damage -and Ogg1 deficiency- exacerbates this phenomenon. The observed cell death resistance under a chronic scenario of genotoxic and oxidative stress may in turn contribute to the carcinogenic effects of i-As.


      PubDate: 2015-07-14T23:58:23Z
       
  • Kinetic analysis of bypass of a basic site by the catalytic core of yeast
           DNA polymerase eta
    • Abstract: Publication date: Available online 9 July 2015
      Source:Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis
      Author(s): Juntang Yang , Rong Wang , Binyan Liu , Qizhen Xue , Mengyu Zhong , Hao Zeng , Huidong Zhang
      Abasic sites (Apurinic/apyrimidinic (AP) sites), produced ∼50,000 times/cell/day, are very blocking and miscoding. To better understand miscoding mechanisms of abasic site for yeast DNA polymerase η, pre-steady-state nucleotide incorporation and LC-MS/MS sequence analysis of extension product were studied using pol ηcore (catalytic core, residues 1-513), which can completely eliminate the potential effects of the C-terminal C2H2 motif of pol η on dNTP incorporation. The extension beyond the abasic site was very inefficient. Compared with incorporation of dCTP opposite G, the incorporation efficiencies opposite abasic site were greatly reduced according to the order of dGTP > dATP >> dCTP and dTTP. Pol ηcore showed no fast burst phase for any incorporation opposite G or abasic site, suggesting that the catalytic step is not faster than the dissociation of polymerase from DNA. LC-MS/MS sequence analysis of extension products showed that 53% products were dGTP misincorporation, 33% were dATP and 14% were -1 frameshift, indicating that Pol ηcore bypasses abasic site by a combined G-rule, A-rule and -1 frameshift deletions. Compared with full-length pol η, pol ηcore relatively reduced the efficiency of incorporation of dCTP opposite G, increased the efficiencies of dNTP incorporation opposite abasic site and the exclusive incorporation of dGTP opposite abasic site, but inhibited the extension beyond abasic site, and increased the priority in extension of A: abasic site relative to G: abasic site. This study provides further understanding in the mutation mechanism of abasic sites for yeast DNA polymerase η.


      PubDate: 2015-07-09T22:55:01Z
       
  • Cadmium delays non-homologous end joining (NHEJ) repair via inhibition of
           DNA-PKcs phosphorylation and downregulation of XRCC4 and Ligase Ⅳ
    • Abstract: Publication date: Available online 9 July 2015
      Source:Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis
      Author(s): Weiwei Li , Xueyan Gu , Xiaoning Zhang , Jinxin Kong , Nan Ding , Yongmei Qi , Yingmei Zhang , Jufang Wang , Dejun Huang
      Although studies have shown that cadmium (Cd) interfered with DNA damage repair (DDR), whether Cd could affect non-homologous end joining (NHEJ) repair remains elusive. To further understand the effect of Cd on DDR, we used X-ray irradiation of Hela cells as an in vitro model system, along with γH2AX and 53BP1 as markers for DNA damage. Results showed that X-ray significantly increased γH2AX and 53BP1 foci in Hela cells (p < 0.01), all of which are characteristic of accrued DNA damage. The number of foci declined rapidly over time (1-8h postirradiation), indicating an initiation of NHEJ process. However, the disappearance of γH2AX and 53BP1 foci was remarkably slowed by Cd pretreatment (p < 0.01), suggesting that Cd reduced the efficiency of NHEJ. To further elucidate the mechanisms of Cd toxicity, several markers of NHEJ pathway including Ku70, DNA-PKcs, XRCC4 and Ligase Ⅳ were examined. Our data showed that Cd altered the phosphorylation of DNA-PKcs, and reduced the expression of both XRCC4 and Ligase Ⅳ in irradiated cells. These observations are indicative of the impairment of NHEJ-dependent DNA repair pathways. In addition, zinc (Zn) mitigated the effects of Cd on NHEJ, suggesting that the Cd-induced NHEJ alteration may partly result from the displacement of Zn or from an interference with the normal function of Zn-containing proteins by Cd. Our findings provide a new insight into the toxicity of Cd on NHEJ repair and its underlying mechanisms in human cells.


      PubDate: 2015-07-09T22:55:01Z
       
  • Dietary folic acid protects against genotoxicity in the red blood cells of
           mice
    • Abstract: Publication date: Available online 29 June 2015
      Source:Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis
      Author(s): Amanda J. MacFarlane , Nathalie A. Behan , Martha S. Field , Andrew Williams , Patrick J. Stover , Carole L. Yauk
      Folate is an essential B vitamin required for the de novo synthesis of purines, thymidylate and methionine. Folate deficiency can lead to mutations and genome instability, and has been shown to exacerbate the genotoxic potential of environmental toxins. We hypothesized that a folic acid (FA) deficient diet would induce genotoxicity in mice as measured by the Pig-a mutant phenotype (CD24-) and micronuclei (MN) in reticulocytes (RET) and red blood cells/normochromatic erythrocytes (RBC/NCE). Male Balb/c mice were fed a FA deficient (0mg/kg), control (2mg/kg) or supplemented (6mg/kg) diet from weaning for 18 wk. Mice fed the deficient diet had 70% lower liver folate (p<0.001), a 1.8 fold increase in MN-RET (p<0.001), and a 1.5 fold increase in MN-NCE (p<0.001) than mice fed the control diet. RETCD24− and RBCCD24− frequencies were not different between mice fed the deficient and control diets. Compared to mice fed the FA supplemented diet, mice fed the deficient diet had 73% lower liver folate (p<0.001), a 2.0 fold increase in MN-RET (p<0.001), a 1.6 fold increase in MN-NCE (p<0.001) and 3.8 fold increase in RBCCD24− frequency (p=0.011). RETCD24− frequency did not differ between mice fed the deficient and supplemented diets. Our data suggest that FA adequacy protects against mutagenesis at the Pig-a locus and MN induction in the red blood cells of mice.


      PubDate: 2015-07-01T14:16:10Z
       
  • Environmental and chemotherapeutic agents induce breakage at genes
           involved in leukemia-causing gene rearrangements in human hematopoietic
           stem/progenitor cells
    • Abstract: Publication date: Available online 27 June 2015
      Source:Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis
      Author(s): Ryan G. Thys , Christine E. Lehman , Levi C.T. Pierce , Yuh-Hwa Wang
      Hematopoietic stem and progenitor cells (HSPCs) give rise to all of the cells that make up the hematopoietic system in the human body, making their stability and resilience especially important. Damage to these cells can severely impact cell development and has the potential to cause diseases, such as leukemia. Leukemia-causing chromosomal rearrangements have largely been studied in the context of radiation exposure and are formed by a multi-step process, including an initial DNA breakage and fusion of the free DNA ends. However, the mechanism for DNA breakage in patients without previous radiation exposure is unclear. Here, we investigate the role of non-cytotoxic levels of environmental factors, benzene and diethylnitrosamine (DEN), and chemotherapeutic agents, etoposide and doxorubicin, in generating DNA breakage at the patient breakpoint hotspots of the MLL and CBFB genes in human HSPCs. These conditions represent exposure to chemicals encountered daily or residual doses from chemotherapeutic drugs. Exposure of HSPCs to non-cytotoxic levels of environmental chemicals or chemotherapeutic agents causes DNA breakage at preferential sites in the human genome, including the leukemia-related genes MLL and CBFB. Though benzene, etoposide, and doxorubicin have previously been linked to leukemia formation, this is the first study to demonstrate a role for DEN in the generation of DNA breakage at leukemia-specific sites. These chemical-induced DNA breakpoints coincide with sites of predicted topoisomerase II cleavage. The distribution of breakpoints by exposure to non-cytotoxic levels of chemicals showed a similar pattern to fusion breakpoints in leukemia patients. Our findings demonstrate that HSPCs exposed to non-cytotoxic levels of environmental chemicals and chemotherapeutic agents are prone to topoisomerase II-mediated DNA damage at the leukemia-associated genes MLL and CBFB. These data suggest a role for long-term environmental chemical or residual chemotherapeutic drug exposure in generation of DNA breakage at sites with a propensity to form leukemia-causing gene rearrangements.


