WorldWideScience

Sample records for dna-damaging agents cell

  1. Parainfluenza Virus Infection Sensitizes Cancer Cells to DNA-Damaging Agents: Implications for Oncolytic Virus Therapy.

    Science.gov (United States)

    Fox, Candace R; Parks, Griffith D

    2018-04-01

    A parainfluenza virus 5 (PIV5) with mutations in the P/V gene (P/V-CPI - ) is restricted for spread in normal cells but not in cancer cells in vitro and is effective at reducing tumor burdens in mouse model systems. Here we show that P/V-CPI - infection of HEp-2 human laryngeal cancer cells results in the majority of the cells dying, but unexpectedly, over time, there is an emergence of a population of cells that survive as P/V-CPI - persistently infected (PI) cells. P/V-CPI - PI cells had elevated levels of basal caspase activation, and viability was highly dependent on the activity of cellular inhibitor-of-apoptosis proteins (IAPs) such as Survivin and XIAP. In challenge experiments with external inducers of apoptosis, PI cells were more sensitive to cisplatin-induced DNA damage and cell death. This increased cisplatin sensitivity correlated with defects in DNA damage signaling pathways such as phosphorylation of Chk1 and translocation of damage-specific DNA binding protein 1 (DDB1) to the nucleus. Cisplatin-induced killing of PI cells was sensitive to the inhibition of wild-type (WT) p53-inducible protein 1 (WIP1), a phosphatase which acts to terminate DNA damage signaling pathways. A similar sensitivity to cisplatin was seen with cells during acute infection with P/V-CPI - as well as during acute infections with WT PIV5 and the related virus human parainfluenza virus type 2 (hPIV2). Our results have general implications for the design of safer paramyxovirus-based vectors that cannot establish PI as well as the potential for combining chemotherapy with oncolytic RNA virus vectors. IMPORTANCE There is intense interest in developing oncolytic viral vectors with increased potency against cancer cells, particularly those cancer cells that have gained resistance to chemotherapies. We have found that infection with cytoplasmically replicating parainfluenza virus can result in increases in the killing of cancer cells by agents that induce DNA damage, and this is linked

  2. The Growing Complexity of Cancer Cell Response to DNA-Damaging Agents: Caspase 3 Mediates Cell Death or Survival?

    Directory of Open Access Journals (Sweden)

    Razmik Mirzayans

    2016-05-01

    Full Text Available It is widely stated that wild-type p53 either mediates the activation of cell cycle checkpoints to facilitate DNA repair and promote cell survival, or orchestrates apoptotic cell death following exposure to cancer therapeutic agents. This reigning paradigm has been challenged by numerous discoveries with different human cell types, including solid tumor-derived cell lines. Thus, activation of the p53 signaling pathway by ionizing radiation and other DNA-damaging agents hinders apoptosis and triggers growth arrest (e.g., through premature senescence in some genetic backgrounds; such growth arrested cells remain viable, secrete growth-promoting factors, and give rise to progeny with stem cell-like properties. In addition, caspase 3, which is best known for its role in the execution phase of apoptosis, has been recently reported to facilitate (rather than suppress DNA damage-induced genomic instability and carcinogenesis. This observation is consistent with an earlier report demonstrating that caspase 3 mediates secretion of the pro-survival factor prostaglandin E2, which in turn promotes enrichment of tumor repopulating cells. In this article, we review these and related discoveries and point out novel cancer therapeutic strategies. One of our objectives is to demonstrate the growing complexity of the DNA damage response beyond the conventional “repair and survive, or die” hypothesis.

