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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  [2563 journals]   [SJR: 0.324]   [H-I: 11]
  • Formaldehyde-induced histone H3 phosphorylation via JNK and the expression
           of proto-oncogenes
    • Abstract: Publication date: Available online 16 September 2014
      Source:Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis
      Author(s): Ikuma Yoshida , Yuko Ibuki
      Formaldehyde (FA) is a very reactive compound that forms DNA adducts and DNA-protein crosslinks, which are known to contribute to FA-induced mutations and carcinogenesis. Post-translational modifications to histones have recently attracted attention due to their link with cancer. In the present study, we examined histone modifications following a treatment with FA. FA significantly phosphorylated histone H3 at serine 10 (H3S10), and at serine 28 (H3S28), the time-course of which was similar to the phosphorylation of H2AX at serine 139 (γ-H2AX), a marker of DNA double strand breaks. The temporal deacetylation of H3 was observed due to the reaction of FA with the lysine residues of histones. The phosphorylation mechanism was then analyzed by focusing on H3S10. The nuclear distribution of the phosphorylation of H3S10 and γ-H2AX did not overlap, and the phosphorylation of H3S10 could not be suppressed with an inhibitor of ATM/ATR, suggesting that the phosphorylation of H3S10 was independent of the DNA damage response. ERK and JNK in the MAPK pathways were phosphorylated by the treatment with FA, in which the JNK pathway was the main target for phosphorylation. The phosphorylation of H3S10 increased at the promoter regions of c-fos and c-jun, indicating a relationship between FA-induced tumor promotion activity and phosphorylation of H3S10. These results suggested that FA both initiates and promotes cancer, as judged by an analysis of histone modifications.
      Graphical abstract image

      PubDate: 2014-09-17T04:04:54Z
       
  • Quantifying murine bone marrow and blood radiation dose response following
           18F-FDG PET with DNA damage biomarkers
    • Abstract: Publication date: Available online 16 September 2014
      Source:Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis
      Author(s): Grainne Manning , Kristina Taylor , Paul Finnon , Jennifer A. Lemon , Douglas R. Boreham , Christophe Badie
      The purpose of this study was to quantify the poorly understood radiation doses to murine bone marrow and blood from whole-body fluorine 18 (18F)-fluorodeoxyglucose (FDG) positron emission tomography (PET), by using specific biomarkers and comparing with whole body external low dose exposures. Groups of 3–5 mice were randomly assigned to 10 groups, each receiving either a different activity of 18F-FDG: 0–37MBq or whole body irradiated with corresponding doses of 0–300mGy X-rays. Blood samples were collected at 24h and at 43h for reticulocyte micronucleus assays and QPCR analysis of gene expression in peripheral blood leukocytes. Blood and bone marrow dose estimates were calculated from injected activities of 18F-FDG and were based on a recommended ICRP model. Doses to the bone marrow corresponding to 33.43mGy and above for internal 18F-FDG exposure and to 25mGy and above for external X-ray exposure, showed significant increases in radiation-induced MN-RET formation relative to controls (P <0.05). Regression analysis showed that both types of exposure produced a linear response with linear regression analysis giving R 2 of 0.992 and 0.999 for respectively internal and external exposure. No significant difference between the two data sets was found with a P-value of 0.493. In vivo gene expression dose–responses at 24h for Bbc3 and Cdkn1 were similar for 18F-FDG and X-ray exposures, with significant modifications occurring for doses over 300mGy for Bbc3 and at the lower dose of 150mGy for Cdkn1a. Both leucocyte gene expression and quantification of MN-RET are highly sensitive biomarkers for reliable estimation of the low doses delivered in vivo to, respectively, blood and bone marrow, following 18F-FDG PET.


      PubDate: 2014-09-17T04:04:54Z
       
  • Phosphorylation of PTEN at STT motif is associated with DNA damage
           response
    • Abstract: Publication date: Available online 4 September 2014
      Source:Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis
      Author(s): Sandip Misra , Ananda Mukherjee , Parimal Karmakar
      Phosphatase and tensin homolog deleted on chromosome Ten (PTEN), a tumor suppressor protein participates in multiple cellular activities including DNA repair. In this work we found a relationship between phosphorylation of carboxy (C)-terminal STT motif of PTEN and DNA damage response. Ectopic expression of C-terminal phospho-mutants of PTEN, in PTEN deficient human glioblastoma cells, U87MG, resulted in reduced viability and DNA repair after etoposide induced DNA damage compared to cells expressing wild type PTEN. Also, after etoposide treatment phosphorylation of PTEN increased at C-terminal serine 380 and threonine 382/383 residues in PTEN positive HEK293T cells and wild type PTEN transfected U87MG cells. One-step further, DNA damage induced phosphorylation of PTEN was confirmed by immunoprecipitation of total PTEN from cellular extract followed by immunobloting with phospho-specific PTEN antibodies. Additionally, phospho-PTEN translocated to nucleus after etoposide treatment as revealed by indirect immunolabeling. Further, phosphorylation dependent nuclear foci formation of PTEN was observed after ionizing radiation or etoposide treatment which colocalized with γH2AX. Additionally, etoposide induced γH2AX, Mre11 and Ku70 foci persisted for a longer period of times in U87MG cells after ectopic expression of PTEN C-terminal phospho-mutant constructs compared to wild type PTEN expressing cells. Thus, our findings strongly suggest that DNA damage induced phosphorylation of C-terminal STT motif of PTEN is necessary for DNA repair.


      PubDate: 2014-09-06T03:06:06Z
       
  • DNA damage and gene therapy of xeroderma pigmentosum, a human DNA
           repair-deficient disease
    • Abstract: Publication date: Available online 6 September 2014
      Source:Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis
      Author(s): Aurélie Dupuy , Alain Sarasin
      Xeroderma pigmentosum (XP) is a genetic disease characterized by hypersensitivity to ultra-violet and a very high risk of skin cancer induction on exposed body sites. This syndrome is caused by germinal mutations on nucleotide excision repair genes. No cure is available for these patients except a complete protection from all types of UV radiations. We reviewed the various techniques to complement or to correct the genetic defect in XP cells. We, particularly, developed the correction of XP-C skin cells using the fidelity of the homologous recombination pathway during repair of double-strand break (DSB) in the presence of XPC wild type sequences. We used engineered nucleases (meganuclease or TALE nuclease) to induce a DSB located at 90bp of the mutation to be corrected. Expression of specific TALE nuclease in the presence of a repair matrix containing a long stretch of homologous wild type XPC sequences allowed us a successful gene correction of the original TG deletion found in numerous North African XP patients. Some engineered nucleases are sensitive to epigenetic modifications, such as cytosine methylation. In case of methylated sequences to be corrected, modified nucleases or demethylation of the whole genome should be envisaged. Overall, we showed that specifically-designed TALE-nuclease allowed us to correct a 2bp deletion in the XPC gene leading to patient's cells proficient for DNA repair and showing normal UV-sensitivity. The corrected gene is still in the same position in the human genome and under the regulation of its physiological promoter. This result is a first step toward gene therapy in XP patients.
      Graphical abstract image

      PubDate: 2014-09-06T03:06:06Z
       
  • Diet and lifestyle factors modify immune/inflammation response genes to
           alter breast cancer risk and prognosis: The Breast Cancer Health
           Disparities Study
    • Abstract: Publication date: Available online 4 September 2014
      Source:Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis
      Author(s): Martha L. Slattery , Abbie Lundgreen , Gabriela Torres-Mejia , Roger K. Wolff , Lisa Hines , Kathy Baumgartner , Esther M. John
      Tumor necrosis factor-α (TNF) and toll-like receptors (TLR) are important mediators of inflammation. We examined 10 of these genes with respect to breast cancer risk and mortality in a genetically admixed population of Hispanic/Native American (NA) (2111 cases, 2597 controls) and non-Hispanic white (NHW) (1481 cases, 1585 controls) women. Additionally, we explored if diet and lifestyle factors modified associations with these genes. Overall, these genes (collectively) were associated with breast cancer risk among women with >70% NA ancestry (PARTP =0.0008), with TLR1 rs7696175 being the primary risk contributor (OR 1.77, 95% CI 1.25, 2.51). Overall, TLR1 rs7696175 (HR 1.40, 95% CI 1.03, 1.91; Padj =0.032), TLR4 rs5030728 (HR 1.96, 95% CI 1.30, 2.95; Padj =0.014), and TNFRSF1A rs4149578 (HR 2.71, 95% CI 1.28, 5.76; Padj =0.029) were associated with increased breast cancer mortality. We observed several statistically significant interactions after adjustment for multiple comparisons, including interactions between our dietary oxidative balance score and CD40LG and TNFSF1A; between cigarette smoking and TLR1, TLR4, and TNF; between body mass index (BMI) among pre-menopausal women and TRAF2; and between regular use of aspirin/non-steroidal anti-inflammatory drugs and TLR3 and TRA2. In conclusion, our findings support a contributing role of certain TNF-α and TLR genes in both breast cancer risk and survival, particularly among women with higher NA ancestry. Diet and lifestyle factors appear to be important mediators of the breast cancer risk associated with these genes.