      PubDate: 2015-07-01T14:16:10Z
       
  • Candida albicans DBF4 gene inducibly duplicated by the mini-Ura-blaster is
           involved in hypha-suppression
    • Abstract: Publication date: Available online 30 June 2015
      Source:Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis
      Author(s): Ting Chien , Tzu-Ling Tseng , Jiun-Yuan Wang , Yi-Ting Shen , Ting-Hui Lin , Jia-Ching Shieh
      The opportunistic human fungal pathogen Candida albicans is a natural diploid that does not have a complete sexual cycle. The ability to switch between diverse cellular forms is important to its virulence. Here, we describe the characterization of the Candida albicans DBF4 gene, a Saccharomyces cerevisiae homolog that encodes a regulatory subunit of Cdc7 kinase that is known to initiate DNA replication. We made a C. albicans strain, with one DBF4 allele deleted by the mini-Ura-blaster and the other controlled by a repressible promoter. We also found a third CaDBF4 copy that was later verified to be inducibly duplicated by targeted recombination with the min-Ura-blaster. Surprisingly, the strain deleted with the third CaDBF4 copy exhibited hyphal growth under repressed conditions. We conclude that the CaDBF4 gene is prone to being duplicated by the mini-Ura-blaster and that it suppresses hyphal growth in C. albicans.


      PubDate: 2015-07-01T14:16:10Z
       
  • Mitochondrial DNA mutations in single human blood cells
    • Abstract: Publication date: Available online 22 June 2015
      Source:Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis
      Author(s): Yong-Gang Yao , Sachiko Kajigaya , Neal S. Young
      Determination mitochondrial DNA (mtDNA) sequences from extremely small amounts of DNA extracted from tissue of limited amounts and/or degraded samples is frequently employed in medical, forensic, and anthropologic studies. Polymerase chain reaction (PCR) amplification followed by DNA cloning is a routine method, especially to examine heteroplasmy of mtDNA mutations. In this review, we compare the mtDNA mutation patterns detected by three different sequencing strategies. Cloning and sequencing methods that are based on PCR amplification of DNA extracted from either single cells or pooled cells yield a high frequency of mutations, partly due to the artifacts introduced by PCR and/or the DNA cloning process. Direct sequencing of PCR product which has been amplified from DNA in individual cells is able to detect the low levels of mtDNA mutations present within a cell. We further summarize the findings in our recent studies that utilized this single cell method to assay mtDNA mutation patterns in different human blood cells. Our data show that many somatic mutations observed in the end-stage differentiated cells are found in hematopoietic stem cells (HSCs) and progenitors within the CD34+ cell compartment. Accumulation of mtDNA variations in the individual CD34+ cells is affected by both aging and family genetic background. Granulocytes harbor higher numbers of mutations compared with the other cells, such as CD34+ cells and lymphocytes. Serial assessment of mtDNA mutations in a population of single CD34+ cells obtained from the same donor over time suggests stability of some somatic mutations. CD34+ cell clones from a donor marked by specific mtDNA somatic mutations can be found in the recipient after transplantation. The significance of these findings is discussed in terms of the lineage tracing of HSCs, aging effect on accumulation of mtDNA mutations and the usage of mtDNA sequence in forensic identification.


      PubDate: 2015-06-26T13:58:47Z
       
  • APE1, the DNA base excision repair protein, regulates the removal of
           platinum adducts in sensory neuronal cultures by NER
    • Abstract: Publication date: Available online 26 June 2015
      Source:Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis
      Author(s): Hyun-Suk Kim , Chunlu Guo , Eric L. Thompson , Yanlin Jiang , Mark R. Kelley , Michael R. Vasko , Suk-Hee Lee
      Peripheral neuropathy is one of the major side effects of treatment with the anticancer drug, cisplatin. One proposed mechanism for this neurotoxicity is the formation of platinum adducts in sensory neurons that could contribute to DNA damage. Although this damage is largely repaired by nuclear excision repair (NER), our previous findings suggest that augmenting the base excision repair pathway (BER) by overexpressing the repair protein APE1 protects sensory neurons from cisplatin-induced neurotoxicity. The question remains whether APE1 contributes to the ability of the NER pathway to repair platinum-damage in neuronal cells. To examine this, we manipulated APE1 expression in sensory neuronal cultures and measured Pt-removal after exposure to cisplatin. When neuronal cultures were treated with increasing concentrations of cisplatin for two or three hours, there was a concentration-dependent increase in Pt-damage that peaked at four hours and returned to near baseline levels after 24h. In cultures where APE1 expression was reduced by ∼80% using siRNA directed at APE1, there was a significant inhibition of Pt-removal over eight hours which was reversed by overexpressing APE1 using a lentiviral construct for human wtAPE1. Reduction in APE1 expression also altered the expression of the NER proteins RPA70 and XPA in sensory neuronal cultures. Overexpressing a mutant APE1 (C65 APE1), which only has DNA repair activity, but not its other significant redox-signaling function, mimicked the effects of wtAPE1. Overexpressing DNA repair activity mutant APE1 (226+177APE1), with only redox activity was ineffective suggesting it is the DNA repair function of APE1 and not its redox-signaling, that restores the Pt-damage removal. Together, these data provide the first evidence that a critical BER enzyme, APE1, helps regulate the NER pathway in the repair of cisplatin damage in sensory neurons.


      PubDate: 2015-06-26T13:58:47Z
       
  • MKP1 phosphatase mediates G1-specific dephosphorylation of H3Serine10P in
           response to DNA damage
    • Abstract: Publication date: August 2015
      Source:Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, Volume 778
      Author(s): Ajit K. Sharma , Shafqat A. Khan , Asmita Sharda , Divya V Reddy , Sanjay Gupta
      Histone mark, H3S10 phosphorylation plays a dual role in a cell by maintaining relaxed chromatin for active transcription in interphase and condensed chromatin state in mitosis. The level of H3S10P has also been shown to alter on DNA damage; however, its cell cycle specific behavior and regulation during DNA damage response is largely unexplored. In the present study, we demonstrate G1 cell cycle phase specific reversible loss of H3S10P in response to IR-induced DNA damage is mediated by opposing activities of phosphatase, MKP1 and kinase, MSK1 of the MAP kinase pathway. We also show that the MKP1 recruits to the chromatin in response to DNA damage and correlates with the decrease of H3S10P, whereas MKP1 is released from chromatin during recovery phase of DDR. Furthermore, blocking of H3S10 dephosphorylation by MKP1 inhibition impairs DNA repair process and results in poor survival of WRL68 cells. Collectively, our data proposes a pathway regulating G1 cell cycle phase specific reversible reduction of H3S10P on IR induced DNA damage and also raises the possibility of combinatorial modulation of H3S10P with specific inhibitors to target the cancer cells in G1-phase of cell cycle.


      PubDate: 2015-06-26T13:58:47Z
       
  • Effects of changes in intracellular iron pool on AlkB-dependent and
           AlkB-independent mechanisms protecting E.coli cells against mutagenic
           action of alkylating agent
    • Abstract: Publication date: August 2015
      Source:Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, Volume 778
      Author(s): Anna Sikora , Agnieszka M. Maciejewska , Jarosław Poznański , Tomasz Pilżys , Michał Marcinkowski , Małgorzata Dylewska , Jan Piwowarski , Wioletta Jakubczak , Katarzyna Pawlak , Elżbieta Grzesiuk
      An Escherichia coli hemH mutant accumulates protoporphyrin IX, causing photosensitivity of cells to visible light. Here, we have shown that intracellular free iron in hemH mutants is double that observed in hemH + strain. The aim of this study was to recognize the influence of this increased free iron concentration on AlkB-directed repair of alkylated DNA by analyzing survival and argE3 →Arg+ reversion induction after λ>320nm light irradiation and MMS-treatment in E. coli AB1157 hemH and alkB mutants. E.coli AlkB dioxygenase constitutes a direct single-protein repair system using non-hem Fe(II) and cofactors 2-oxoglutarate (2OG) and oxygen (O2) to initiate oxidative dealkylation of DNA/RNA bases. We have established that the frequency of MMS-induced Arg+ revertants in AB1157 alkB + hemH– /pMW1 strain was 40 and 26% reduced comparing to the alkB + hemH– and alkB + hemH+ /pMW1, respectively. It is noteworthy that the effect was observed only when bacteria were irradiated with λ >320nm light prior MMS-treatment. This finding indicates efficient repair of alkylated DNA in photosensibilized cells in the presence of higher free iron pool and AlkB concentrations. Interestingly, a 31% decrease in the level of Arg+ reversion was observed in irradiated and MMS-treated hemH – alkB – cells comparing to the hemH + alkB – strain. Also, the level of Arg+ revertants in the irradiated and MMS treated hemH– alkB– mutant was significantly lower (by 34%) in comparison to the same strain but MMS-treated only. These indicate AlkB-independent repair involving Fe ions and reactive oxygen species. According to our hypothesis it may be caused by non-enzymatic dealkylation of alkylated dNTPs in E. coli cells. In in vitro studies, the absence of AlkB protein in the presence of iron ions allowed etheno(ε) dATP and εdCTP to spontaneously convert to dAMP and dCMP, respectively. Thus, hemH – intra-cellular conditions may favor Fe-dependent dealkylation of modified dNTPs.