  3. Loss of Atrx sensitizes cells to DNA damaging agents through p53-mediated death pathways.

    Directory of Open Access Journals (Sweden)

    Damiano Conte

    Full Text Available Prevalent cell death in forebrain- and Sertoli cell-specific Atrx knockout mice suggest that Atrx is important for cell survival. However, conditional ablation in other tissues is not associated with increased death indicating that diverse cell types respond differently to the loss of this chromatin remodeling protein. Here, primary macrophages isolated from Atrx(f/f mice were infected with adenovirus expressing Cre recombinase or β-galactosidase, and assayed for cell survival under different experimental conditions. Macrophages survive without Atrx but undergo rapid apoptosis upon lipopolysaccharide (LPS activation suggesting that chromatin reorganization in response to external stimuli is compromised. Using this system we next tested the effect of different apoptotic stimuli on cell survival. We observed that survival of Atrx-null cells were similar to wild type cells in response to serum withdrawal, anti-Fas antibody, C2 ceramide or dexamethasone treatment but were more sensitive to 5-fluorouracil (5-FU. Cell survival could be rescued by re-introducing Atrx or by removal of p53 demonstrating the cell autonomous nature of the effect and its p53-dependence. Finally, we demonstrate that multiple primary cell types (myoblasts, embryonic fibroblasts and neurospheres were sensitive to 5-FU, cisplatin, and UV light treatment. Together, our results suggest that cells lacking Atrx are more sensitive to DNA damaging agents and that this may result in enhanced death during development when cells are at their proliferative peak. Moreover, it identifies potential treatment options for cancers associated with ATRX mutations, including glioblastoma and pancreatic neuroendocrine tumors.

  4. FANCG knockout CHO cells display sensitivity to diverse DNA damaging agents and possible genomic instability

    International Nuclear Information System (INIS)

    Hinz, J.M.; Tebbs, R.S.; Yamada, N.A.; Salazar, E.P.; Kopf, V.L.; Thompson, L.H.

    2003-01-01

    Full text: The function of the proteins encoded by the genes responsible for the disease Fanconi anemia (FA) have not been elucidated. Several of these proteins (FancA, C, E, F, and G) form a complex in the nucleus, and cells deficient in any one of these proteins are sensitive to crosslinking agents, suggesting a possible role for these proteins in some aspect of DNA repair, chromosomal maintenance, or replication. We constructed a FancG knockout mutant (FGKO40) in CHO AA8 cells, as well as FancG-corrected FGKO40 cells (KO40BP6, which is a pool of six BAC-clone transformants). FGKO40 cells are sensitive to a wide variety of DNA damaging agents. Sensitivity to the cross-linking agents MMC (3x) and chloroethyl-nitrosourea (3x) does not exceed that of methyl methanesulfonate (MMS) (4x), methyl-nitrosourea (4x), or ethyl-nitrosourea (3x), or the purine analog 6-thioguanine (5x). Other agents show mild sensitivity in FGKO40 cells: ionizing radiation (1.2x), UV-C (1.5x), hydroxyurea (1.2x), camptothecin (1.2x), and excess thymidine (normal). The length of S phase was carefully measured by monitoring the progression of highly synchronous G1 cells obtained by centrifugal elutriation. FGKO40 cells traversed S phase normally but had a slightly longer G2 phase than parental cells. Treatment of synchronized G1 cells with MMS did not increase S phase in parental or mutant cells, but again G2 was slightly longer for FGKO40. Mutation rates were measured at the hrpt and aprt loci, where gene inactivation confers resistance to 6-thioguanine or 8-azaadenine, respectively. FGKO40 had a slightly reduced mutation rate for hprt mutants, suggesting reduced recovery of large deletions at this locus. Moreover, the rate of methotrexate resistance was elevated 2.5-fold in mutant cells compared to controls. Resistance to this drug is generally associated with amplification of the dhfr locus, suggesting FancG plays a role in this aspect of genome stability. We suggest that the defect in FGKO

  5. Poly (ADP-ribose) catabolism in mammalian cells exposed to DNA-damaging agents

    International Nuclear Information System (INIS)

    Alvarez-Gonzalez, R.; Althaus, F.R.