      PubDate: 2014-09-06T03:06:06Z
       
  • Optimal standard regimen and predicting response to docetaxel therapy
    • Abstract: Publication date: Available online 27 August 2014
      Source:Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis
      Author(s): Emad Y. Moawad
      The purpose of this research is optimizing and predicting the potent activity of docetaxel through an efficient regimen to settle down a new protocol for the treatment of cancer. Effectiveness of docetaxel was examined in vivo in several mouse models engrafted either subcutaneously or intravenously with several types of cell lines. The effects of 147–5040mg/L of docetaxel in treatments of different regimens in those xenograft growths were monitored and quantified to identify energy of those doses as described before in earlier studies. Mock processes were performed on untreated groups of mice for controls. Docetaxel had significant influence on all sizes of treated tumors compared to the control animals. The longer the induced tumor doubling time intraday to more than half the time period from the start of therapy to the time of delivery of the dose, the higher the energy of docetaxel doses and hence the effectiveness of the treatment and vice versa. The energy yield by drug doses in optimal standard regimens was perfectly power correlated (r =1) with the drug dose. An efficient dose-energy model with a perfect fit (R 2 =1) estimating the energy yield by docetaxel doses in optimal standard regimens has been established to administer the personalized dose. Administration of docetaxel doses should be patient-specific and sufficient for the suggested regimen. Time periods from the start of therapy to the time of dose delivery of the efficient regimen are shorter than twice the tumor doubling time intraday on time of dose delivery. Patients with tumors of lower mitotic index may particularly benefit more from optimal standard regimens, whereas metronomic regimens would be more efficient in patients with tumors of higher mitotic index that need lower doses of docetaxel.
      Graphical abstract image

      PubDate: 2014-09-02T02:25:43Z
       
  • Effect of drinking water disinfection by-products in human peripheral
           blood lymphocytes and sperm
    • Abstract: Publication date: Available online 26 August 2014
      Source:Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis
      Author(s): Aftab Ali , Malgorzata Kurzawa-Zegota , Mojgan Najafzadeh , Rajendran C. Gopalan , Michael J. Plewa , Diana Anderson
      Background Drinking water disinfection by-products (DBPs) are generated by the chemical disinfection of water and may pose hazards to public health. Two major classes of DBPs are found in finished drinking water: haloacetic acids (HAAs) and trihalomethanes (THMs). HAAs are formed following disinfection with chlorine, which reacts with iodide and bromide in the water. Previously the HAAs were shown to be cytotoxic, genotoxic, mutagenic, teratogenic and carcinogenic. Objectives To determine the effect of HAAs in human somatic and germ cells and whether oxidative stress is involved in genotoxic action. In the present study both somatic and germ cells have been examined as peripheral blood lymphocytes and sperm. Methods The effects of three HAA compounds: iodoacetic acid (IAA), bromoacetic acid (BAA) and chloroacetic acid (CAA) were investigated. After determining appropriate concentration responses, oxygen radical involvement with the antioxidants, butylated hydroxanisole (BHA) and the enzyme catalase, were investigated in the single cell gel electrophoresis (Comet) assay under alkaline conditions, >pH 13 and the micronucleus assay. Results In the Comet assay, BHA and catalase were able to reduce DNA damage in each cell type compared to HAA alone. In the micronucleus assay, micronuclei (MNi) were found in peripheral lymphocytes exposed to all three HAAs and catalase and BHA were in general, able to reduce MNi induction, suggesting oxygen radicals play a role in both assays. Conclusion These observations are of concern to public health since both human somatic and germ cells show similar genotoxic responses.


      PubDate: 2014-09-02T02:25:43Z
       
  • Oxidative stress preferentially induces a subtype of micronuclei and
           mediates the genomic instability caused by p53 dysfunction
    • Abstract: Publication date: Available online 27 August 2014
      Source:Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis
      Author(s): Bing Xu , Wenxing Wang , Haiyang Guo , Zhaoliang Sun , Zhao Wei , Xiyu Zhang , Zhaojian Liu , Jay A. Tischfield , Yaoqin Gong , Changshun Shao
      Reactive oxygen species (ROS) are known to cause many types of DNA lesions that could be converted into cancer-promoting genetic alterations. Evidence showed that tumor suppressor p53 plays an important role in regulating the generation of cellular ROS, either by reducing oxidative stress under physiological and mildly stressed conditions, or by promoting oxidative stress under highly stressed conditions. In this report we characterized the effect of oxidative stress on the induction of micronuclei, especially the subclass marked by pan-staining of γ-H2AX or MN-γ-H2AX (+). We found that MN-γ-H2AX (+) were more responsive to hydrogen peroxide (H2O2) than the MN-γ-H2AX (−). In human and mouse cells that are deficient in p53, the frequency of MN-γ-H2AX (+) is significantly elevated, but can be attenuated by antioxidant N-acetylcysteine (NAC). Depletion of p53-regulated antioxidant gene SESN1 by RNA interference also resulted in an elevation of MN-γ-H2AX (+). Furthermore, we found that in cells that were depleted of p400 by RNAi, and therefore were experiencing increased ROS, the frequency of MN-γ-H2AX (+), but not that of MN-γ-H2AX (−), was significantly induced. We further demonstrated that the induction of MN-γ-H2AX (+) by replication stress can also be attenuated by NAC, and that H2O2 also leads to increased phosphorylation of Chk1 and Rad17 that mimics replication stress, suggesting that replication stress and oxidative stress are intertwined and may reinforce each other in driving genomic instability. Our findings illustrate the importance of p53-regulated redox level in the maintenance of genomic stability.
      Graphical abstract image

      PubDate: 2014-09-02T02:25:43Z
       
  • High levels of γ-H2AX foci and cell membrane oxidation in adolescents
           with type 1 diabetes
    • Abstract: Publication date: Available online 27 August 2014
      Source:Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis
      Author(s): Caterina Giovannini , Simona Piaggi , Giovanni Federico , Roberto Scarpato
      Oxidative stress caused by an excess of free radicals is implicated in the pathogenesis and development of type 1 diabetes mellitus (T1DM) and, in turn, it can lead to genome damage, especially in the form of DNA double-strand break (DSB). The DNA DSB is a potentially carcinogenic lesion for human cells. Thus, we aimed to evaluate whether the level of oxidative stress was increased in peripheral blood lymphocytes of a group of affected adolescents. In 35 T1DM adolescents and 19 healthy controls we assessed: (1) spontaneous and H2O2-induced oxidation of cell membrane using a fluorescence lipid probe; (2) spontaneous and LPS-induced expression of iNOS protein and indirect NO determination via cytofluorimetric analysis of O2 −; (3) immunofluorescent detection of the basal level of histone H2AX phosphorylation (γ-H2AX foci), a well-validated marker of DNA DSB. In T1DM, the frequencies of oxidized cells, both spontaneous and H2O2-induced (47.13±0.02) were significantly higher than in controls (35.90±0.03). Patients showed, in general, both a reduced iNOS expression and production of NO. Furthermore, the level of spontaneous nuclear damage, quantified as γ-H2AX foci, was markedly increased in T1DM adolescents (6.15±1.08% of γ-H2AX+ cells; 8.72±2.14 γ-H2AXF/n; 9.26±2.37 γ-H2AXF/np), especially in females. In the present study, we confirmed the role that oxidative stress plays in the disease damaging lipids of cell membrane and, most importantly, causing genomic damage in circulating white blood cells of affected adolescents. This also indicates that oxidative stress can affect several tissues in the body. However, although the observed DNA damage is a clear indication that the proper DNA repair mechanisms are activated, the risk for young T1DM subjects of developing not only cardiovascular complications but also some type of cancer cannot be ruled out. In this view, females, probably due to hormonal imbalance typical of adolescence, might represent a more susceptible population.


      PubDate: 2014-09-02T02:25:43Z
       
  • In vitro cytotoxicity of silver nanoparticles and zinc oxide nanoparticles
           to human epithelial colorectal adenocarcinoma (Caco-2) cells
    • Abstract: Publication date: Available online 12 August 2014
      Source:Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis
      Author(s): Yijuan Song , Rongfa Guan , Fei Lyu , Tianshu Kang , Yihang Wu , Xiaoqiang Chen
      With the increasing applications of silver nanoparticles (Ag NPs) and zinc oxide nanoparticles (ZnO NPs) in foods and cosmetics, the concerns about the potential toxicities to human have been raised. The aims of this study are to observe the cytotoxicity of Ag NPs and ZnO NPs to human epithelial colorectal adenocarcinoma (Caco-2) cells in vitro, and to discover the toxicity mechanism of nanoparticles on Caco-2 cells. Caco-2 cells were exposed to 10, 25, 50, 100, 200μg/mL of Ag NPs and ZnO NPs (90nm). AO/EB double staining was used to characterize the morphology of the treated cells. The cell counting kit-8 (CCK-8) assay was used to detect the proliferation of the cells. Reactive oxygen species (ROS), superoxide dismutase (SOD) and glutathione (GSH) assay were used to explore the oxidative damage of Caco-2 cells. The results showed that Ag NPs and ZnO NPs (0-200μg/mL) had highly significant effect on the Caco-2 cells activity. ZnO NPs exerted higher cytotoxicity than Ag NPs in the same concentration range. ZnO NPs have dose-depended toxicity. The LD50 of ZnO NPs in Caco-2 cells is 0.431mg/L. Significant depletion of SOD level, variation in GSH level and release of ROS in cells treated by ZnO NPs were observed, which suggests that cytotoxicity of ZnO NPs in intestine cells might be mediated through cellular oxidative stress. While Caco-2 cells treated with Ag NPs at all experimental concentrations showed no cellular oxidative damage. Moreover, the cells’ antioxidant capacity increased, and reached the highest level when the concentration of Ag NPs was 50μg/mLmL. Therefore, it can be concluded that Ag NPs are safer antibacterial material in food packaging materials than ZnO NPs.


      PubDate: 2014-08-16T01:24:40Z
       
  • MutY-Glycosylase: an overview on mutagenesis and activities beyond the GO
           system
    • Abstract: Publication date: Available online 13 August 2014
      Source:Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis
      Author(s): Ana Helena Sales de Oliveira , Acarízia Eduardo da Silva , Iuri Marques de Oliveira , João Antônio Pegas Henriques , Lucymara Fassarella Agnez-Lima
      MutY is a glycosylase known for its role in DNA base excision repair (BER). It is critically important in the prevention of DNA mutations derived from 7,8-dihydro-8-oxoguanine (8-oxoG), which are the major lesions resulting from guanine oxidation. MutY has been described as a DNA repair enzyme in the GO system responsible for removing adenine residues misincorporated in 8-oxoG:A mispairs, avoiding G:C to T:A mutations. Further studies have shown that this enzyme binds to other mispairs, interacts with several enzymes, avoids different transversions/transitions in DNA, and is involved in different repair pathways. Additional activities have been reported for MutY, such as the repair of replication errors in newly synthesized DNA strands through its glycosylase activity. Moreover, MutY is a highly conserved enzyme present in several prokaryotic and eukaryotic organisms. MutY defects are associated with a hereditary colorectal cancer syndrome termed MUTYH-associated polyposis (MAP). Here, we have reviewed the roles of MutY in the repair of mispaired bases in DNA as well as its activities beyond the GO system.