      PubDate: 2015-06-26T13:58:47Z
       
  • Nrf2 facilitates repair of radiation induced DNA damage through homologous
           recombination repair pathway in a ROS independent manner in cancer cells
    • Abstract: Publication date: Available online 22 June 2015
      Source:Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis
      Author(s): Sundarraj Jayakumar , Debojyoti Pal , Santosh K. Sandur
      Nrf2 is a redox sensitive transcription factor that is involved in the co-ordinated transcription of genes involved in redox homeostasis. But the role of Nrf2 in DNA repair is not investigated in detail. We have employed A549 and MCF7 cells to study the role of Nrf2 on DNA repair by inhibiting Nrf2 using all-trans retinoic acid (ATRA) or by knock down approach prior to radiation exposure (4Gy). DNA damage and repair analysis was studied by γH2AX foci formation and comet assay. Results suggested that the inhibition of Nrf2 in A549 or MCF7 cells led to significant slowdown in DNA repair as compared to respective radiation controls. The persistence of residual DNA damage even in the presence of free radical scavenger N-acetyl cysteine, suggested that the influence of Nrf2 on DNA repair was not linked to its antioxidant functions. Further, its influence on non-homologous end joining repair pathway was studied by inhibiting both Nrf2 and DNA-PK together. This led to synergistic reduction of survival fraction, indicating that Nrf2 may not be influencing the NHEJ pathway. To investigate the role of homologous recombination repair (HR) pathway, RAD51 foci formation was monitored. There was a significant reduction in the foci formation in cells treated with ATRA or shRNA against Nrf2 as compared to their respective radiation controls. Further, Nrf2 inhibition led to significant reduction in mRNA levels of RAD51. BLAST analysis was also performed on upstream regions of DNA repair genes to identify antioxidant response element and found that many repair genes that are involved in HR pathway may be regulated by Nrf2. Together, these results suggest the involvement of Nrf2 in DNA repair, a hitherto unknown function of Nrf2, putatively through its influence on HR pathway.


      PubDate: 2015-06-26T13:58:47Z
       
  • Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) interacts with
           apurinic/apyrimidinic sites in DNA
    • Abstract: Publication date: Available online 17 June 2015
      Source:Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis
      Author(s): Anastasiya A. Kosova , Svetlana N. Khodyreva , Olga I. Lavrik
      Apurinic/apyrimidinic (AP) sites are some of the most frequent DNA damages and the key intermediates of base excision repair. Certain proteins can interact with the deoxyribose of the AP site to form a Schiff base, which can be stabilized by NaBH4 treatment. Several types of DNA containing the AP site were used to trap proteins in human cell extracts by this method. In the case of single-stranded AP DNA and AP DNA duplex with both 5ʹ and 3ʹ dangling ends, the major crosslinking product had apparent molecular mass of 45 kDa. Using peptide mass mapping based on mass spectrometry data, we identified the protein forming this adduct as an isoform of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) called “uracil-DNA glycosylase”. GAPDH is a glycolytic enzyme with many additional putative functions, which include interaction with nucleic acids, different DNA damages and DNA repair enzymes. We investigated interaction of GAPDH purified from HeLa cells and rabbit muscles with different AP DNAs. In spite of the ability to form a Schiff-base intermediate with the deoxyribose of the AP site, GAPDH does not display the AP lyase activity. In addition, along with borohydride-dependent adducts with AP DNA containing single-stranded regions, GAPDH was shown to form also stable borohydride-independent crosslinks with these DNA. GAPDH was proven to crosslink preferentially to AP DNA cleaved via β-elimination mechanism (spontaneously or by AP lyases) as compared to DNA containing the intact AP site. The level of GAPDH–AP DNA adduct formation depends on oxidation of the protein SH-groups; disulfide bond reduction in GAPDH leads to loss of its ability to form adducts with AP DNA. Possible role of formation of stable adducts with AP sites by GAPDH is discussed.
      Graphical abstract image

      PubDate: 2015-06-21T13:07:03Z
       
  • Increased Human AP Endonuclease 1 Level Confers Protection Against the
           Paternal Age Effect in Mice
    • Abstract: Publication date: Available online 20 June 2015
      Source:Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis
      Author(s): Jamila R. Sanchez , Traci L. Reddick , Marissa Perez , Victoria E. Centonze , Sankar Mitra , Tadahide Izumi , C.Alex McMahan , Christi A. Walter.
      Increased paternal age is associated with a greater risk of producing children with genetic disorders originating from de novo germline mutations. Mice mimic the human condition by displaying an age-associated increase in spontaneous mutant frequency in spermatogenic cells. The observed increase in mutant frequency appears to be associated with a decrease in the DNA repair protein, AP endonuclease 1 (APEX1) and Apex1 heterozygous mice display an accelerated paternal age effect as young adults. In this study, we directly tested if APEX1 over-expression in cell lines and transgenic mice could prevent increases in mutagenesis. Cell lines with ectopic expression of APEX1 had increased APEX1 activity and lower spontaneous and induced mutations in the lacI reporter gene relative to the control. Spermatogenic cells obtained from mice transgenic for human APEX1 displayed increased APEX1 activity, were protected from the age-dependent increase in spontaneous germline mutagenesis, and exhibited increased apoptosis in the spermatogonial cell population. These results directly indicate that increases in APEX1 level confer protection against the murine paternal age effect, thus highlighting the role of APEX1 in preserving reproductive health with increasing age and in protection against genotoxin-induced mutagenesis in somatic cells.


      PubDate: 2015-06-21T13:07:03Z
       
  • The BAH domain of BAF180 is required for PCNA ubiquitination
    • Abstract: Publication date: Available online 17 June 2015
      Source:Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis
      Author(s): Atsuko Niimi , Suzanna R Hopkins , Jessica A Downs , Chikahide Masutani
      Monoubiquitination of proliferating cell nuclear antigen (PCNA) is a critical regulator of Post Replication Repair (PRR). The depletion of BAF180, a unique subunit of the PBAF chromatin remodeling complex in human cells results in reduced PCNA ubiquitination leading to less efficient fork progression following DNA damage, but little is known about the mechanism. Here, we report that the expression of exogenous BAF180 in cells promotes PCNA ubiquitination during S-phase after UV irradiation and it persists for many hours. No correlation was observed between the protein level of ubiquitin-specific protease 1 (USP1) and ubiquitinated PCNA in BAF180 expressing cells. Analysis of cells expressing BAF180 deletion mutants showed that the bromo-adjacent homology (BAH) domains are responsible for this effect. Surprisingly, a deletion construct encoding only the BAH domain region is able to increase the level of ubiquitinated PCNA, even though it is unable to be assembled into the PBAF complex. These results suggest that the ATPase-dependent chromatin remodeling activity of PBAF is not necessary, but instead the BAH domains are sufficient to promote PCNA ubiquitination.