    1989-01-01

    DNA damage inflicted by the alkylating agens N-methyl-N-nitro-N-nitrosoquanidine, or by UV stimulated the catabolism of protein-bound poly (ADP-ribose) in the chromatin of cultured hepatocytes. The stimulation was highest at the largest doses of DNA-damaging treatment. As a consequence, the half-life of ADP-ribosyl polymers may drop to less than 41 s. This rapid turnover contrasts with the slow catabolism of a constitutive fraction of polymers exhibiting a half-life of 7.7 h. These data suggest that post-incisional stimulation of poly (ADP-ribose) biosynthesis in DNA-excision repair is coupled with an adaptation of poly (ADP-ribose) catabolism in mammalian cells. (Author). 37 refs.; 3 figs

  6. Targeting neddylation induces DNA damage and checkpoint activation and sensitizes chronic lymphocytic leukemia B cells to alkylating agents.

    Science.gov (United States)

    Paiva, C; Godbersen, J C; Berger, A; Brown, J R; Danilov, A V

    2015-07-09

    Microenvironment-mediated upregulation of the B-cell receptor (BCR) and nuclear factor-κB (NF-κB) signaling in CLL cells resident in the lymph node and bone marrow promotes apoptosis evasion and clonal expansion. We recently reported that MLN4924 (pevonedistat), an investigational agent that inhibits the NEDD8-activating enzyme (NAE), abrogates stromal-mediated NF-κB pathway activity and CLL cell survival. However, the NAE pathway also assists degradation of multiple other substrates. MLN4924 has been shown to induce DNA damage and cell cycle arrest, but the importance of this mechanism in primary neoplastic B cells has not been studied. Here we mimicked the lymph node microenvironment using CD40 ligand (CD40L)-expressing stroma and interleukin-21 (IL-21) to find that inducing proliferation of the primary CLL cells conferred enhanced sensitivity to NAE inhibition. Treatment of the CD40-stimulated CLL cells with MLN4924 resulted in deregulation of Cdt1, a DNA replication licensing factor, and cell cycle inhibitors p21 and p27. This led to DNA damage, checkpoint activation and G2 arrest. Alkylating agents bendamustine and chlorambucil enhanced MLN4924-mediated DNA damage and apoptosis. These events were more prominent in cells stimulated with IL-21 compared with CD40L alone, indicating that, following NAE inhibition, the culture conditions were able to direct CLL cell fate from an NF-κB inhibition to a Cdt1 induction program. Our data provide insight into the biological consequences of targeting NAE in CLL and serves as further rationale for studying the clinical activity of MLN4924 in CLL, particularly in combination with alkylating agents.

  7. Sensitization of human cells by inhibitors of DNA synthesis following the action of DNA-damaging agents

    Energy Technology Data Exchange (ETDEWEB)

    Filatov, M.V.; Noskin, L.A. (Leningrad Inst. of Nuclear Physics, Gatchina (USSR))

    1983-08-01

    Inhibitors of DNA synthesis 1-..beta..-arabinofuranosylcytosine (Ac) and hydroxyurea (Hu) taken together drastically sensitized human cells to the killing effect of DNA-damaging agents. For UV-irradiation this sensitization depended on the cells' ability for excision repair. By using viscoelastometric methods of measurement of double-strand breaks (DSB) in the genome, it was established that the first DSB were generated after incubation of the damaged cells in the mixture of inhibitors at about the same dose when sensitization appeared. A scheme is proposed to describe molecular events associated with the phenomenon studied. 35 refs.

  8. Quantitative Analyses of Synergistic Responses between Cannabidiol and DNA-Damaging Agents on the Proliferation and Viability of Glioblastoma and Neural Progenitor Cells in Culture

    OpenAIRE

    Deng, Liting; Ng, Lindsay; Ozawa, Tatsuya; Stella, Nephi

    2017-01-01

    Evidence suggests that the nonpsychotropic cannabis-derived compound, cannabidiol (CBD), has antineoplastic activity in multiple types of cancers, including glioblastoma multiforme (GBM). DNA-damaging agents remain the main standard of care treatment available for patients diagnosed with GBM. Here we studied the antiproliferative and cell-killing activity of CBD alone and in combination with DNA-damaging agents (temozolomide, carmustine, or cisplatin) in several human GBM cell lines and in mo...