      PubDate: 2014-08-16T01:24:40Z
       
  • Occurrence of low frequency PIK3CA and AKT2 mutations in gastric cancer
    • Abstract: Publication date: Available online 30 July 2014
      Source:Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis
      Author(s): Qing-Ying Zhang , Wen-Xiang Cheng , Wen-Mei Li , William Au , You-Yong Lu
      The PI3K/AKT signal transduction pathway have distinct functional roles in tumor progression. PIK3CA was reported to harbor the hot-spot in many types of tumor. Akt, the downstream of PI3K, its family members especially AKT2 activation in human cancer has been extensively studied, but its activation by mutation was less reported. The occurrence of PIK3CA and AKT2 mutations in a variety of cancers indicates their important involvement in carcinogenesis. Therefore, we investigated their mutation frequencies in gastric cancer (GC) in China. In our study, we selected hot-spot related exons 9, 18 and 20 of PIK3CA and kinase domain exons 6∼14 of AKT2 genes were screened in 10 GC cell lines, 100 advanced primary GC and matched normal tissues. Denaturing high performance liquid chromatography (DHPLC) and DNA sequencing were used to analyze the mutations in the two genes. Two point mutations in the PIK3CA gene were identified in 4 of 10 GC cell lines and in 4 of 100 GC primary tumors. Two polymorphisms in AKT2 were detected in 19 of 100 GC primary tumors. One point mutation in AKT2 was detected in 1 of 10 GC cell lines and 3 of 100 GC primary tumors but no hot spot variation was detected. Our results indicate that PIK3CA and AKT2 mutations occurred at low frequency in GC, and suggest that the PIK3CA/AKT2 pathway might engage other events during gastric carcinogenesis.


      PubDate: 2014-08-03T23:47:20Z
       
  • Conserved motifs of MutL proteins
    • Abstract: Publication date: Available online 31 July 2014
      Source:Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis
      Author(s): Michał Banasik , Paweł Sachadyn
      The MutL protein is best known for its function in DNA mismatch repair (MMR). However, there is evidence to suggest that MutL is not only the linker connecting the functions of MutS and MutH in MMR, but that it also participates in other repair systems, such as Very Short Patch (VSP), Base Excision (BER) and Nucleotide Excision Repair (NER). This study set out to identify the most highly conserved amino acid sequence motifs in MutL proteins. We analyzed 208 MutL amino acid sequences of 199 representative prokaryotic species belonging to 28 classes of bacteria and archaea. The analysis revealed 16 conserved motifs situated in the ATPase and endonuclease domains, as well as within the disordered loop, and in the MutL regions interacting with the β clamp of DNA polymerase III. The conserved sequence motifs thus determined constitute a structural definition of MutL and they may be used in site-directed mutagenesis studies. We found conserved residues within the potential regions where binding with MutS occurs. However, the existing data does not provide clues as to the possible sites of MutL interactions with the proteins involved in other DNA repair systems such as NER, BER and VSP. We determined the 57 most highly conserved amino acid residues, including 43 which were identical in all the sequences analyzed. The greater part of the most predominantly conserved amino acid residues identified in MutL are identical to the corresponding residues reported as mutational hot-spots in one of its human homologues, MLH1, but not in the other, PMS2. This is the first study to present the conserved sequence motifs of MutL widespread in bacteria and archaea and the classification of MutLs into five groups distinguished on the basis of differences in the C-terminal region. Our analysis is of use in better understanding MutL functions.


      PubDate: 2014-08-03T23:47:20Z
       
  • The analysis of S. cerevisiae cells deleted for mitotic cyclin Clb2
           reveals a novel requirement of Sgs1 DNA helicase and Exonuclease 1 when
           replication forks break in the presence of alkylation damage
    • Abstract: Publication date: Available online 1 August 2014
      Source:Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis
      Author(s): Laurence Signon , Marie Noelle Simon
      In this study, we report the effects of deleting the principal mitotic cyclin, Clb2, in different repair deficient contexts on sensitivity to the alkylating DNA damaging agent, methyl methanesulphonate (MMS). A yeast clb2 mutant is sensitive to MMS and displays synergistic effect when combined with inactivation of numerous genes involved in DNA recombination and replication. In contrast, clb2 has basically no additional effect with deletion of the RecQ helicase SGS1, the exonuclease EXO1 and the protein kinase RAD53 suggesting that Clb2 functions in these pathways. In addition, clb2 increases the viability of the mec1 kinase deficient mutant, suggesting Mec1 inhibits a deleterious Clb2 activity. Interestingly, we found that the rescue by EXO1 deletion of rad53K227 mutant, deficient in checkpoint activation, requires Sgs1, suggesting a role for Rad53, independent of its checkpoint function, in regulating an ordered recruitment of Sgs1 and Exo1 to fork structure. Overall, our data suggest that Clb2 affects recombinant structure of replication fork blocked by alkykating DNA damage at numerous steps and could regulate Sgs1 and Exo1 activity. In addition, we found novel requirement of Sgs1 DNA helicase and Exonuclease 1 when replication forks breaks in the presence of alkylation damage. Models for the functional interactions of mitotic cyclin Clb2, Sgs1 and Exo1 with replication fork stabilization are proposed.


      PubDate: 2014-08-03T23:47:20Z
       
  • The functional polymorphism of NBS1 p. Glu185Gln is associated with an
           increased risk of lung cancer in Chinese populations: case-control and a
           meta-analysis
    • Abstract: Publication date: Available online 1 August 2014
      Source:Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis
      Author(s): Wenxiang Fang , Fuman Qiu , Lisha Zhang , Jieqiong Deng , Haibo Zhang , Lei Yang , Yifeng Zhou , Jiachun Lu
      NBS1 plays pivotal roles in maintaining genomic stability and cancer development. The exon variant rs1805794G>C (p. Glu185Gln) of NBS1 has been frequency studied in several association studies. However, the results were conflicting. Also, the function of this variant has never been well studied. In the current study, we performed a two centers case-control study and function assays to investigate the effect of the variant rs1805794G>C on lung cancer risk in Chinese, and a meta-analysis to summarize the data on the association between rs1805794G>C and cancer risk. We found that compared with the rs1805794GG genotype, the C genotypes (CG/CC) conferred a significantly increased risk of lung cancer in Chinese (OR=1.40, 95%CI=1.21–1.62) and interacted with medical ionizing radiation exposure on increasing cancer risk (Pinteraction =0.015). The lymphocyte cells from the C genotypes individuals developed more chromatid breaks than those from the GG genotype carriers after the X-ray radiation (P =0.036). Moreover, the rs1805794C allele encoding p.185Gln attenuated NBS1's ability to repair DNA damage as that the cell lines transfected with NBS1 cDNA expression vector carrying rs1805794C allele had significantly higher DNA breaks than those transfected with NBS1 cDNA expression vector carrying rs1805794G allele (P < 0.05). The meta-analysis further confirmed the association between the variant rs1805794G>C and lung cancer risk, that compared with the GG genotype, the carriers of C genotypes had a 1.30-fold risk of cancer (95% CI = 1.14–1.49, p = 8.49×10-5). These findings suggest that the rs1805794G>C of NBS1 may be a functional genetic biomarker for lung cancer.


      PubDate: 2014-08-03T23:47:20Z
       
  • Suppression of DNA-dependent protein kinase sensitize cells to radiation
           without affecting DSB repair
    • Abstract: Publication date: November 2014
      Source:Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, Volume 769
      Author(s): Ann-Sofie Gustafsson , Andris Abramenkovs , Bo Stenerlöw
      Efficient and correct repair of DNA double-strand break (DSB) is critical for cell survival. Defects in the DNA repair may lead to cell death, genomic instability and development of cancer. The catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) is an essential component of the non-homologous end joining (NHEJ) which is the major DSB repair pathway in mammalian cells. In the present study, by using siRNA against DNA-PKcs in four human cell lines, we examined how low levels of DNA-PKcs affected cellular response to ionizing radiation. Decrease of DNA-PKcs levels by 80–95%, induced by siRNA treatment, lead to extreme radiosensitivity, similar to that seen in cells completely lacking DNA-PKcs and low levels of DNA-PKcs promoted cell accumulation in G2/M phase after irradiation and blocked progression of mitosis. Surprisingly, low levels of DNA-PKcs did not affect the repair capacity and the removal of 53BP1 or γ-H2AX foci and rejoining of DSB appeared normal. This was in strong contrast to cells completely lacking DNA-PKcs and cells treated with the DNA-PKcs inhibitor NU7441, in which DSB repair were severely compromised. This suggests that there are different mechanisms by which loss of DNA-PKcs functions can sensitize cells to ionizing radiation. Further, foci of phosphorylated DNA-PKcs (T2609 and S2056) co-localized with DSB and this was independent of the amount of DNA-PKcs but foci of DNA-PKcs was only seen in siRNA-treated cells. Our study emphasizes on the critical role of DNA-PKcs for maintaining survival after radiation exposure which is uncoupled from its essential function in DSB repair. This could have implications for the development of therapeutic strategies aiming to radiosensitize tumors by affecting the DNA-PKcs function.


      PubDate: 2014-07-26T23:04:48Z
       
  • 15-Deoxy-Δ12,14-prostaglandin J2 induces expression of
           15-hydroxyprostaglandin dehydrogenase through Elk-1 activation in human
           breast cancer MDA-MB-231 cells
    • Abstract: Publication date: Available online 24 June 2014
      Source:Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis
      Author(s): Hye-Rim Kim , Ha-Na Lee , Kyu Lim , Young-Joon Surh , Hye-Kyung Na
      Overproduction of prostaglandin E2 (PGE2) has been reported to be implicated in carcinogenesis. The intracellular level of PGE2 is maintained not only by its biosynthesis, but also by inactivation/degradation. 15-Hydroxyprostaglandin dehydrogenase (15-PGDH) is the key enzyme that catalyzes the conversion of oncogenic PGE2 to a biologically inactive keto metabolite. In the present study, we demonstrate that 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2), one of the terminal products of cyclooxygenase-2, updregulates the expression and the activity of 15-PGDH in human breast cancer MDA-MB-231 cells. By using deletion constructs of the 15-PGDH promoter, we have found that E-twenty six (Ets) is the most essential determinant for 15-PGDH induction. 15d-PGJ2 induced phosphorylation of Elk-1, one of Ets transcription factor family members, in the nucleus. Knockdown of Elk-1 abolished the ability of 15d-PGJ2 to upregulate 15-PGDH expression. Furthermore, 15d-PGJ2-mediated activation of Elk-1 was found to be dependent on activation of extracellular-signal related kinase (ERK) 1/2. Treatment of U0126, a pharmacological inhibitor of MEK1/2-ERK, abolished phosphorylation and DNA binding of Elk-1 as well as 15-PGDH induction in 15d-PGJ2-treated MDA-MB-231 cells. Moreover, 15d-PGJ2 generated reactive oxygen species (ROS), which contribute to the expression of 15-PGDH as well as phosphorylation of ERK1/2 and Elk-1. 15d-PGJ2 inhibited the migration of MDA-MB-231 cells, which was attenuated by transient transfection with 15-PGDH siRNA. Taken together, these findings suggest that 15d-PGJ2 induces the expression of 15-PGDH through ROS-mediated activation of ERK1/2 and subsequently Elk-1 in the MDA-MB-231 cells, which may contribute to tumor suppressive activity of this cyclopentenone prostaglandin.