      PubDate: 2015-06-21T13:07:03Z
       
  • HUMAN CIRCULATING PLASMA DNA SIGNIFICANTLY DECREASES WHILE LYMPHOCYTE DNA
           DAMAGE INCREASES UNDER CHRONIC OCCUPATIONAL EXPOSURE TO LOW-DOSE
           GAMMA-NEUTRON AND TRITIUM β-RADIATION
    • Abstract: Publication date: Available online 15 May 2015
      Source:Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis
      Author(s): Inna B. Korzeneva , Svetlana V. Kostuyk , Liza S. Ershova , Andrian N. Osipov , Veronika F. Zhuravleva , Galina V. Pankratova , Lev N. Porokhovnik , Natalia N. Veiko
      The blood plasma of healthy people contains cell-fee (circulating) DNA (cfDNA). Apoptotic cells are the main source of the cfDNA. The cfDNA concentration increases in case of the organism's cell death rate increase, for example in case of exposure to high-dose ionizing radiation (IR). The objects of the present research are the blood plasma and blood lymphocytes of people, who contacted occupationally with the sources of external gamma/neutron radiation or internal β-radiation of tritium (N=176). As the controls (references), blood samples of people, who had never been occupationally subjected to the IR sources, were used (N=109). With respect to the plasma samples of each donor there were defined: the cfDNA concentration (the cfDNA index), DNase1 activity (the DNase1 index) and titre of antibodies to DNA (the Ab DNA index). The general DNA damage in the cells was defined (using the Comet assay, the tail moment (TM) index). A chronic effect of the low-dose ionizing radiation on a human being is accompanied by the enhancement of the DNA damage in lymphocytes along with a considerable cfDNA content reduction, while the DNase1 content and concentration of antibodies to DNA (Ab DNA) increase. All the aforementioned changes were also observed in people, who had not worked with the IR sources for more than a year. The ratio cfDNA/(DNase1×Ab DNA×TM) is proposed to be used as a marker of the chronic exposure of a person to the external low-dose IR. It was formulated the assumption that the joint analysis of the cfDNA, DNase1, Ab DNA and TM values may provide the information about the human organism's cell resistivity to chronic exposure to the low-dose IR and about the development of the adaptive response in the organism that is aimed, firstly, at the effective cfDNA elimination from the blood circulation, and, secondly – at survival of the cells, including the cells with the damaged DNA.


      PubDate: 2015-06-14T11:42:17Z
       
  • A combinatorial role for MutY and Fpg DNA glycosylases in mutation
           avoidance in Mycobacterium smegmatis
    • Abstract: Publication date: Available online 9 June 2015
      Source:Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis
      Author(s): Farzanah Hassim , Andrea O. Papadopoulos , Bavesh D. Kana , Bhavna G. Gordhan
      Hydroxyl radical (OH) among reactive oxygen species cause damage to nucleobases with thymine being the most susceptible, whilst in contrast, the singlet oxygen (102) targets only guanine bases. The high GC content of mycobacterial genomes predisposes these organisms to oxidative damage of guanine. The exposure of cellular DNA to OH and one-electron oxidants results in the formation of two main degradation products, the pro-mutagenic 8-oxo-7,8-dihydroguanine (8-oxoGua) and the cytotoxic 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua). These lesions are repaired through the base excision repair (BER) pathway and we previously, demonstrated a combinatorial role for the mycobacterial Endonuclease III (Nth) and the Nei family of DNA glycosylases in mutagenesis. In addition, the Formamidopyrimidine (Fpg/MutM) and MutY DNA glycosylases have also been implicated in mutation avoidance and BER in mycobacteria. In this study, we further investigate the combined role of MutY and the Fpg/Nei DNA glycosylases in Mycobacterium smegmatis and demonstrate that deletion of mutY resulted in enhanced sensitivity to oxidative stress, an effect which was not exacerbated in Δfpg1 Δfpg2 or Δnei1 Δnei2 double mutant backgrounds. However, combinatorial loss of the mutY, fpg1 and fpg2 genes resulted in a significant increase in mutation rates suggesting interplay between these enzymes. Consistent with this, there was a significant increase in C → A mutations with a corresponding change in cell morphology of rifampicin resistant mutants in the Δfpg1 Δfpg2 ΔmutY deletion mutant. In contrast, deletion of mutY together with the nei homologues did not result in any growth/survival defects or changes in mutation rates. Taken together these data indicate that the mycobacterial MutY, in combination with the Fpg DNA N-glycosylases, plays an important role in controlling mutagenesis under oxidative stress.


      PubDate: 2015-06-14T11:42:17Z
       
  • The different radiation response and radiation-induced bystander effects
           in colorectal carcinoma cells differing in p53 status
    • Abstract: Publication date: Available online 10 June 2015
      Source:Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis
      Author(s): Maria Widel , Anna Lalik , Aleksandra Krzywon , Jan Poleszczuk , Krzysztof Fujarewicz , Joanna Rzeszowska-Wolny
      Radiation-induced bystander effect, appearing as different biological changes in cells that are not directly exposed to ionizing radiation but are under the influence of molecular signals secreted by irradiated neighbors, have recently attracted considerable interest due to their possible implication for radiotherapy. However, various cells present diverse radiosensitivity and bystander responses that depend, inter alia, on genetic status including TP53, the gene controlling the cell cycle, DNA repair and apoptosis. Here we compared the ionizing radiation and bystander responses of human colorectal carcinoma HCT116 cells with wild type or knockout TP53 using a transwell co-culture system. The viability of exposed to X-rays (0-8Gy) and bystander cells of both lines showed a roughly comparable decline with increasing dose. The frequency of micronuclei was also comparable at lower doses but at higher increased considerably, especially in bystander TP53-/- cells. Moreover, the TP53-/- cells showed a significantly elevated frequency of apoptosis, while TP53+/+ counterparts expressed high level of senescence. The cross-matched experiments where irradiated cells of one line were co-cultured with non-irradiated cells of opposite line show that both cell lines were also able to induce bystander effects in their counterparts, however different endpoints revealed with different strength. Potential mediators of bystander effects, IL-6 and IL-8, were also generated differently in both lines. The knockout cells secreted IL-6 at lower doses whereas wild type cells only at higher doses. Secretion of IL-8 by TP53-/- control cells was many times lower than that by TP53+/+ but increased significantly after irradiation. Transcription of the NFκBIA was induced in irradiated TP53+/+ mainly, but in bystanders a higher level was observed in TP53-/- cells, suggesting that TP53 is required for induction of NFκB pathway after irradiation but another mechanism of activation must operate in bystander cells.


      PubDate: 2015-06-14T11:42:17Z
       
  • A eudesmane-type sesquiterpene isolated from Pluchea odorata (L.) Cass.
           combats three hallmarks of cancer cells: Unrestricted proliferation,
           escape from apoptosis and early metastatic outgrowth in vitro
    • Abstract: Publication date: July 2015
      Source:Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, Volume 777
      Author(s): Michael Blaschke , Ruxandra McKinnon , Chi Huu Nguyen , Silvio Holzner , Martin Zehl , Atanas Georgiev Atanasov , Karin Schelch , Sigurd Krieger , Rene Diaz , Richard Frisch , Björn Feistel , Walter Jäger , Gerhard F. Ecker , Verena M. Dirsch , Michael Grusch , Istvan Zupko , Ernst Urban , Brigitte Kopp , Georg Krupitza
      Pluchea odorata is ethno pharmaceutically used to treat inflammation-associated disorders. The dichloromethane extract (DME) was tested in the carrageenan-induced rat paw oedema assay investigating its effect on inflammation that was inhibited by 37%. Also an in vitro anti-neoplastic potential was reported. However, rather limited information about the bio-activity of purified compounds and their cellular mechanisms are available. Therefore, two of the most abundant eudesmanes in P. odorata were isolated and their anti-neoplastic and anti-intravasative activities were studied. HL-60 cells were treated with P. odorata compounds and metabolic activity, cell number reduction, cell cycle progression and apoptosis induction were correlated with relevant protein expression. Tumour cell intravasation through lymph endothelial monolayers was measured and potential causal mechanisms were analyzed by Western blotting. Compound PO-1 decreased the metabolic activity of HL-60 cells (IC50 =8.9μM after 72h) and 10μM PO-1 induced apoptosis, while PO-2 showed just weak anti-neoplastic activities at concentrations beyond 100μM. PO-1 arrested the cell cycle in G1 and this correlated with induction of JunB expression. Independent of this mechanism 25μM PO-1 decreased MCF-7 spheroid intravasation through the lymph endothelial barrier. Hence, PO-1 inhibits an early step of metastasis, impairs unrestricted proliferation and induces apoptosis at low micromolar concentrations. These results warrant further testing in vivo to challenge the potential of PO-1 as novel lead compound.