  9. Cross-sensitivity of X-ray-hypersensitive cells derived from LEC strain rats to DNA-damaging agents

    International Nuclear Information System (INIS)

    Okui, T.; Endoh, D.; Arai, S.; Isogai, E.; Hayashi, M.

    1996-01-01

    The cross-sensitivity of X-ray-hypersensitive lung fibroblasts from LEC strain (LEC) rats to other DNA-damaging agents was examined. The LEC cells were 2- to 3-fold more sensitive to bleomycin (BLM) that induces DNA double-strand breaks, and to a cross-linking agent, mitomycin C, than the cells from WKAH strain (WKAH) rats, while they were slightly sensitive to alkylating agents, ethyl nitrosourea and N-methyl-N'-nitro-N-nitrosoguanidine, but not to UV-irradiation. Although no difference was observed in the initial yields of DNA double-strand breaks induced by BLM between LEC and WKAH cells, the repair process of DNA double-strand breaks was significantly slower in LEC cells than in WKAH cells

  10. DNA excision repair in cell extracts from human cell lines exhibiting hypersensitivity to DNA-damaging agents

    International Nuclear Information System (INIS)

    Hansson, J.; Keyse, S.M.; Lindahl, T.; Wood, R.D.

    1991-01-01

    Whole cell extracts from human lymphoid cell lines can perform in vitro DNA repair synthesis in plasmids damaged by agents including UV or cis-diamminedichloroplatinum(II) (cis-DDP). Extracts from xeroderma pigmentosum (XP) cells are defective in repair synthesis. We have now studied in vitro DNA repair synthesis using extracts from lymphoblastoid cell lines representing four human hereditary syndromes with increased sensitivity to DNA-damaging agents. Extracts of cell lines from individuals with the sunlight-sensitive disorders dysplastic nevus syndrome or Cockayne's syndrome (complementation groups A and B) showed normal DNA repair synthesis in plasmids with UV photoproducts. This is consistent with in vivo measurements of the overall DNA repair capacity in such cell lines. A number of extracts were prepared from two cell lines representing the variant form of XP (XP-V). Half of the extracts prepared showed normal levels of in vitro DNA repair synthesis in plasmids containing UV lesions, but the remainder of the extracts from the same cell lines showed deficient repair synthesis, suggesting the possibility of an unusually labile excision repair protein in XP-V. Fanconi's anemia (FA) cells show cellular hypersensitivity to cross-linking agents including cis-DDP. Extracts from cell lines belonging to two different complementation groups of FA showed normal DNA repair synthesis in plasmids containing cis-DDP or UV adducts. Thus, there does not appear to be an overall excision repair defect in FA, but the data do not exclude a defect in the repair of interstrand DNA cross-links

  11. Quantitative Analyses of Synergistic Responses between Cannabidiol and DNA-Damaging Agents on the Proliferation and Viability of Glioblastoma and Neural Progenitor Cells in Culture.

    Science.gov (United States)

    Deng, Liting; Ng, Lindsay; Ozawa, Tatsuya; Stella, Nephi

    2017-01-01

    Evidence suggests that the nonpsychotropic cannabis-derived compound, cannabidiol (CBD), has antineoplastic activity in multiple types of cancers, including glioblastoma multiforme (GBM). DNA-damaging agents remain the main standard of care treatment available for patients diagnosed with GBM. Here we studied the antiproliferative and cell-killing activity of CBD alone and in combination with DNA-damaging agents (temozolomide, carmustine, or cisplatin) in several human GBM cell lines and in mouse primary GBM cells in cultures. This activity was also studied in mouse neural progenitor cells (NPCs) in culture to assess for potential central nervous system toxicity. We found that CBD induced a dose-dependent reduction of both proliferation and viability of all cells with similar potencies, suggesting no preferential activity for cancer cells. Hill plot analysis indicates an allosteric mechanism of action triggered by CBD in all cells. Cotreatment regimens combining CBD and DNA-damaging agents produced synergistic antiproliferating and cell-killing responses over a limited range of concentrations in all human GBM cell lines and mouse GBM cells as well as in mouse NPCs. Remarkably, antagonistic responses occurred at low concentrations in select human GBM cell lines and in mouse GBM cells. Our study suggests limited synergistic activity when combining CBD and DNA-damaging agents in treating GBM cells, along with little to no therapeutic window when considering NPCs. Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.