      PubDate: 2014-07-26T23:04:48Z
       
  • Enhanced DNA repair of bleomycin-induced 3′-phosphoglycolate termini
           at the transcription start sites of actively transcribed genes in human
           cells
    • Abstract: Publication date: Available online 8 July 2014
      Source:Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis
      Author(s): Vincent Murray , Jon K. Chen , Anne M. Galea
      The anti-tumour agent, bleomycin, cleaves DNA to give 3′-phosphoglycolate and 5′-phosphate termini. The removal of 3′-phosphoglycolate to give 3′-OH ends is a very important step in the DNA repair of these lesions. In this study, next-generation DNA sequencing was utilised to investigate the repair of these 3′-phosphoglycolate termini at the transcription start sites (TSSs) of genes in HeLa cells. The 143,600 identified human TSSs in HeLa cells comprised 82,596 non-transcribed genes and 61,004 transcribed genes; and the transcribed genes were divided into quintiles of 12,201 genes comprising the top 20%, 20–40%, 40–60%, 60–80%, 80–100% of expressed genes. Repair of bleomycin-induced 3′-phosphoglycolate termini was enhanced at actively transcribed genes. The top 20% and 20–40% quintiles had a very similar level of enhanced repair, the 40–60% quintile was intermediate, while the 60–80% and 80–100% quintiles were close to the low level of enhancement found in non-transcribed genes. There were also interesting differences regarding bleomycin repair on the sense and antisense strands of DNA at TSSs. The sense strand had highly enhanced repair between 0 and 250bp relative to the TSS, while for the antisense strand highly enhanced repair was between 150 and 450bp. Repair of DNA damage is a major mechanism of resistance to anti-tumour drugs and this study provides an insight into this process in human tumour cells.
      Graphical abstract image

      PubDate: 2014-07-26T23:04:48Z
       
  • A common genetic variation in the promoter of miR-107 is associated with
           gastric adenocarcinoma susceptibility and survival
    • Abstract: Publication date: Available online 16 July 2014
      Source:Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis
      Author(s): Shizhi Wang , Chunye Lv , Hua Jin , Ming Xu , Meiyun Kang , Haiyan Chu , Na Tong , Dongmei Wu , Haixia Zhu , Weida Gong , Qinghong Zhao , Guoquan Tao , Jianwei Zhou , Zhengdong Zhang , Meilin Wang
      Background Global miRNA expression profile has been widely used to characterize human cancers. It is well established that genetic variants in miRNAs can modulate miRNA biogenesis and disease risk. Methods Genome-wide miRNA microarray was employed for assessment of miRNA expression profile of gastric adenocarcinoma (GAC). The variants of significantly dysregulated miRNA were genotyped in test (715 cases and 804 controls) and validation (940 cases and 1050 controls) subject sets. Results MiRNA microarray revealed that 12 miRNAs including miR-107 significantly dysregulated in GAC tissues. The sequencing of the promoter of miR-107 identified 3 SNPs (rs11185777, rs78591545, and rs2296616) with minor allele frequency (MAF)>5%. Analyzing their association with GAC risk and prognosis revealed that the C allele of rs2296616 (T>C) was significantly associated with the decreased risk of GAC among the test, validation and combined sets (TC/CC vs. TT, adjusted OR=0.39, 95% CI=0.31–0.49 for the combined set). However, the C allele was related to an unfavorable prognosis of Cardia GAC (CGAC) (adjusted HR=1.49, 95% CI=1.01–2.20). In vivo evidence showed that the individuals with the rs2296616C allele had lower miR-107 expression compared with the homozygous T allele carriers. Conclusion miR-107 is dysregulated in GAC pathogenesis and the SNP rs2296616 may play a role in the process.


      PubDate: 2014-07-26T23:04:48Z
       
  • Benzo[a]pyrene-induced cell cycle arrest in HepG2 cells is associated with
           delayed induction of mitotic instability
    • Abstract: Publication date: Available online 19 July 2014
      Source:Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis
      Author(s): Dimitris Stellas , Vassilis L. Souliotis , Margarita Bekyrou , Despina Smirlis , Micheline Kirsch-Volders , Francesca Degrassi , Enrico Cundari , Soterios A. Kyrtopoulos
      The environmental carcinogen benzo[a]pyrene (B[a]P) after being metabolised by cytochrome P450 enzymes forms DNA adducts. This abnormal situation induces changes in the cell cycle, DNA damage, chromosomal and mitotic aberrations, all of which may be related to carcinogenesis. In order to further investigate the mechanistic basis of these effects, HepG2 cells were treated with 3μM B[a]P for various time periods, followed by further incubation in the absence of B[a]P for up to 192h. B[a]P treatment led initially to S-phase arrest followed by recovery and subsequent induction of G2/M arrest, indicating activation of the corresponding DNA damage checkpoints. Immunofluorescence-based studies revealed accumulation of B[a]P-induced DNA adducts and chromosomal damage which persisted beyond mitosis and entry into a new cycle, thus giving rise to a new round of activation of the S-phase checkpoint. Prolonged further cultivation of the cells in the absence of B[a]P resulted in high frequencies of various abnormal mitotic events. Abrogation of the B[a]P-induced S-phase arrest by the Chk1 inhibitor UCN-01 triggered a strong apoptotic response but also dramatically decreased the frequency of mitotic abnormalities in the surviving cells, suggesting that events occurring during S-phase arrest contribute to the formation of delayed mitotic damage. Overall, our data demonstrate that, although S-phase arrest serves as a mechanism by which the cells reduce their load of genetic damage, its prolonged activation may also have a negative impact on the balance between cell death and heritable genetic damage.


      PubDate: 2014-07-26T23:04:48Z
       
  • Mutations in BALB mitochondrial DNA induce CCL20 up-regulation promoting
           tumorigenic phenotypes
    • Abstract: Publication date: Available online 19 July 2014
      Source:Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis
      Author(s): James Sligh , Jaroslav Janda , Jana Jandova
      mtDNA mutations are common in human cancers and are thought to contribute to the process of neoplasia. We examined the role of mtDNA mutations in skin cancer by generating fibroblast cybrids harboring a mutation in the gene encoding the mitochondrial tRNA for arginine. This somatic mutation (9821insA) was previously reported in UV-induced hyperkeratotic skin tumors in hairless mice and confers specific tumorigenic phenotypes to mutant cybrids. Microarray analysis revealed and RT-PCR along with Western blot analysis confirmed the up-regulation of CCL20 and its receptor CCR6 in mtBALB haplotype containing the mt-Tr 9821insA allele compared to wild type mtB6 haplotype. Based on reported role of CCL20 in cancer progression we examined whether the hyper-proliferation and enhanced motility of mtBALB haplotype would be associated with CCL20 levels. Treatment of both genotypes with recombinant CCL20 (rmCCL20) resulted in enhanced growth and motility of mtB6 cybrids. Furthermore, the acquired somatic alteration increased the in vivo tumor growth of mtBALB cybrids through the up-regulation of CCL20 since neutralizing antibody significantly decreased in vivo tumor growth of these cells; and tumors from anti-CCL20 treated mice injected with mtBALB cybrids showed significantly decreased CCL20 levels. When rmCCL20 or mtBALB cybrids were used as chemotactic stimuli, mtB6 cybrids showed increased motility while anti-CCL20 antibody decreased the migration and in vivo tumor growth of mtBALB cybrids. Moreover, the inhibitors of MAPK signaling and NF-κB activation inhibited CCL20 expression in mtBALB cybrids and decreased their migratory capabilities. Thus, acquired mtDNA mutations may promote tumorigenic phenotypes through up-regulation of chemokine CCL20.


      PubDate: 2014-07-26T23:04:48Z
       
  • IL-13 Overexpression in Mouse Lungs Triggers Systemic Genotoxicity in
           Peripheral Blood
    • Abstract: Publication date: Available online 24 July 2014
      Source:Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis
      Author(s): Aaron M. Chapman , Daniel J. Malkin , Jessica Camacho , Robert H. Schiestl
      Asthma is a common heterogeneous disease with both genetic and environmental factors that affects millions of individuals worldwide. Activated type 2 helper T cells secrete a panel of cytokines, including IL-13, a central immune regulator of many of the hallmark type 2 disease characteristics found in asthma. IL-13 has been directly implicated as a potent stimulator of asthma induced airway remodeling. Although IL-13 is known to play a major role in the development and persistence of asthma, the complex combination of environmental and genetic origin of the disease obfuscate the solitary role of IL-13 in the disease. We therefore, used a genetically modified mouse model which conditionally overexpresses IL-13 in the lungs to study the independent role of IL-13 in the progression of asthma. Our results demonstrate IL-13 is associated with a systemic induction of genotoxic parameters such as oxidative DNA damage, single and double DNA strand breaks, micronucleus formation, and protein nitration. Furthermore we show that inflammation induced genotoxicity found in asthma extends beyond the primary site of the lung to circulating leukocytes and erythroblasts in the bone marrow eliciting systemic effects driven by IL-13 over-expression.


      PubDate: 2014-07-26T23:04:48Z
       
  • Induction and repair of DNA damage measured by the comet assay in human T
           lymphocytes separated by immunomagnetic cell sorting
    • Abstract: Publication date: Available online 21 July 2014
      Source:Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis
      Author(s): Julia Bausinger , Günter Speit
      The comet assay is widely used in human biomonitoring to measure DNA damage in whole blood or isolated peripheral blood mononuclear cells (PBMC) as a marker of exposure to genotoxic agents. Cytogenetic assays with phytohemagglutinin (PHA)-stimulated cultured T lymphocytes are also frequently performed in human biomonitoring. Cytogenetic effects (micronuclei, chromosome aberrations, sister chromatid exchanges) may be induced in vivo but also occur ex vivo during the cultivation of lymphocytes as a consequence of DNA damage present in lymphocytes at the time of sampling. To better understand whether DNA damage measured by the comet assay in PBMC is representative for DNA damage in T cells, we comparatively investigated DNA damage and its repair in PBMC and T cells obtained by immunomagnetic cell sorting. PBMC cultures and T cell cultures were exposed to mutagens with different modes of genotoxic action and DNA damage was measured by the comet assay after the end of a 2h exposure and after 18h post-incubation. The mutagens tested were methyl methanesulfonate (MMS), (±)-anti-B[a]P-7,8-dihydrodiol-9,10-epoxide (BPDE), 4-nitroquinoline-1-oxide (4NQO), styrene oxide and potassium bromate. MMS and potassium bromate were also tested by the modified comet assay with formamido pyrimidine glycosylase (FPG) protein. The results indicate that the mutagens tested induce DNA damage in PBMC and T cells in the same range of concentrations and removal of induced DNA lesions occurs to a comparable extent. Based on these results, we conclude that the comet assay with PBMC is suited to predict DNA damage and its removal in T cells.