      PubDate: 2015-06-14T11:42:17Z
       
  • The effect of age at exposure on the inactivating mechanisms and relative
           contributions of key tumor suppressor genes in radiation-induced mouse
           T-cell lymphomas
    • Abstract: Publication date: Available online 10 June 2015
      Source:Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis
      Author(s): Masaaki Sunaoshi , Yoshiko Amasaki , Shinobu Hirano-Sakairi , Benjamin J. Blyth , Takamitsu Morioka , Mutsumi Kaminishi , Yi Shang , Mayumi Nishimura , Yoshiya Shimada , Akira Tachibana , Shizuko Kakinuma
      Children are considered more sensitive to radiation-induced cancer than adults, yet any differences in genomic alterations associated with age-at-exposure and their underlying mechanisms remain unclear. We assessed genome-wide DNA copy number and mutation of key tumor suppressor genes in T-cell lymphomas arising after weekly irradiation of female B6C3F1 mice with 1.2Gy X-rays for 4 consecutive weeks starting during infancy (1 week old), adolescence (4 weeks old) or as young adults (8 weeks old). Although T-cell lymphoma incidence was similar, loss of heterozygosity at Cdkn2a on chromosome 4 and at Ikaros on chromosome 11 was more frequent in the two older groups, while loss at the Pten locus on chromosome 19 was more frequent in the infant-irradiated group. Cdkn2a and Ikaros mutation/loss was a common feature of the young adult–irradiation group, with Ikaros frequently (60%) incurring multiple independent hits (including deletions and mutations) or suffering a single hit predicted to result in a dominant negative protein (such as those lacking exon 4, an isoform we have designated Ik12, which lacks two DNA binding zinc–finger domains). Conversely, Pten mutations were more frequent after early irradiation (60%) than after young adult–irradiation (30%). Homozygous Pten mutations occurred without DNA copy number change after irradiation starting in infancy, suggesting duplication of the mutated allele by chromosome mis-segregation or mitotic recombination. Our findings demonstrate that while deletions on chromosomes 4 and 11 affecting Cdkn2a and Ikaros are a prominent feature of young adult irradiation–induced T-cell lymphoma, tumors arising after irradiation from infancy suffer a second hit in Pten by mis-segregation or recombination. This is the first report showing an influence of age-at-exposure on genomic alterations of tumor suppressor genes and their relative involvement in radiation-induced T-cell lymphoma. These data are important for considering the risks associated with childhood exposure to radiation.


      PubDate: 2015-06-14T11:42:17Z
       
  • MicroRNA transcriptome analysis identifies miR-365 as a novel negative
           regulator of cell proliferation in Zmpste24-deficient mouse embryonic
           fibroblasts
    • Abstract: Publication date: July 2015
      Source:Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, Volume 777
      Author(s): Xing-dong Xiong , Hwa Jin Jung , Saurabh Gombar , Jung Yoon Park , Chun-long Zhang , Huiling Zheng , Jie Ruan , Jiang-bin Li , Matt Kaeberlein , Brian K. Kennedy , Zhongjun Zhou , Xinguang Liu , Yousin Suh
      Zmpste24 is a metalloproteinase responsible for the posttranslational processing and cleavage of prelamin A into mature laminA. Zmpste24 −/− mice display a range of progeroid phenotypes overlapping with mice expressing progerin, an altered version of lamin A associated with Hutchinson-Gilford progeria syndrome (HGPS). Increasing evidence has demonstrated that miRNAs contribute to the regulation of normal aging process, but their roles in progeroid disorders remain poorly understood. Here we report the miRNA transcriptomes of mouse embryonic fibroblasts (MEFs) established from wild type (WT) and Zmpste24 −/− progeroid mice using a massively parallel sequencing technology. With data from 19.5×106 reads from WT MEFs and 16.5×106 reads from Zmpste24 −/− MEFs, we discovered a total of 306 known miRNAs expressed in MEFs with a wide dynamic range of read counts ranging from 10 to over 1 million. A total of 8 miRNAs were found to be significantly down-regulated, with only 2 miRNAs upregulated, in Zmpste24 −/− MEFs as compared to WT MEFs. Functional studies revealed that miR-365, a significantly down-regulated miRNA in Zmpste24 −/− MEFs, modulates cellular growth phenotypes in MEFs. Overexpression of miR-365 in Zmpste24 −/− MEFs increased cellular proliferation and decreased the percentage of SA-β-gal-positive cells, while inhibition of miR-365 function led to an increase of SA-β-gal-positive cells in WT MEFs. Furthermore, we identified Rasd1, a member of the Ras superfamily of small GTPases, as a functional target of miR-365. While expression of miR-365 suppressed Rasd1 3′ UTR luciferase-reporter activity, this effect was lost with mutations in the putative 3′ UTR target-site. Consistently, expression levels of miR-365 were found to inversely correlate with endogenous Rasd1 levels. These findings suggest that miR-365 is down-regulated in Zmpste24 −/− MEFs and acts as a novel negative regulator of Rasd1. Our comprehensive miRNA data provide a resource to study gene regulatory networks in MEFs.


      PubDate: 2015-06-14T11:42:17Z
       
  • Cytogenetic heterogeneity and their serial dynamic changes during
           
    • Abstract: Publication date: July 2015
      Source:Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, Volume 777
      Author(s): Jung-Ah Kim , Kyong Ok Im , Si Nae Park , Ji Seok Kwon , Seon Young Kim , Keunhee Oh , Dong-Sup Lee , Min Kyung Kim , Seong Who Kim , Mi Jang , Gene Lee , Yeon-Mok Oh , Sang Do Lee , Dong Soon Lee
      To minimize the risk of tumorigenesis in mesenchymal stem cells (MSCs), G-banding analysis is widely used to detect chromosomal aberrations in MSCs. However, a critical limitation of G-banding is that it only reflects the status of metaphase cells, which can represent as few as 0.01% of tested cells. During routine cytogenetic testing in MSCs, we often detect chromosomal aberrations in minor cell populations. Therefore, we aimed to investigate whether such a minority of cells can expand over time or if they ultimately disappear during MSC passaging. We passaged MSCs serially while monitoring quantitative changes for each aberrant clone among heterogeneous MSCs. To investigate the cytogenetic status of interphase cells, which represent the main population, we also performed interphase FISH analysis, in combination with G-banding and telomere length determination. In human adipose tissue-derived MSCs, 4 types of chromosomal aberrations were found during culturing, and in umbilical cord MSCs, 2 types of chromosomal aberrations were observed. Sequential dynamic changes among heterogeneous aberrant clones during passaging were similar to the dynamic changes observed in cancer stem cells during disease progression. Throughout all passages, the quantitative G-banding results were inconsistent with those of the interphase FISH analysis. Interphase FISH revealed hidden aberrations in stem cell populations with normal karyotypes by G-banding analysis. We found that telomere length gradually decreased during passaging until the point at which cytogenetic aberrations appeared. The present study demonstrates that rare aberrant clones at earlier passages can become predominant clones during later passages. Considering the risk of tumorigenesis due to aberrant MSCs, we believe that our results will help to establish proper safety guidelines for MSC use. In particular, we believe it is critical to test for chromosomal aberrations using both G-banding and FISH to ensure the safety of human stem cells that are manufactured in vitro for clinical applications.