  12. Increased genome instability is not accompanied by sensitivity to DNA damaging agents in aged yeast cells

    NARCIS (Netherlands)

    Novarina, Daniele; Mavrova, Sara N.; Janssens, Georges E.; Rempel, Irina L.; Veenhoff, Liesbeth M.; Chang, Michael

    The budding yeast Saccharomyces cerevisiae divides asymmetrically, producing a new daughter cell from the original mother cell. While daughter cells are born with a full lifespan, a mother cell ages with each cell division and can only generate on average 25 daughter cells before dying. Aged yeast

  13. BAG3 sensitizes cancer cells exposed to DNA damaging agents via direct interaction with GRP78.

    Science.gov (United States)

    Kong, De-Hui; Zhang, Qiang; Meng, Xin; Zong, Zhi-Hong; Li, Chao; Liu, Bao-Qin; Guan, Yifu; Wang, Hua-Qin

    2013-12-01

    Bcl-2 associated athanogene 3 (BAG3) has a modular structure that contains a BAG domain, a WW domain, a proline-rich (PxxP) domain to mediate potential interactions with chaperons and other proteins that participate in more than one signal transduction. In search for novel interacting partners, the current study identified that 78kDa glucose-regulated protein (GRP78) was a novel partner interacting with BAG3. Interaction between GRP78 and BAG3 was confirmed by coimmunoprecipitation and glutathione S-transferase (GST) pulldown. We also identified that the ATPase domain of GRP78 and BAG domain of BAG3 mediated their interaction. Counterintuitive for a prosurvival protein, BAG3 was found to promote the cytotoxicity of breast cancer MCF7, thyroid cancer FRO and glioma U87 cells subjected to genotoxic stress. In addition, the current study demonstrated that BAG3 interfered with the formation of the antiapoptotic GRP78-procaspase-7 complex, which resulted in an increased genotoxic stress-induced cytotoxicity in cancer cells. Furthermore, overexpression of GRP78 significantly blocked the enhancing effects of BAG3 on activation of caspase-7 and induction of apoptosis by genotoxic stress. Overall, these results suggested that through direct interaction BAG3 could prevent the antiapoptotic effect of GRP78 upon genotoxic stress. © 2013.

  14. Sensitization of melanoma cells to alkylating agent-induced DNA damage and cell death via orchestrating oxidative stress and IKKβ inhibition.

    Science.gov (United States)

    Tse, Anfernee Kai-Wing; Chen, Ying-Jie; Fu, Xiu-Qiong; Su, Tao; Li, Ting; Guo, Hui; Zhu, Pei-Li; Kwan, Hiu-Yee; Cheng, Brian Chi-Yan; Cao, Hui-Hui; Lee, Sally Kin-Wah; Fong, Wang-Fun; Yu, Zhi-Ling

    2017-04-01

    Nitrosourea represents one of the most active classes of chemotherapeutic alkylating agents for metastatic melanoma. Treatment with nitrosoureas caused severe systemic side effects which hamper its clinical use. Here, we provide pharmacological evidence that reactive oxygen species (ROS) induction and IKKβ inhibition cooperatively enhance nitrosourea-induced cytotoxicity in melanoma cells. We identified SC-514 as a ROS-inducing IKKβ inhibitor which enhanced the function of nitrosoureas. Elevated ROS level results in increased DNA crosslink efficiency triggered by nitrosoureas and IKKβ inhibition enhances DNA damage signals and sensitizes nitrosourea-induced cell death. Using xenograft mouse model, we confirm that ROS-inducing IKKβ inhibitor cooperates with nitrosourea to reduce tumor size and malignancy in vivo. Taken together, our results illustrate a new direction in nitrosourea treatment, and reveal that the combination of ROS-inducing IKKβ inhibitors with nitrosoureas can be potentially exploited for melanoma therapy. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