      PubDate: 2014-07-26T23:04:48Z
       
  • Optimization of the Divergent method for genotyping single nucleotide
           variations using SYBR Green-based single-tube real-time PCR
    • Abstract: Publication date: August–September 2014
      Source:Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, Volumes 766–767
      Author(s): Fabio Gentilini , Maria E. Turba
      A novel technique, called Divergent, for single-tube real-time PCR genotyping of point mutations without the use of fluorescently labeled probes has recently been reported. This novel PCR technique utilizes a set of four primers and a particular denaturation temperature for simultaneously amplifying two different amplicons which extend in opposite directions from the point mutation. The two amplicons can readily be detected using the melt curve analysis downstream to a closed-tube real-time PCR. In the present study, some critical aspects of the original method were specifically addressed to further implement the technique for genotyping the DNM1 c.G767T mutation responsible for exercise-induced collapse in Labrador retriever dogs. The improved Divergent assay was easily set up using a standard two-step real-time PCR protocol. The melting temperature difference between the mutated and the wild-type amplicons was approximately 5°C which could be promptly detected by all the thermal cyclers. The upgraded assay yielded accurate results with 157pg of genomic DNA per reaction. This optimized technique represents a flexible and inexpensive alternative to the minor grove binder fluorescently labeled method and to high resolution melt analysis for high-throughput, robust and cheap genotyping of single nucleotide variations.


      PubDate: 2014-07-26T23:04:48Z
       
  • Functional characterization of genetic polymorphisms in the H2AFX distal
           promoter
    • Abstract: Publication date: August–September 2014
      Source:Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, Volumes 766–767
      Author(s): Karla L. Bretherick , Stephen Leach , Angela R. Brooks-Wilson
      Due to the critical role of the H2AX histone variant in double-strand break repair, genetic variants in the H2AX gene, H2AFX, may influence cancer susceptibility. Genetic association studies have correlated H2AFX upstream variants with cancer risk; however it is unclear if any are causal. H2AFX has at least two alternate transcripts that encode the same reading frame; a short 0.6kb transcript that lacks an intron or poly-A tail and is predicted to be highly expressed during the replication stage of the cell cycle, and a long 1.6kb poly-A tailed transcript that is expressed in a replication-independent manner. To examine the functional impact of the rs643788, rs8551, rs7759, and rs2509049 upstream variants, we characterized their influence on gene expression, cell survival after DNA assault, and transcription factor binding. Analysis of allelic imbalance using quantitative sequencing of cDNA from lymphoblast cell lines did not reveal any difference in expression of the 1.6kb polyadenylated transcript between the common H2AFX upstream haplotypes. We did, however, identify a previously unreported 197 base pair intron in the H2AFX 3′untranslated region that appears to be present regardless of haplotype. Assessment of cell survival after irradiation treatment did not show any difference in survival between cell lines of different haplotypes. Gel shift assays revealed that the rs643788 C allele disrupts YY1 transcription factor binding and the rs2509049 C allele binds more strongly to a protein complex than does the rs2509049 T allele. Though we did not identify any differences in expression or survival between haplotypes, differential protein binding at two of the polymorphisms suggests further functional analyses may reveal a role for these variants in influencing gene expression at specific points of the cell cycle or in specific tissues.


      PubDate: 2014-07-26T23:04:48Z
       
  • Deletion of BRCA2 exon 27 causes defects in response to both stalled and
           collapsed replication forks
    • Abstract: Publication date: August–September 2014
      Source:Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, Volumes 766–767
      Author(s): Tae Moon Kim , Mi Young Son , Sherry Dodds , Lingchuan Hu , Paul Hasty
      BRCA2 is a tumor suppressor that maintains genomic integrity through double strand break (DSB) repair and replication fork protection. The BRC motifs and an exon 27-encoded domain (Ex27) of BRCA2 interact with the recombinase RAD51 to, respectively, facilitate the formation and stability of a RAD51 filament on single strand DNA. The BRC-RAD51 associations enable DSB repair while the Ex27-RAD51 association protects the nascent replication strand from MRE11-mediated degradation. MRE11 is a nuclease that facilitates the generation of 3′ overhangs needed for homologous recombination (HR)-mediated DSB repair. Here we report the dynamics of replication fork maintenance in mouse embryonic stem (ES) cells deleted for Ex27 (brca2 lex1/lex2 ) after exposure to hydroxyurea (HU) that depletes nucleotides. HU conditions were varied from mild to severe. Mild conditions induce an ATR-response to replication fork stalling while severe conditions induce a DNA-PKCS-response to replication fork collapse and a DSB. These responses were differentiated by replication protein A (RPA) phosphorylation. We found that Ex27 deletion reduced MRE11 localization to stalled, but not collapsed, replication forks and that Ex27-deletion caused a proportionately more severe phenotype with HU dose. Therefore, the BRCA2 exon 27 domain maintains chromosomal integrity at both stalled and collapsed replication forks consistent with involvement in both replication fork maintenance and double strand break repair.


      PubDate: 2014-07-26T23:04:48Z
       
  • Direct observation of preferential processing of clustered abasic DNA
           damages with APE1 in TATA box and CpG island by reaction kinetics and
           fluorescence dynamics
    • Abstract: Publication date: August–September 2014
      Source:Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, Volumes 766–767
      Author(s): Vandana Singh , Bhavini Kumari , Banibrata Maity , Debabrata Seth , Prolay Das
      Sequences like the core element of TATA box and CpG island are frequently encountered in the genome and related to transcription. The fate of repair of clustered abasic sites in such sequences of genomic importance is largely unknown. This prompted us to investigate the sequence dependence of cleavage efficiency of APE1 enzyme at abasic sites within the core sequences of TATA box and CpG island using fluorescence dynamics and reaction kinetics. Simultaneous molecular dynamics study through steady state and time resolved fluorescence spectroscopy using unique ethidium bromide dye release assay confirmed an elevated amount of abasic site cleavage of the TATA box sequence as compared to the core CpG island. Reaction kinetics showed that catalytic efficiency of APE1 for abasic site cleavage of core CpG island sequence was ∼4 times lower as compared to that of the TATA box. Higher value of K m was obtained from the core CpG island sequence than the TATA box sequence. This suggests a greater binding effect of APE1 enzyme on TATA sequence that signifies a prominent role of the sequence context of the DNA substrate. Evidently, a faster response from APE1 was obtained for clustered abasic damage repair of TATA box core sequences than CpG island consensus sequences. The neighboring bases of the abasic sites in the complementary DNA strand were found to have significant contribution in addition to the flanking bases in modulating APE1 activity. The repair refractivity of the bistranded clustered abasic sites arise from the slow processing of the second abasic site, consequently resulting in decreased overall production of potentially lethal double strand breaks.


      PubDate: 2014-07-26T23:04:48Z
       
  • Curcumin and trans-resveratrol exert cell cycle-dependent radioprotective
           or radiosensitizing effects as elucidated by the PCC and G2-assay
    • Abstract: Publication date: August–September 2014
      Source:Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, Volumes 766–767
      Author(s): N. Sebastià , A. Montoro , D. Hervás , G. Pantelias , V.I. Hatzi , J.M. Soriano , J.I. Villaescusa , G.I. Terzoudi
      Curcumin and trans-resveratrol are well-known antioxidant polyphenols with radiomodulatory properties, radioprotecting non-cancerous cells while radiosensitizing tumor cells. This dual action may be the result of their radical scavenging properties and their effects on cell-cycle checkpoints that are activated in response to radiation-induced chromosomal damage. It could be also caused by their effect on regulatory pathways with impact on detoxification enzymes, the up-regulation of endogenous protective systems, and cell-cycle-dependent processes of DNA damage. This work aims to elucidate the mechanisms underlying the dual action of these polyphenols and investigates under which conditions they exhibit radioprotecting or radiosensitizing properties. The peripheral blood lymphocyte test system was used, applying concentrations ranging from 1.4 to 140μM curcumin and 2.2 to 220μM trans-resveratrol. The experimental design focuses first on their radioprotective effects in non-cycling lymphocytes, as uniquely visualized using cell fusion-mediated premature chromosome condensation, excluding, thus, cell-cycle interference to repair processes and activation of checkpoints. Second, the radiosensitizing potential of these chemicals on the induction of chromatid breaks in cultured lymphocytes following G2-phase irradiation was evaluated by a standardized G2-chromosomal radiosensitivity predictive assay. This assay uses caffeine for G2-checkpoint abrogation and it was applied to obtain an internal control for radiosensitivity testing, which simulates conditions similar to those of the highly radiosensitive lymphocytes of AT patients. The results demonstrate for the first time the cell-cycle-dependent action of these polyphenols. When non-cycling cells are irradiated, the radioprotective properties of curcumin and trans-resveratrol are more prominent. However, when cycling cells are irradiated during G2-phase, the radiosensitizing features of these compounds are more pronounced. This observation offers a new biological basis for the mechanisms underlying the action of these polyphenols in cancer radiotherapy.


      PubDate: 2014-07-26T23:04:48Z
       
  • TITLE PAGE (EDI BOARD)
    • Abstract: Publication date: August–September 2014
      Source:Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, Volumes 766–767




      PubDate: 2014-07-26T23:04:48Z
       
  • Ethyl methanesulfonate induces mutations in Caenorhabditis elegans embryos
           at a high frequency
    • Abstract: Publication date: August–September 2014
      Source:Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, Volumes 766–767
      Author(s): Phil S. Hartman , James Barry , Whitney Finstad , Numan Khan , Masayuki Tanaka , Kayo Yasuda , Naoaki Ishii
      Mutagenesis protocols typically call for exposure of late-stage larvae or adults to a mutagen with the intention of inducing mutations in a robust germ line. Instead, ca. 16,000 CB665 [unc-58(e665)] one- to four-cell embryos of the nematode Caenorhabditis elegans were hand selected and exposed to ethyl methanesulfonate (EMS) for 50min. Twenty-one reversion mutants were recovered, of which 17 were intragenic suppressors of the e665 mutation. The mutation frequency was 6.5-fold higher than when CB665 adults were similarly mutagenized, which was predicted given that cell-cycle checkpoints are muted in C. elegans embryos. The mutation spectrum was similar to that obtained after standard EMS mutagenesis.