      PubDate: 2015-06-14T11:42:17Z
       
  • Role of chromatin structure modulation by the histone deacetylase
           inhibitor trichostatin A on the radio-sensitivity of ataxia telangiectasia
           
    • Abstract: Publication date: July 2015
      Source:Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, Volume 777
      Author(s): Roberta Meschini , Elisa Morucci , Andrea Berni , Wilner Lopez-Martinez , Fabrizio Palitti
      At present, a lot is known about biochemical aspects of double strand breaks (DBS) repair but how chromatin structure affects this process and the sensitivity of DNA to DSB induction is still an unresolved question. Ataxia telangiectasia (A-T) patients are characterised by very high sensitivity to DSB-inducing agents such as ionising radiation. This radiosensitivity is revealed with an enhancement of chromosomal instability as a consequence of defective DNA repair for a small fraction of breaks located in the heterochromatin, where they are less accessible. Besides, recently it has been reported that Ataxia Telangiectasia Mutated (ATM) mediated signalling modifies chromatin structure. In order to study the impact of chromatin compaction on the chromosomal instability of A-T cells, the response to trichostatin-A, an histone deacetylase inhibitor, in normal and A-T lymphoblastoid cell lines was investigated testing its effect on chromosomal aberrations, cell cycle progression, DNA damage and repair after exposure to X-rays. The results suggest that the response to both trichostatin-A pre- and continuous treatments is independent of the presence of either functional or mutated ATM protein, as the reduction of chromosomal damage was found also in the wild-type cell line. The presence of trichostatin-A before exposure to X-rays could give rise to prompt DNA repair functioning on chromatin structure already in an open conformation. Differently, trichostatin-A post-treatment causing hyperacetylation of histone tails and reducing the heterochromatic DNA content might diminish the requirement for ATM and favour DSBs repair reducing chromosomal damage only in A-T cells. This fact could suggest that trichostatin-A post-treatment is favouring the slow component of DSB repair pathway, the one impaired in absence of a functionally ATM protein. Data obtained suggest a fundamental role of chromatin compaction on chromosomal instability in A-T cells.


      PubDate: 2015-06-14T11:42:17Z
       
  • Transmission of clonal chromosomal abnormalities in human hematopoietic
           stem and progenitor cells surviving radiation exposure
    • Abstract: Publication date: July 2015
      Source:Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, Volume 777
      Author(s): Daniela Kraft , Sylvia Ritter , Marco Durante , Erhard Seifried , Claudia Fournier , Torsten Tonn
      In radiation-induced acute myeloid leukemia (rAML), clonal chromosomal abnormalities are often observed in bone marrow cells of patients, suggesting that their formation is crucial in the development of the disease. Since rAML is considered to originate from hematopoietic stem and progenitor cells (HSPC), we investigated the frequency and spectrum of radiation-induced chromosomal abnormalities in human CD34+ cells. We then measured stable chromosomal abnormalities, a possible biomarker of leukemia risk, in clonally expanded cell populations which were grown for 14 days in a 3D-matrix (CFU-assay). We compared two radiation qualities used in radiotherapy, sparsely ionizing X-rays and densely ionizing carbon ions (29 and 60–85keV/μm, doses between 0.5 and 4Gy). Only a negligible number of de novo arising, unstable aberrations (≤0.05 aberrations/cell, 97% breaks) were measured in the descendants of irradiated HSPC. However, stable aberrations were detected in colonies formed by irradiated HSPC. All cells of the affected colonies exhibited one or more identical aberrations, indicating their clonal origin. The majority of the clonal rearrangements (92%) were simple exchanges such as translocations (77%) and pericentric inversions (15%), which are known to contribute to the development of rAML. Carbon ions were more efficient in inducing cell killing (maximum of ∼30–35% apoptotic cells for 2Gy carbon ions compared to ∼25% for X-rays) and chromosomal aberrations in the first cell-cycle after exposure (∼70% and ∼40% for 1Gy of carbon ions and X-rays, respectively), with a higher fraction of non-transmissible aberrations. In contrast, for both radiation qualities the percentage of clones with chromosomal abnormalities was similar (40%). Using the frequency of colonies with clonal aberrations as a surrogate marker for the leukemia risk following radiotherapy of solid tumors, charged particle therapy is not expected to lead to an increased risk of leukemia in patients.


      PubDate: 2015-06-14T11:42:17Z
       
  • γ-H2AX induced by linear alkylbenzene sulfonates is due to
           deoxyribonuclease-1 translocation to the nucleus via actin disruption
    • Abstract: Publication date: July 2015
      Source:Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, Volume 777
      Author(s): Xiaoxu Zhao , Tatsushi Toyooka , Toru Kubota , Guang Yang , Yuko Ibuki
      Phosphorylation of histone H2AX (γ-H2AX) occurs following formation of DNA double strand breaks (DSBs). Other types of DNA damage also generate DSBs through DNA replication and repair, leading to the production of γ-H2AX. In the present study, we demonstrated that linear alkylbenzene sulfonates (LAS), the most widely used and non-genotoxic anionic surfactants, could generate γ-H2AX via a novel pathway. Breast adenocarcinoma MCF-7 cells were treated with five kinds of LAS with alkyl chains ranging from 10 to 14 carbon units (C10–C14LAS). The generation of DSBs and subsequent production of γ-H2AX increased in a manner that depended on the number of carbon units in LAS. γ-H2AX could also be generated with non-cytotoxic doses of LAS and was independent of the cell cycle, indicating the non-apoptotic and DNA replication-independent formation of DSBs. The generation of γ-H2AX could be attenuated by EGTA and ZnCl2, deoxyribonuclease-1 (DNase I) inhibitors, as well as by the knockdown of DNase I. LAS weakened the interaction between DNase I and actin, and the enhanced release of DNase I was dependent on the number of carbon units in LAS. DNase I released by the LAS treatment translocated to the nucleus, in which DNase I attacked DNA and generated γ-H2AX. These results suggested that the LAS-induced generation of γ-H2AX could be attributed to the translocation of DNase I to the nucleus through the disruption of actin, and not to LAS-induced DNA damage.
      Graphical abstract image

      PubDate: 2015-06-14T11:42:17Z
       
  • Methods for comparing mutation rates using fluctuation assay data
    • Abstract: Publication date: July 2015
      Source:Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, Volume 777
      Author(s): Qi Zheng
      Comparing microbial mutation rates via the fluctuation assay protocol is an important routine task in the laboratory. However, methods for the comparison of mutation rates are still scarce and not well understood. This paper proposes new methods to address this situation. First, it provides a likelihood ratio test. Second, it explores the use of confidence intervals that can be readily computed. Third, it uses simulations to assess the new and existing methods. Both the likelihood ratio test and the use of confidence intervals were found to be superior to the general-purpose Mann–Whitney test, while the t-test was found to be unsuitable for fluctuation assay data. In addition, the method based on confidence intervals is suitable for comparison of experiments in which the terminal cell population sizes differ.


      PubDate: 2015-06-14T11:42:17Z
       
  • Inhibition of Bcl-2 or IAP proteins does not provoke mutations in
           surviving cells
    • Abstract: Publication date: July 2015
      Source:Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, Volume 777
      Author(s): Tanmay M. Shekhar , Maja M. Green , David M. Rayner , Mark A. Miles , Suzanne M. Cutts , Christine J. Hawkins
      Chemotherapy and radiotherapy can cause permanent damage to the genomes of surviving cells, provoking severe side effects such as second malignancies in some cancer survivors. Drugs that mimic the activity of death ligands, or antagonise pro-survival proteins of the Bcl-2 or IAP families have yielded encouraging results in animal experiments and early phase clinical trials. Because these agents directly engage apoptosis pathways, rather than damaging DNA to indirectly provoke tumour cell death, we reasoned that they may offer another important advantage over conventional therapies: minimisation or elimination of side effects such as second cancers that result from mutation of surviving normal cells. Disappointingly, however, we previously found that concentrations of death receptor agonists like TRAIL that would be present in vivo in clinical settings provoked DNA damage in surviving cells. In this study, we used cell line model systems to investigate the mutagenic capacity of drugs from two other classes of direct apoptosis-inducing agents: the BH3-mimetic ABT-737 and the IAP antagonists LCL161 and AT-406. Encouragingly, our data suggest that IAP antagonists possess negligible genotoxic activity. Doses of ABT-737 that were required to damage DNA stimulated Bax/Bak-independent signalling and exceeded concentrations detected in the plasma of animals treated with this drug. These findings provide hope that cancer patients treated by BH3-mimetics or IAP antagonists may avoid mutation-related illnesses that afflict some cancer survivors treated with conventional DNA-damaging anti-cancer therapies.
      Graphical abstract image