  15. Sensitization of melanoma cells to alkylating agent-induced DNA damage and cell death via orchestrating oxidative stress and IKKβ inhibition

    Directory of Open Access Journals (Sweden)

    Anfernee Kai-Wing Tse

    2017-04-01

    Full Text Available Nitrosourea represents one of the most active classes of chemotherapeutic alkylating agents for metastatic melanoma. Treatment with nitrosoureas caused severe systemic side effects which hamper its clinical use. Here, we provide pharmacological evidence that reactive oxygen species (ROS induction and IKKβ inhibition cooperatively enhance nitrosourea-induced cytotoxicity in melanoma cells. We identified SC-514 as a ROS-inducing IKKβ inhibitor which enhanced the function of nitrosoureas. Elevated ROS level results in increased DNA crosslink efficiency triggered by nitrosoureas and IKKβ inhibition enhances DNA damage signals and sensitizes nitrosourea-induced cell death. Using xenograft mouse model, we confirm that ROS-inducing IKKβ inhibitor cooperates with nitrosourea to reduce tumor size and malignancy in vivo. Taken together, our results illustrate a new direction in nitrosourea treatment, and reveal that the combination of ROS-inducing IKKβ inhibitors with nitrosoureas can be potentially exploited for melanoma therapy.

  16. DNA damage induced in mouse peritoneal exudate cells after in vivo administration of chemical and physical agents as determined by alkaline elution

    International Nuclear Information System (INIS)

    Nishi, Yoshisuke; Miyanaga, Kumiko; Sato, Sei-ichi; Inui, Naomichi

    1990-01-01

    The alkaline elution technique for detecting DNA strand breaks has been applied to the study of DNA damage in mouse peritoneal exudate cells resulting from the in vivo administration of chemical and physical agents. The direct methylating agents methyl methanesulphonate and N-methyl-N-nitrosourea induced extensive breakage in samples taken 2 h after administration. The direct ethylating agents ethyl methanesulphonate and N-ethyl-N-nitrosourea also induced DNA strand breaks, but to a lesser extent than the methylating agents. The indirect methylating agent dimethylnitrosamine showed hardly any effect in this system. A weak but positive response was observed upon treatment with the anti-neoplastic alkylating agent procarbazine hydrochloride. The whole-body irradiation of mice with 60 Co γ-rays also induced DNA strand breaks. The elution profiles for γ-ray irradiation were different from those of alkylating agents, and indicate that alkylating agents produce many more secondary lesions leading to DNA strand breaks than γ-rays. N-methyl-N-nitrosourea produced slightly more DNA strand breaks in mutagen-sensitive mice, which are derived from the CD-1 strain, than in ICR mice. (Author)

  17. DNA damage induced in mouse peritoneal exudate cells after in vivo administration of chemical and physical agents as determined by alkaline elution

    Energy Technology Data Exchange (ETDEWEB)

    Nishi, Yoshisuke (Japan Tobacco Inc., Yokohama (Japan). Central Research Inst.); Miyanaga, Kumiko; Sato, Sei-ichi (Japan Tobacco Inc., Hatano, Kanagawa (Japan). Toxicology Research Lab.); Inui, Naomichi (Japan Tobacco Inc., Yokohama, Kanagawa (Japan). Pharmaceutical Research Labs.)

    1990-01-01

    The alkaline elution technique for detecting DNA strand breaks has been applied to the study of DNA damage in mouse peritoneal exudate cells resulting from the in vivo administration of chemical and physical agents. The direct methylating agents methyl methanesulphonate and N-methyl-N-nitrosourea induced extensive breakage in samples taken 2 h after administration. The direct ethylating agents ethyl methanesulphonate and N-ethyl-N-nitrosourea also induced DNA strand breaks, but to a lesser extent than the methylating agents. The indirect methylating agent dimethylnitrosamine showed hardly any effect in this system. A weak but positive response was observed upon treatment with the anti-neoplastic alkylating agent procarbazine hydrochloride. The whole-body irradiation of mice with {sup 60}Co {gamma}-rays also induced DNA strand breaks. The elution profiles for {gamma}-ray irradiation were different from those of alkylating agents, and indicate that alkylating agents produce many more secondary lesions leading to DNA strand breaks than {gamma}-rays. N-methyl-N-nitrosourea produced slightly more DNA strand breaks in mutagen-sensitive mice, which are derived from the CD-1 strain, than in ICR mice. (Author).