      PubDate: 2014-07-26T23:04:48Z
       
  • BsmI polymorphism of vitamin D receptor gene and cancer risk: A
           comprehensive meta-analysis
    • Abstract: Publication date: November 2014
      Source:Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, Volume 769
      Author(s): Sara Raimondi , Elena Pasquali , Patrizia Gnagnarella , Davide Serrano , Davide Disalvatore , Harriet A. Johansson , Sara Gandini
      The VDR gene is an important regulator of the vitamin D pathway, and the role of some of its polymorphisms on cancer risk was previously investigated. A trend of cancer risk reduction with the VDR BsmI B allele was observed for many cancer sites. We performed a comprehensive meta-analysis to investigate the role of VDR BsmI polymorphism on cancer risk, even according to different ethnicities. Summary odds ratios (SORs) were calculated with random-effects models and maximum likelihood estimation. We categorized studies into three groups (“moderate”, “high” and “very high confidence”) according to departure from Hardy–Weinberg equilibrium in controls, reported minor allele frequency and genotyping quality controls. The meta-analysis included 73 studies with 45,218 cases and 52,057 controls. We found a significant 6–7% reduction of cancer risk at any site respectively for carriers of Bb genotype (SOR; 95%CI: 0.94; 0.90–0.99) and for carriers of BsmI BB genotype (SOR; 95%CI: 0.93; 0.89–0.98) compared to bb carriers, and they remain statistically significant when we restricted the analysis to at least “high confidence” studies. For skin cancer, a significant risk reduction was observed for Bb carriers (SOR; 95%CI: 0.86; 0.76–0.98). We also found a significant reduction of colorectal cancer risk for BB and Bb + BB genotypes carriers, but these SORs were no more significant when we restricted the analysis to studies with “high confidence”. When the analysis was stratified by ethnicity, we still observed a significant decreased risk for both Bb and BB compared to bb genotype among Caucasians: SORs (95%CI) for any cancer site were 0.97 (0.93–1.00) and 0.95 (0.91–0.99), respectively. Among other ethnic groups the inverse association was still present, but did not reach statistical significance. In conclusion, we suggest a weak effect of BsmI B allele in reducing cancer risk at any site, especially of the skin.


      PubDate: 2014-07-26T23:04:48Z
       
  • Deficiency of the DNA repair protein nibrin increases the basal but not
           the radiation induced mutation frequency in vivo
    • Abstract: Publication date: November 2014
      Source:Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, Volume 769
      Author(s): Petra Wessendorf , Jan Vijg , André Nussenzweig , Martin Digweed
      Nibrin (NBN) is a member of a DNA repair complex together with MRE11 and RAD50. The complex is associated particularly with the repair of DNA double strand breaks and with the regulation of cell cycle check points. Hypomorphic mutation of components of the complex leads to human disorders characterised by radiosensitivity and increased tumour occurrence, particularly of the lymphatic system. We have examined here the relationship between DNA damage, mutation frequency and mutation spectrum in vitro and in vivo in mouse models carrying NBN mutations and a lacZ reporter plasmid. We find that NBN mutation leads to increased spontaneous DNA damage in fibroblasts in vitro and high basal mutation rates in lymphatic tissue of mice in vivo. The characteristic mutation spectrum is dominated by single base transitions rather than the deletions and complex rearrangements expected after abortive repair of DNA double strand breaks. We conclude that in the absence of wild type nibrin, the repair of spontaneous errors, presumably arising during DNA replication, makes a major contribution to the basal mutation rate. This applies also to cells heterozygous for an NBN null mutation. Mutation frequencies after irradiation in vivo were not increased in mice with nibrin mutations as might have been expected considering the radiosensitivity of NBS patient cells in vitro. Evidently apoptosis is efficient, even in the absence of wild type nibrin.


      PubDate: 2014-07-26T23:04:48Z
       
  • Inherited variability in a master regulator polymorphism (rs4846126)
           associates with survival in 5-FU treated colorectal cancer patients
    • Abstract: Publication date: Available online 7 June 2014
      Source:Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis
      Author(s): Barbara Pardini , Justo Lorenzo Bermejo , Alessio Naccarati , Cornelia Di Gaetano , Fabio Rosa , Carine Legrand , Jan Novotny , Pavel Vodicka , Rajiv Kumar
      Background Treatment with 5-fluorouracil (5-FU) is known to improve survival in many cancers including colorectal cancer. Response to the treatment, overall survival and recurrence show inter-individual variation. Methods In this study we employed a strategy to search eQTL variants influencing the expression of a large number of genes. We identified four single nucleotide polymorphisms, defined as master regulators of transcription, and genotyped them in a set of 218 colorectal cancer patients undergoing adjuvant 5-FU based therapy. Results Our results showed that the minor allele variant of the rs4846126 polymorphism was associated with poor overall and progression-free survival. Patients that were homozygous for the variant allele showed an over two fold increased risk of death (HR 2.20 95%CI 1.05-4.60) and progression (HR 2.88, 95% 1.47-5.63). The integration of external information from publicly available gene expression repositories suggested that the rs4846126 polymorphism deserves further investigation. This variant potentially regulates the gene expression of 273 genes with some of them possibly associated to the patient's response to 5-FU treatment or colorectal cancer. Conclusions Present results show that mining of public data repositories in combination with own data can be a fruitful approach to identify markers that affect therapy outcome. In particular, a genetic screen of master regulators may help in order to search for the polymorphisms involved in treatment response in cancer patients.


      PubDate: 2014-06-08T16:18:34Z
       
  • Impact of abasic site orientation within nucleosomes on human APE1
           endonuclease activity
    • Abstract: Publication date: Available online 6 June 2014
      Source:Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis
      Author(s): John M. Hinz
      Glycosylases responsible for recognizing DNA lesions and initiating Base Excision Repair (BER) are impeded by the presence of histones, which are essential for compaction of the genetic material in the nucleus. Abasic sites are an abundant mutagenic lesion in the DNA, arising spontaneously and as the product of glycosylase activity, making it a common intermediate in BER. The apurinic/apyrimidinic endonuclease 1 (APE1) recognizes abasic sites and cleaves the DNA backbone adjacent to the lesion, creating the single-strand break essential for the subsequent steps of BER. In this study the endonuclease activity of human APE1 was measured on reconstituted nucleosome core particles (NCPs) with DNA containing enzymatically-created abasic sites (AP) or the abasic site analog tetrahydrofuran (TF) at different rotational positions relative to the histone core surface. The presence of histones on the DNA reduced APE1 activity overall, and the magnitude was greatly influenced by differences in orientation of the lesions along the DNA gyre relative to the histone core. Abasic moieties oriented with their phosphate backbones adjacent to the underlying histones (In) were cleaved less efficiently than those oriented away from the histone core (Out) or between the In and Out orientations (Mid). The impact on APE1 at each orientation was very similar between the AP and TF lesions, highlighting the dependability of the TF abasic analog in APE1 activity measurements in nucleosomes. Measurement of APE1 binding to the NCP substrates reveals a substantial reduction in its interaction with nucleosomes compared to naked DNA, also in a lesion orientation-dependent manner, reinforcing the concept that reduction in APE1 activity on nucleosomes is due to occlusion from its abasic DNA substrate by the histones. These results suggest that APE1 activity in nucleosomes, like BER glycosylases, is primarily regulated by its chance interactions with transiently exposed lesions


      PubDate: 2014-06-08T16:18:34Z
       
  • Acute and chronic administration of gold nanoparticles cause dna damage in
           the cerebral cortex of adult rats
    • Abstract: Publication date: Available online 6 June 2014
      Source:Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis
      Author(s): Eria Cardoso , Gislaine Tezza Rezin , Elton Torres Zanoni , Frederico de Souza Notoya , Daniela Dimer Leffa , Adriani Paganini Damiani , Francine Daumann , Juan Carlos Ortiz Rodriguez , Roberto Benavides , Luciano da Silva , Vanessa M. Andrade , Marcos Marques da Silva Paula
      The use of gold nanoparticles is increasing in medicine; however, their toxic effects remain to be elucidated. Studies show that gold nanoparticles can cross the blood–brain barrier, as well as accumulate in the brain. Therefore, this study was undertaken to better understand the effects of gold nanoparticles on rat brains. DNA damage parameters were evaluated in the cerebral cortex of adult rats submitted to acute and chronic administration of gold nanoparticles of two different diameters: 10 and 30nm. During acute administration, adult rats received a single intraperitoneal injection of either gold nanoparticles or saline solution. During chronic administration, adult rats received a daily single injection for 28 days of the same gold nanoparticles or saline solution. Twenty-four hours after either single (acute) or last injection (chronic), the rats were euthanized by decapitation, their brains removed, and the cerebral cortices isolated for evaluation of DNA damage parameters. Our study showed that acute administration of gold nanoparticles in adult rats presented higher levels of damage frequency and damage index in their DNA compared to the control group. It was also observed that gold nanoparticles of 30nm presented higher levels of damage frequency and damage index in the DNA compared to the 10nm ones. When comparing the effects of chronic administration of gold nanoparticles of 10 and 30nm, we observed that occured significant different index and frequency damage, comparing with control group. However, there is no difference between the 10 and 30nm groups in the levels of DNA damage for both parameters of the Comet assay. Results suggest that gold nanoparticles for both sizes cause DNA damage for chronic as well as acute treatments, although a higher damage was observed for the chronic one


      PubDate: 2014-06-08T16:18:34Z
       
  • Expression and Methylation of DNA Repair Genes in Lens Epithelium Cells of
           Age-related Cataract
    • Abstract: Publication date: Available online 6 June 2014
      Source:Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis
      Author(s): Fei Li , Yong Wang , Guowei Zhang , Jing Zhou , Ling Yang , Huaijin Guan
      The development of age-related cataract (ARC) is associated with DNA damage of the lens epithelial cells (LECs). This study aimed to investigate the expression level of DNA repair genes in LECs of ARC and examine whether any altered expression observed could result from DNA methylation of the promoter region of the genes. The expression levels of DNA repair genes were evaluated by microarray analysis. The results were further confirmed by qRT-PCR. DNA methylation of genes with altered expression was determined by Bisulfite-specific (BSP) PCR. The mRNA levels of 10 DNA repair genes were decreased and the level of 1 DNA repair gene was increased in LECs of ARC patients compared with controls. The promoter region of the MGMT gene was hypermethylated in ARC tissue compared to controls. The data provides evidence that altered expression of DNA repair genes is associated with pathogenesis of ARC. DNA methylation of MGMT may regulate the expression of the gene and be involved in the development of ARC.