      PubDate: 2015-06-14T11:42:17Z
       
  • Electrophysiologic and cellular characteristics of cardiomyocytes after
           X-ray irradiation
    • Abstract: Publication date: July 2015
      Source:Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, Volume 777
      Author(s): Johannes L. Frieß , Anja Heselich , Sylvia Ritter , Angelina Haber , Nicole Kaiser , Paul G. Layer , Christiane Thielemann
      The aim of this study was to investigate possible effects of ionizing irradiation on the electrophysiological functionality of cardiac myocytes in vitro. Primary chicken cardiomyocytes with spontaneous beating activity were irradiated with X-rays (dose range of 0.5–7Gy). Functional alterations of cardiac cell cultures were evaluated up to 7 days after irradiation using microelectrode arrays. As examined endpoints, cell proliferation, apoptosis, reactive oxygen species (ROS) and DNA damage were evaluated. The beat rate of the cardiac networks increased in a dose-dependent manner over one week. The duration of single action potentials was slightly shortened. Additionally, we observed lower numbers of mitotic and S-phase cells at certain time points after irradiation. Also, the number of cells with γH2AX foci increased as a function of the dose. No significant changes in the level of ROS were detected. Induction of apoptosis was generally negligibly low. This is the first report to directly show alterations in cardiac electrophysiology caused by ionizing radiation, which were detectable up to one week after irradiation.


      PubDate: 2015-06-14T11:42:17Z
       
  • Functional interaction between hMYH and hTRADD in the TNF-α-mediated
           survival and death pathways of HeLa cells
    • Abstract: Publication date: July 2015
      Source:Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, Volume 777
      Author(s): An Hue Vy Tran , Soo-Hyun Hahm , Se Hee Han , Ji Hyung Chung , Geon Tae Park , Ye Sun Han
      The tumor necrosis factor (TNF) signaling pathway is a classical immune system pathway that plays a key role in regulating cell survival and apoptosis. The TNF receptor-associated death domain (TRADD) protein is recruited to the death domain of TNF receptor 1 (TNFR1), where it interacts with TNF receptor-associated factor 2 (TRAF2) and receptor-interacting protein (RIP) for the induction of apoptosis, necrosis, nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB), and mitogen-activated protein (MAP) kinase activation. In this study, we found that the human MutY homolog (hMYH) interacted with human TRADD (hTRADD) via the C-terminal domain of hMYH. Moreover, under conditions promoting TNF-α-induced cell death or survival in HeLa cells, this interaction was weakened or enhanced, respectively. The interaction between hMYH and hTRADD was important for signaling pathways mediated by TNF-α. Our results also suggested that the hTRADD–hMYH association was involved in the nuclear translocation of NFκB and formation of the TNFR1–TRADD complex. Thus, this study identified a novel mechanism through which the hMYH–hTRADD interaction may affect the TNF-α signaling pathway. Implications In HeLa cells, the hTRADD–hMYH interaction functioned in both cell survival and apoptosis pathways following TNF-α stimulation.


      PubDate: 2015-06-14T11:42:17Z
       
  • TITLE PAGE (EDI BOARD)
    • Abstract: Publication date: July 2015
      Source:Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, Volume 777




      PubDate: 2015-06-14T11:42:17Z
       
  • The genetic consequences of paternal acrylamide exposure and potential for
           amelioration
    • Abstract: Publication date: July 2015
      Source:Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, Volume 777
      Author(s): Aimee L. Katen , Shaun D. Roman
      Acrylamide is a toxin that humans are readily exposed to due to its formation in many carbohydrate rich foods cooked at high temperatures. Acrylamide is carcinogenic, neurotoxic and causes reproductive toxicity when high levels of exposure are reached in mice and rats. Acrylamide induced effects on fertility occur predominantly in males. Acrylamide exerts its reproductive toxicity via its metabolite glycidamide, a product which is only formed via the cytochrome P450 detoxifying enzyme CYP2E1. Glycidamide is highly reactive and forms adducts with DNA. Chronic low dose acrylamide exposure in mice relevant to human exposure levels results in significantly increased levels of DNA damage in terms of glycidamide adducts in spermatocytes, the specific germ cell stage where Cyp2e1 is expressed. Since cells in the later stages of spermatogenesis are unable to undergo DNA repair, and this level of acrylamide exposure causes no reduction in fertility, there is potential for this damage to persist until sperm maturation and fertilisation. Cyp2e1 is also present within epididymal cells, allowing for transiting spermatozoa to be exposed to glycidamide. This could have consequences for future generations in terms of predisposition to diseases such as cancer, with growing indications that paternal DNA damage can be propagated across multiple generations. Since glycidamide is the major contributor to DNA damage, a mechanism for preventing these effects is inhibiting the function of Cyp2e1. Resveratrol is an example of an inhibitor of Cyp2e1 which has shown success in reducing damage caused by acrylamide treatment in mice.


      PubDate: 2015-06-14T11:42:17Z
       
  • Prognostic role of APC and RASSF1A promoter methylation status in cell
           free circulating DNA of operable gastric cancer patients
    • Abstract: Publication date: August 2015
      Source:Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, Volume 778
      Author(s): I. Balgkouranidou , D. Matthaios , A. Karayiannakis , H. Bolanaki , P. Michailidis , N. Xenidis , K. Amarantidis , L. Chelis , G. Trypsianis , E. Chatzaki , E.S. Lianidou , S. Kakolyris
      Gastric carcinogenesis is a multistep process including not only genetic mutations but also epigenetic alterations. The best known and more frequent epigenetic alteration is DNA methylation affecting tumor suppressor genes that may be involved in various carcinogenetic pathways. The aim of the present study was to investigate the methylation status of APC promoter 1A and RASSF1A promoter in cell free DNA of operable gastric cancer patients. Using methylation specific PCR, we examined the methylation status of APC promoter 1A and RASSF1A promoter in 73 blood samples obtained from patients with gastric cancer. APC and RASSF1A promoters were found to be methylated in 61 (83.6%) and 50 (68.5%) of the 73 gastric cancer samples examined, but in none of the healthy control samples (p <0.001). A significant association between methylated RASSF1A promoter status and lymph node positivity was observed (p =0.005). Additionally, a significant correlation between a methylated APC promoter and elevated CEA (p =0.033) as well as CA-19.9 (p =0.032) levels, was noticed. The Kaplan–Meier estimates of survival, significantly favored patients with a non-methylated APC promoter status (p =0.008). No other significant correlations between APC and RASSF1A methylation status and different tumor variables examined was observed. Serum RASSF1A and APC promoter hypermethylation is a frequent epigenetic event in patients with early operable gastric cancer. The observed correlations between APC promoter methylation status and survival as well as between a hypermethylated RASSF1A promoter and nodal positivity may be indicative of a prognostic role for those genes in early operable gastric cancer. Additional studies, in a larger cohort of patients are required to further explore whether these findings could serve as potential molecular biomarkers of survival and/or response to specific treatments.


      PubDate: 2015-06-14T11:42:17Z
       
  • The role of base excision repair in the development of primary open angle
           glaucoma in the Polish population
    • Abstract: Publication date: August 2015
      Source:Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, Volume 778
      Author(s): Magda Cuchra , Lukasz Markiewicz , Bartosz Mucha , Dariusz Pytel , Katarzyna Szymanek , Janusz Szemraj , Jerzy Szaflik , Jacek P. Szaflik , Ireneusz Majsterek
      Glaucoma is a leading cause of irreversible blindness in developing countries. Previous data have shown that progressive loss of human TM cells may be connected with chronic exposure to oxidative stress. This hypothesis may suggest a role of the base excision repair (BER) pathway of oxidative DNA damage in primary open angle glaucoma (POAG) patients. The aim of our study was to evaluate an association of BER gene polymorphism with a risk of POAG. Moreover, an association of clinical parameters was examined including cup disk ratio (c/d), rim area (RA) and retinal nerve fiber layer (RNFL) with glaucoma progression according to BER gene polymorphisms. Our research included 412 patients with POAG and 454 healthy controls. Gene polymorphisms were analyzed by PCR-RFLP. Heidelberg Retinal Tomography (HRT) clinical parameters were also analyzed. The 399Arg/Gln genotype of the XRCC1 gene (OR 1.38; 95% CI 1.02–1.89 p =0.03) was associated with an increased risk of POAG occurrence. It was indicated that the 399Gln/Gln XRCC1 genotype might increase the risk of POAG progression according to the c/d ratio (OR 1.67; 95% CI 1.07–2.61 P =0.02) clinical parameter. Moreover, the association of VF factor with 148Asp/Glu of APE1 genotype distribution and POAG progression (OR 2.25; 95% CI 1.30–3.89) was also found. Additionally, the analysis of the 324Gln/His MUTYH polymorphism gene distribution in the patient group according to RNFL factor showed that it might decrease the progression of POAG (OR 0.47; 95% CI 0.30–0.82 P =0.005). We suggest that the 399Arg/Gln polymorphism of the XRCC1 gene may serve as a predictive risk factor of POAG.