  18. Balancing repair and tolerance of DNA damage caused by alkylating agents

    OpenAIRE

    Fu, Dragony; Calvo, Jennifer A.; Samson, Leona D.

    2012-01-01

    Alkylating agents constitute a major class of frontline chemotherapeutic drugs that inflict cytotoxic DNA damage as their main mode of action, in addition to collateral mutagenic damage. Numerous cellular pathways, including direct DNA damage reversal, base excision repair (BER) and mismatch repair (MMR), respond to alkylation damage to defend against alkylation-induced cell death or mutation. However, maintaining a proper balance of activity both within and between these pathways is crucial ...

  19. SERIES: Genomic instability in cancer Balancing repair and tolerance of DNA damage caused by alkylating agents

    OpenAIRE

    Fu, Dragony; Calvo, Jennifer A.; Samson, Leona D

    2012-01-01

    Alkylating agents comprise a major class of frontline chemotherapeutic drugs that inflict cytotoxic DNA damage as their main mode of action, in addition to collateral mutagenic damage. Numerous cellular pathways, including direct DNA damage reversal, base excision repair (BER), and mismatch repair (MMR) respond to alkylation damage to defend against alkylation-induced cell death or mutation. However, maintaining a proper balance of activity both within and between these pathways is crucial fo...

  20. DNA Damage Induced by Alkylating Agents and Repair Pathways

    Science.gov (United States)

    Kondo, Natsuko; Takahashi, Akihisa; Ono, Koji; Ohnishi, Takeo

    2010-01-01

    The cytotoxic effects of alkylating agents are strongly attenuated by cellular DNA repair processes, necessitating a clear understanding of the repair mechanisms. Simple methylating agents form adducts at N- and O-atoms. N-methylations are removed by base excision repair, AlkB homologues, or nucleotide excision repair (NER). O6-methylguanine (MeG), which can eventually become cytotoxic and mutagenic, is repaired by O6-methylguanine-DNA methyltransferase, and O6MeG:T mispairs are recognized by the mismatch repair system (MMR). MMR cannot repair the O6MeG/T mispairs, which eventually lead to double-strand breaks. Bifunctional alkylating agents form interstrand cross-links (ICLs) which are more complex and highly cytotoxic. ICLs are repaired by complex of NER factors (e.g., endnuclease xeroderma pigmentosum complementation group F-excision repair cross-complementing rodent repair deficiency complementation group 1), Fanconi anemia repair, and homologous recombination. A detailed understanding of how cells cope with DNA damage caused by alkylating agents is therefore potentially useful in clinical medicine. PMID:21113301

  1. Activation of ATM by DNA Damaging Agents

    National Research Council Canada - National Science Library

    Kurz, Ebba U; Lees-Miller, Susan P

    2004-01-01

    Ataxia-telangiectasia mutated (ATM) is a serine/threonine protein kinase that acts as a master switch controlling the cell cycle in response to ionizing radiation-induced DNA double-strand breaks (DSBs...

  2. Activation of ATM by DNA Damaging Agents

    National Research Council Canada - National Science Library

    Kurz, Ebba U; Lees-Miller, Susan P

    2005-01-01

    Ataxia-telangiectasia mutated (ATM) is a serine/threonine protein kinase that acts as a master switch controlling the cell cycle in response to ionizing radiation-induced DNA double-strand breaks (DSBs...