      PubDate: 2014-06-08T16:18:34Z
       
  • TITLE PAGE (EDI BOARD)
    • Abstract: Publication date: July 2014
      Source:Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, Volume 765




      PubDate: 2014-06-02T15:10:06Z
       
  • A functional perspective of Nitazoxanide as a potential anticancer drug
    • Abstract: Publication date: Available online 1 June 2014
      Source:Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis
      Author(s): Nicola Di Santo , Jessie Ehrisman
      Cancer is a group of diseases characterized by uncontrolled cell proliferation, evasion of cell death and the ability to invade and disrupt vital tissue function. The classic model of carcinogenesis describes successive clonal expansion driven by the accumulation of mutations that eliminate restraints on proliferation and cell survival. It has been proposed that during cancer's development, the loose-knit colonies of only partially differentiated cells display some unicellular/prokaryotic behavior reminiscent of robust ancient life forms. The seeming “regression” of cancer cells involves changes within metabolic machinery and survival strategies. This atavist change in physiology enables cancer cells to behave as selfish “neo-endo-parasites” that exploit the tumor stromal cells in order to extract nutrients from the surrounding microenvironment. In this framework, it is conceivable that anti-parasitic compounds might serve as promising anticancer drugs. Nitazoxanide (NTZ), a thiazolide compound, has shown antimicrobial properties against anaerobic bacteria, as well as against helminths and protozoa. NTZ has also been successfully used to promote Hepatitis C virus (HCV) elimination by improving interferon signaling and promoting autophagy. More compelling however are the potential anti-cancer properties that have been observed. NTZ seems to be able to interfere with crucial metabolic and pro-death signaling such as drug detoxification, unfolded protein response (UPR), autophagy, anti-cytokine activities and c-Myc inhibition. In this article, we review the ability of NTZ to interfere with integrated survival mechanisms of cancer cells and propose that this compound might be a potent addition to the current chemotherapeutic strategy against cancer.


      PubDate: 2014-06-02T15:10:06Z
       
  • Mechanistic perspectives on cancer chemoprevention/chemotherapeutic
           effects of thymoquinone
    • Abstract: Publication date: Available online 27 May 2014
      Source:Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis
      Author(s): Juthika Kundu , Kyung-Soo Chun , Okezie I. Aruoma , Joydeb Kumar Kundu
      The bioactive natural products (secondary metabolites) are widely known to possess therapeutic value for the prevention and treatment of various chronic diseases including cancer. Thymoquinone (2-methyl-5-isopropyl-1,4-benzoquinone; TQ), a monoterpene present in black cumin seeds, exhibits pleiotropic pharmacological activities including antioxidant, anti-inflammatory, antidiabetic and antitumor effects. TQ inhibits experimental carcinogenesis in a wide range of animal models and has been shown to arrest the growth of various cancer cells in culture as well as xenograft tumors in vivo. The mechanistic basis of anticancer effects of TQ includes the inhibition of carcinogen metabolizing enzyme activity and oxidative damage of cellular macromolecules, attenuation of inflammation, induction of cell cycle arrest and apoptosis in tumor cells, blockade of tumor angiogenesis, and suppression of migration, invasion and metastasis of cancer cells. TQ shows synergistic and/or potentiating anticancer effects when combined with clinically used chemotherapeutic agents. At the molecular level, TQ targets various components of intracellular signaling pathways, particularly a variety of upstream kinases and transcription factors, which are aberrantly activated during the course of tumorigenesis.


      PubDate: 2014-06-02T15:10:06Z
       
  • Noninvasive measurement of aristolochic acid-DNA adducts in urine samples
           from aristolochic acid-treated rats by liquid chromatography coupled
           tandem mass spectrometry: Evidence for DNA repair by nucleotide-excision
           repair mechanisms
    • Abstract: Publication date: Available online 29 May 2014
      Source:Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis
      Author(s): Elvis M.K. Leung , Wan Chan
      Nephrotoxic aristolochic acids (AAs) form covalently bonded DNA adducts upon metabolic activation. In this work, a non-invasive approach to detect AAs exposure by quantifying urinary excreted DNA-AA adducts is presented. The developed method entails solid-phase extraction (SPE) enrichment of the urine-excreted DNA-AAs adducts, addition of internal standard, and quantification by liquid chromatography coupled tandem mass spectrometric (LC–MS/MS) analysis. Quantitative analysis revealed 7-(deoxyadenosine-N 6-yl)-aristolactam II and 7-(deoxyguanosine-N 2-yl)-aristolactam I that were previously detected as major DNA-AA adducts in different organs of AA-dosed rats, were detected as the major urine excreted adducts. Lower levels of 7-(deoxyadenosine-N 6-yl)-aristolactam I and 7-(deoxyguanosine-N 2-yl)-aristolactam II were also detected in the collected urine samples. The identities of the detected urinary DNA-AA adducts were confirmed by comparing chromatographic retention time with synthetic standards, by high-accuracy MS, and MS/MS analyses. LC–MS/MS analysis of the urine samples collected from the AAs-dosed rats demonstrated a time-dependent decrease in the urinary adduct levels, indicating the urinary DNA-AA adduct levels were reflective of the tissue adduct levels. It is expected that the developed approach of detecting urinary DNA-AA adducts will facilitate further carcinogenesis investigations of AAs.


      PubDate: 2014-06-02T15:10:06Z
       
  • Silibinin inhibits fibronectin induced motility, invasiveness and survival
           in human prostate carcinoma PC3 cells via targeting integrin signaling
    • Abstract: Publication date: Available online 23 May 2014
      Source:Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis
      Author(s): Gagan Deep , Rahul Kumar , Anil K. Jain , Chapla Agarwal , Rajesh Agarwal
      Prostate cancer (PCA) is the 2nd leading cause of cancer-related deaths among men in the United States. Preventing or inhibiting metastasis-related events through non-toxic agents could be a useful approach for lowering high mortality among PCA patients. We have earlier reported that natural flavonoid silibinin possesses strong anti-metastatic efficacy against PCA however, mechanism/s of its action still remains largely unknown. One of the major events during metastasis is the replacement of cell-cell interaction with integrins-based cell-matrix interaction that controls motility, invasiveness and survival of cancer cells. Accordingly, here we examined silibinin effect on advanced human PCA PC3 cells’ interaction with extracellular matrix component fibronectin. Silibinin (50-200μM) treatment significantly decreased the fibronectin (5μg/ml)-induced motile morphology via targeting actin cytoskeleton organization in PC3 cells. Silibinin also decreased the fibronectin-induced cell proliferation and motility but significantly increased cell death in PC3 cells. Silibinin also inhibited the PC3 cells invasiveness in Transwell invasion assays with fibronectin or cancer associated fibroblasts (CAFs) serving as chemoattractant. Importantly, PC3-luc cells cultured on fibronectin showed rapid dissemination and localized in lungs following tail vein injection in athymic male nude mice; however, in silibinin-treated PC3-luc cells, dissemination and lung localization was largely compromised. Molecular analyses revealed that silibinin treatment modulated the fibronectin-induced expression of integrins (α5, αV, β1 and β3), actin-remodeling (FAK, Src, GTPases, ARP2 and cortactin), apoptosis (cPARP and cleaved caspase 3), EMT (E-cadherin and β-catenin), and cell survival (survivin and Akt) related signaling molecules in PC3 cells. Furthermore, PC3-xenograft tissue analyses confirmed the inhibitory effect of silibinin on fibronectin and integrins expression. Together, these results showed that silibinin targets PCA cells’ interaction with fibronectin and inhibits their motility, invasiveness and survival; thus further supporting silibinin use in PCA intervention including its metastatic progression.


      PubDate: 2014-05-26T00:41:08Z
       
  • TITLE PAGE (EDI BOARD)
    • Abstract: Publication date: May–June 2014
      Source:Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, Volumes 763–764




      PubDate: 2014-05-15T17:48:12Z
       
  • DNA damage and S phase arrest induced by Ochratoxin A in human embryonic
           kidney cells (HEK 293)
    • Abstract: Publication date: Available online 9 May 2014
      Source:Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis
      Author(s): Qian Yang , Xiaoyun He , Xiaohong Li , Wentao Xu , Yunbo Luo , Xuan Yang , Yan Wang , Yingcong Li , Kunlun Huang
      Ochratoxin A (OTA) is a ubiquitous mycotoxin with potential nephrotoxic, hepatotoxic and immunotoxic effects. The mechanisms underlying the nephrotoxicity of OTA remain obscure. To investigate DNA damage and the changes of the cell cycle distribution induced by OTA, human embryonic kidney cells (HEK 293 cells) were incubated with various concentrations of OTA for 24h in vitro. The results indicated that OTA treatment led to the production of reactive oxygen species (ROS) and to a decrease of the mitochondrial membrane potential (ΔΨm). OTA-induced DNA damage in HEK 293 cells was evidenced by DNA comet tail formation and increased expression of γ-H2AX. In addition, OTA could induce cell cycle arrest at the S phase in HEK 293 cells. The expression of key cell cycle regulatory factors that were critical to the S phase, including cyclin A2, cyclin E1 and CDK2, were further detected. The expression of cyclin A2, cyclin E1 and CDK2 were significantly decreased by OTA treatment at both the mRNA and protein levels. The apoptosis of HEK 293 cells after OTA treatment was observed using Hoechst 33342 staining. The results confirmed that OTA did induce apoptosis in HEK 293 cells. In conclusion, our results provided new insights into the molecular mechanisms by which OTA might promote nephrotoxicity.


      PubDate: 2014-05-10T12:42:58Z
       
  • Dose- and time-dependent changes of micronucleus frequency and gene
           expression in the progeny of irradiated cells: Two components in
           radiation-induced genomic instability'
    • Abstract: Publication date: Available online 3 May 2014
      Source:Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis
      Author(s): Katriina Huumonen , Merja Korkalainen , Eeva Boman , Janne Heikkilä , Anne Höytö , Tapani Lahtinen , Jukka Luukkonen , Matti Viluksela , Jonne Naarala , Jukka Juutilainen
      Murine embryonic C3H/10T½ fibroblasts were exposed to X-rays at doses of 0.2, 0.5, 1, 2 or 5Gy. To follow the development of radiation-induced genomic instability (RIGI), the frequency of micronuclei was measured with flow cytometry at 2 days after exposure and in the progeny of the irradiated cells at 8 and 15 days after exposure. Gene expression was measured at the same points in time by PCR arrays profiling the expression of 84 cancer-relevant genes. The micronucleus results showed a gradual decrease in the slope of the dose–response curve between days 2 and 15. The data were consistent with a model assuming two components in RIGI. The first component is characterized by dose-dependent increase in micronuclei. It may persist more than ten cell generations depending on dose, but eventually disappears. The second component is more persistent and independent of dose above a threshold higher than 0.2Gy. Gene expression analysis 2 days after irradiation at 5Gy showed consistent changes in genes that typically respond to DNA damage. However, the consistency of changes decreased with time, suggesting that non-specificity and increased heterogeneity of gene expression are characteristic to the second, more persistent component of RIGI.