      PubDate: 2015-06-14T11:42:17Z
       
  • Genetic variants of H2AX gene were associated with PM2.5-modulated DNA
           damage levels in Chinese Han populations
    • Abstract: Publication date: August 2015
      Source:Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, Volume 778
      Author(s): Chongqi Sun , Minjie Chu , Weihong Chen , Guangfu Jin , Jianhang Gong , Meng Zhu , Jing Yuan , Juncheng Dai , Meilin Wang , Yun Pan , Yuanchao Song , Xiaojie Ding , Mulong Du , Jing Dong , Zhengdong Zhang , Zhibin Hu , Tangchun Wu , Hongbing Shen
      Exposure to particulate matter 2.5 (PM2.5) may result in DNA damage. Histone variant H2AX phosphorylation plays a central role in the response to damaged chromatin. In the current study, we investigated whether H2AX gene polymorphisms account for PM2.5-modulated DNA damage levels. A total of 307 healthy urban residents were collected from three cities in southern, central, and northern China, Zhuhai, Wuhan, and Tianjin, respectively. The dust mass concentrations of PM2.5 were detected by Gilian 5000 pumps, and the DNA damage levels were measured using comet assay. Seven potentially functional single nucleotide polymorphisms (SNPs) of H2AX gene were selected and genotyped by Illumina Infinium® BeadChip. We found that three SNPs (rs10790283 G>A, rs604714 C>A and rs7759 A>G) were significantly associated with DNA damage levels (adjusted P =0.002, 0.018 and 0.027, respectively). Significant interactions (P <0.05) were observed between certain genetic polymorphisms and PM2.5-modulated DNA damage levels. These results suggested that genetic variations of H2AX might be associated with the DNA damage levels in urban residents with different exposure to PM2.5. Further studies with large sample size in independent populations merit validating these findings.


      PubDate: 2015-06-14T11:42:17Z
       
  • Are effects of common ragwort in the Ames test caused by pyrrolizidine
           alkaloids'
    • Abstract: Publication date: August 2015
      Source:Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, Volume 778
      Author(s): Toine F.H. Bovee , Richard J.R. Helsdingen , Ron L.A.P. Hoogenboom , Monique W.C.M. de Nijs , Xiaojie Liu , Klaas Vrieling , Peter G.L. Klinkhamer , Ad A.C.M. Peijnenburg , Patrick P.J. Mulder
      It has previously been demonstrated by others that acetone extracts of Senecio jacobaea (syn. Jacobaea vulgaris, common or tansy ragwort) test positive in the Salmonella/microsome mutagenicity test (Ames test). Pyrrolizidine alkaloids (PAs) are thought to be responsible for these mutagenic effects. However, it was also observed that the major PA present in common ragwort, jacobine, produced a negative response (with and without the addition of rat liver S9) in Salmonella test strains TA98, TA100, TA1535 and TA1537. To investigate which compounds in the plant extracts were responsible for the positive outcome, the present study investigated the contents and mutagenic effects of methanol and acetone extracts prepared from dried ground S. jacobaea and Senecio inaequidens (narrow-leafed ragwort). Subsequently, a fractionation approach was set up in combination with LC-MS/MS analysis of the fractions. It was shown that the positive Ames test outcomes of S. jacobaea extracts are unlikely to be caused by PAs, but rather by the flavonoid quercetin. This study also demonstrates the importance of identifying compounds responsible for positive test results in bioassays.


      PubDate: 2015-06-14T11:42:17Z
       
  • Genetic determinants of quantitative traits associated with cardiovascular
           disease risk
    • Abstract: Publication date: August 2015
      Source:Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, Volume 778
      Author(s): Božena Smolková , Stefano Bonassi , Verona Buociková , Mária Dušinská , Alexandra Horská , Daniel Kuba , Zuzana Džupinková , Katarína Rašlová , Juraj Gašparovič , Ivan Slíž , Marcello Ceppi , Branislav Vohnout , Ladislava Wsólová , Katarína Volkovová
      Established risk factors for cardiovascular diseases (CVD) may be moderated by genetic variants. In 2403 unrelated individuals from general practice (mean age 40.5 years), we evaluated the influence of 15 variants in 12 candidate genes on quantitative traits (QT) associated with CVD (body mass index, abdominal obesity, glucose, serum lipids, and blood pressure). Prior to multiple testing correction, univariate analysis associated APOE rs429358, rs7412 and ATG16L1 rs2241880 variants with serum lipid levels, while LEPR rs1137100 and ATG16L1 rs2241880 variants were linked to obesity related QTs. After taking into account confounding factors and correcting for multiple comparisons only APOE rs429358 and rs7412 variants remained significantly associated with risk of dyslipidemia. APOE rs429358 variant almost tripled the risk in homozygous subjects (OR=2.97; 95% CI 1.09–8.10, p <0.03) and had a lesser but still highly significant association also in heterozygous individuals (OR=1.67; 95% CI 1.24–2.10; p <0.001). Associations with hypertension, diabetes mellitus, and metabolic syndrome were not significant after Bonferroni correction. The influence of genetic variation is more evident in dyslipidemia than in other analyzed QTs. These results may contribute to strategic research aimed at including genetic variation in the set of data required to identify subjects at high risk of CVD.


      PubDate: 2015-06-14T11:42:17Z
       
  • Why the DNA self-depurination mechanism operates in HB-β but not in
           β-globin paralogs HB-δ, HB-ɛ1, HB-γ1 and HB-γ2
    • Abstract: Publication date: August 2015
      Source:Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, Volume 778
      Author(s): Olga Amosova , Juan R. Alvarez-Dominguez , Jacques R. Fresco
      The human β-globin, δ-globin and ɛ-globin genes contain almost identical coding strand sequences centered about codon 6 having potential to form a stem-loop with a 5′GAGG loop. Provided with a sufficiently stable stem, such a structure can self-catalyze depurination of the loop 5′G residue, leading to a potential mutation hotspot. Previously, we showed that such a hotspot exists about codon 6 of β-globin, with by far the highest incidence of mutations across the gene, including those responsible for 6 anemias (notably Sickle Cell Anemia) and β-thalassemias. In contrast, we show here that despite identical loop sequences, there is no mutational hotspot in the δ- or ɛ1-globin potential self-depurination sites, which differ by only one or two base pairs in the stem region from that of the β-globin gene. These differences result in either one or two additional mismatches in the potential 7-base pair-forming stem region, thereby weakening its stability, so that either DNA cruciform extrusion from the duplex is rendered ineffective or the lifetime of the stem-loop becomes too short to permit self-catalysis to occur. Having that same loop sequence, paralogs HB-γ1 and HB-γ2 totally lack stem-forming potential. Hence the absence in δ- and ɛ1-globin genes of a mutational hotspot in what must now be viewed as non-functional homologs of the self-depurination site in β-globin. Such stem-destabilizing variants appeared early among vertebrates and remained conserved among mammals and primates. Thus, this study has revealed conserved sequence determinants of self-catalytic DNA depurination associated with variability of mutation incidence among human β-globin paralogs.


      PubDate: 2015-06-14T11:42:17Z
       
 
 
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