      PubDate: 2014-05-06T04:19:40Z
       
  • Nucleotide excision repair related gene polymorphisms and genetic
           susceptibility, chemotherapeutic sensitivity and prognosis of gastric
           cancer
    • Abstract: Publication date: Available online 24 April 2014
      Source:Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis
      Author(s): Jingwei Liu , Caiyun He , Chengzhong Xing , Yuan Yuan
      Human genomic DNA is in a dynamic balance of damage and repair. Cells employ multiple and specific repair pathways, such as nucleotide excision repair (NER), as unrepaired DNA damage has deleterious consequences and could give rise to carcinogenesis. Gene polymorphisms play a crucial role in predicting the risk and prognosis of cancer. Polymorphisms of NER-related genes could alter the ability of NER to effectively monitor and repair DNA damage, and thus may be associated with genetic susceptibility, chemotherapeutic sensitivity and prognosis of cancer. In recent years, increasing studies have focused on the association between polymorphisms of NER genes and gastric cancer, the world's fourth most common cancer and the second most common cause for cancer-related death. Here we reviewed the recent studies on the associations between polymorphisms of NER genes and gastric cancer from perspectives of genetic susceptibility, chemotherapeutic sensitivity and prognosis.


      PubDate: 2014-04-26T04:20:20Z
       
  • Alpha particle induced DNA damage and repair in normal cultured thyrocytes
           of different proliferation status
    • Abstract: Publication date: Available online 21 April 2014
      Source:Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis
      Author(s): Madeleine Nordén Lyckesvärd , Ulla Delle , Helena Kahu , Sture Lindegren , Holger Jensen , Tom Bäck , John Swanpalmer , Kecke Elmroth
      Childhood exposure to ionizing radiation increases the risk of developing thyroid cancer later in life and this is suggested to be due to higher proliferation of the young thyroid. The interest of using high-LET alpha particles from Astatine-211 (211At), concentrated in the thyroid by the same mechanism as 131I [1], in cancer treatment has increased during recent years because of its high efficiency in inducing biological damage and beneficial dose distribution when compared to low-LET radiation. Most knowledge of the DNA damage response in thyroid is from studies using low-LET irradiation and much less is known of high-LET irradiation. In this paper we investigated the DNA damage response and biological consequences to photons from Cobolt-60 (60Co) and alpha particles from 211At in normal primary thyrocytes of different cell cycle status. For both radiation qualities the intensity levels of γH2AX decreased during the first 24h in both cycling and stationary cultures and complete repair was seen in all cultures but cycling cells exposed to 211At. Compared to stationary cells alpha particles were more harmful for cycling cultures, an effect also seen at the pChk2 levels. Increasing ratios of micronuclei per cell nuclei were seen up to 1Gy 211At. We found that primary thyrocytes were much more sensitive to alpha particle exposure compared with low-LET photons. Calculations of the relative biological effectiveness yielded higher RBE for cycling cells compared with stationary cultures at a modest level of damage, clearly demonstrating that cell cycle status influences the relative effectiveness of alpha particles.


      PubDate: 2014-04-26T04:20:20Z
       
  • Individual telomere length decay in patients with spondyloarthritis
    • Abstract: Publication date: Available online 24 April 2014
      Source:Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis
      Author(s): María Tamayo , Sonia Pértega , Alejandro Mosquera , Montserrat Rodríguez , Francisco J. Blanco , José Luis Fernández-Sueiro , Jaime Gosálvez , José Luis Fernández
      Telomere length was sequentially determined in peripheral blood leukocytes (PBL) from patients with ankylosing spondylitis (AS; n=44) and psoriatic arthritis (PsA; n=42) followed through 2.93±0.99 years, using a quantitative PCR (qPCR) assay. The initial telomere size from PsA patients was higher than those with cutaneous psoriasis only (n=53), possibly due to the inflammatory condition. The qPCR assay was sensitive enough to evidence a significant telomere length shortening in PBL from practically all subjects and PsA patients showed a higher rate of loss of telomere sequence than patients with AS during the follow-up time.


      PubDate: 2014-04-26T04:20:20Z
       
  • The association between personal sun exposure, serum vitamin D and global
           methylation in human lymphocytes in a population of healthy adults in
           South Australia
    • Abstract: Publication date: Available online 12 April 2014
      Source:Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis
      Author(s): Visalini Nair-Shalliker , Varinderpal Dhillon , Mark Clements , Bruce K. Armstrong , Michael Fenech
      Background There is a positive association between solar UV exposure and micronucleus frequency in peripheral blood lymphocytes (PBL) and this association may be stronger when serum vitamin D (25(OH)D) levels are insufficient (<50nmol/L). Micronucleus formation can result from global hypomethylation of DNA repeat sequences. The aim of this analysis was to evaluate the relationship between solar UV exposure and methylation pattern in LINE-1 repetitive elements in PBL DNA and to see if serum 25(OH)D levels modify it. Method Personal solar UV exposure was estimated from hours of outdoor exposure over 6 weeks recalled at the time of blood collection in 208 male and female participants living in South Australia. Methylation in LINE-1 repetitive elements was assessed in PBL using pyrosequencing. Results Methylation in LINE-1 decreased with increasing solar UV exposure (% decrease=0.5% per doubling of sUV; 95%CI:-0.7-0.2 p value =0.00003). Although there was no correlation between LINE-1 methylation and micronucleus frequency, there was a 4.3% increase (95% CI:0.6-8.1 p-value=0.02) in nucleoplasmic bridges and a 4.3% increase in necrosis (CI:1.9-6.8 p-value=0.0005) for every 1% increase in LINE-1 methylation. Serum 25(OH)D was not associated with DNA methylation; nor did it modify the association of solar UV with DNA methylation. Conclusion Exposure to solar UV radiation may reduce DNA methylation in circulating lymphocytes. This association does not appear to be influenced or mediated by vitamin D status.


      PubDate: 2014-04-15T23:03:45Z
       
  • Reciprocal bystander effect between α-irradiated macrophage and
           hepatocyte is mediated by cAMP through a membrane signaling pathway
    • Abstract: Publication date: Available online 20 March 2014
      Source:Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis
      Author(s): Mingyuan He , Chen Dong , Yuexia Xie , Jitao Li , Dexiao Yuan , Yang Bai , Chunlin Shao
      Irradiated cells can induce biological effects on vicinal non-irradiated bystander cells, meanwhile the bystander cells may rescue the irradiated cells through a feedback signal stress. To elucidate the nature of this reciprocal effect, we examined the interaction between α-irradiated human macrophage cells U937 and its bystander HL-7702 hepatocyte cells using a cell co-culture system. Results showed that after 6h of cell co-culture, mitochondria depolarization corresponding to apoptosis was significantly induced in the HL-7702 cells, but the formation of micronuclei in the irradiated U937 cells was markedly decreased compared to that without cell co-culture treatment. This reciprocal effect was not observed when the cell membrane signaling pathway was blocked by filipin that inhibited cAMP transmission from bystander cells to irradiated cells. After treatment of cells with exogenous cAMP, forskolin (an activator of cAMP) or KH-7 (an inhibitor of cAMP), respectively, it was confirmed that cAMP communication from bystander cells to targeted cells could mitigate radiation damage in U739 cells, and this cAMP insufficiency in the bystander cells contributed to the enhancement of bystander apoptosis. Moreover, the bystander apoptosis in HL-7702 cells was aggravated by cAMP inhibition but it could not be evoked when p53 of HL-7702 cells was knocked down no matter of forskolin and KH-7 treatment. In conclusion, this study disclosed that cAMP could be released from bystander HL-7702 cells and compensated to α-irradiated U937 cells through a membrane signaling pathway and this cAMP communication played a profound role in regulating the reciprocal bystander effects.


      PubDate: 2014-03-21T23:21:09Z
       
  • Bipyridine (2,2′-dipyridyl) potentiates Escherichia coli lethality
           induced by nitrogen mustard mechlorethamine
    • Abstract: Publication date: Available online 13 March 2014
      Source:Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis
      Author(s): T.A.M. De Alencar , T.C. Wilmart-Gonçalves , L.S. Vidal , R.S. Fortunato , A.C. Leitão , C. Lage
      Alkylating agents are used in anti-tumor chemotherapy because they bind covalently to DNA and generate adducts that may lead to cell death. Bifunctional (HN2) and monofunctional (HN1) nitrogen are two such agents, and HN2 was the first drug successfully employed in anti-leukemia chemotherapy. Currently, HN2 is used either alone or combined with other drugs to treat Hodgkin's disease. It is well known that several crosslinking agents require metabolic activation via reactive oxygen species (ROS) to exert their lethal effects. The objective of this work was therefore to determine whether the abovementioned mustards would also require metabolic activation to exert lethal action against Escherichia coli. For this purpose, we measured survival following exposure to HN2 in E. coli strains that were deficient in nucleotide excision repair (uvrA NER mutant), base excision repair (xthA nfo nth fpg BER mutant) or superoxide dismutase (sodAB mutant) activity. We also performed the same experiments in cells pretreated with an iron chelator (2,2’-dipyridyl, DIP). The NER and BER mutants were only sensitive to HN2 treatment (survival rates similar to those of the wild-type were achieved with 5-fold lower HN2 doses). However, wild-type and sodAB strains were not sensitive to treatment with HN2. In all tested strains, survival dropped by 2.5-fold following pretreatment with DIP compared to treatment with HN2 alone. Furthermore, DIP treatment increased ROS generation in both wild type and sodAB-deficient strains. Based on these data and on the survival of the SOD-deficient strain, we suggest that the increased production of ROS caused by Fe2+ chelation may potentiate the lethal effects of HN2 but not HN1. This potentiation may arise as a consequence of enhancement in the number of or modification of the type of lesions formed. No sensitization was observed for the non-crosslinkable HN2 analog, HN1.


      PubDate: 2014-03-16T20:15:18Z
       
 
 
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