  3. Mechanisms of dealing with DNA damage in terminally differentiated cells

    Energy Technology Data Exchange (ETDEWEB)

    Fortini, P. [Department of Environment and Primary Prevention, Istituto Superiore di Sanita, Viale Regina Elena 299, 00161 Rome (Italy); Dogliotti, E., E-mail: eugenia.dogliotti@iss.it [Department of Environment and Primary Prevention, Istituto Superiore di Sanita, Viale Regina Elena 299, 00161 Rome (Italy)

    2010-03-01

    To protect genomic integrity living cells that are continuously exposed to DNA-damaging insults are equipped with an efficient defence mechanism termed the DNA damage response. Its function is to eliminate DNA damage through DNA repair and to remove damaged cells by apoptosis. The DNA damage response has been investigated mainly in proliferating cells, in which the cell cycle machinery is integrated with the DNA damage signalling. The current knowledge of the mechanisms of DNA repair, DNA damage signalling and cell death of post-mitotic cells that have undergone irreversible cell cycle withdrawal will be reviewed. Evidence will be provided that the protection of the genome integrity in terminally differentiated cells is achieved by different strategies than in proliferating cells.

  4. Mechanisms of dealing with DNA damage in terminally differentiated cells

    International Nuclear Information System (INIS)

    Fortini, P.; Dogliotti, E.

    2010-01-01

    To protect genomic integrity living cells that are continuously exposed to DNA-damaging insults are equipped with an efficient defence mechanism termed the DNA damage response. Its function is to eliminate DNA damage through DNA repair and to remove damaged cells by apoptosis. The DNA damage response has been investigated mainly in proliferating cells, in which the cell cycle machinery is integrated with the DNA damage signalling. The current knowledge of the mechanisms of DNA repair, DNA damage signalling and cell death of post-mitotic cells that have undergone irreversible cell cycle withdrawal will be reviewed. Evidence will be provided that the protection of the genome integrity in terminally differentiated cells is achieved by different strategies than in proliferating cells.

  5. SERIES: Genomic instability in cancer Balancing repair and tolerance of DNA damage caused by alkylating agents

    Science.gov (United States)

    Fu, Dragony; Calvo, Jennifer A.; Samson, Leona D

    2013-01-01

    Alkylating agents comprise a major class of frontline chemotherapeutic drugs that inflict cytotoxic DNA damage as their main mode of action, in addition to collateral mutagenic damage. Numerous cellular pathways, including direct DNA damage reversal, base excision repair (BER), and mismatch repair (MMR) respond to alkylation damage to defend against alkylation-induced cell death or mutation. However, maintaining a proper balance of activity both within and between these pathways is crucial for an organism's favorable response to alkylating agents. Furthermore, an individual's response to alkylating agents can vary considerably from tissue to tissue and from person to person, pointing to genetic and epigenetic mechanisms that modulate alkylating agent toxicity. PMID:22237395

  6. Metformin (dimethyl-biguanide induced DNA damage in mammalian cells

    Directory of Open Access Journals (Sweden)

    Rubem R. Amador

    2012-01-01

    Full Text Available Metformin (dimethyl-biguanide is an insulin-sensitizing agent that lowers fasting plasma-insulin concentration, wherefore it's wide use for patients with a variety of insulin-resistant and prediabetic states, including impaired glucose tolerance. During pregnancy it is a further resource for reducing first-trimester pregnancy loss in women with the polycystic ovary syndrome. We tested metformin genotoxicity in cells of Chinese hamster ovary, CHO-K1 (chromosome aberrations; comet assays and in mice (micronucleus assays. Concentrations of 114.4 µg/mL and 572 µg/mL were used in in vitro tests, and 95.4 mg/kg, 190.8 mg/kg and 333.9 mg/kg in assaying. Although the in vitro tests revealed no chromosome aberrations in metaphase cells, DNA damage was detected by comet assaying after 24 h of incubation at both concentrations. The frequency of DNA damage was higher at concentrations of 114.4 µg/mL. Furthermore, although mortality was not observed in in vitro tests, the highest dose of metformin suppressed bone marrow cells. However, no statistically significant differences were noted in micronuclei frequencies between treatments. In vitro results indicate that chronic metformin exposure may be potentially genotoxic. Thus, pregnant woman undergoing treatment with metformin should be properly evaluated beforehand, as regards vulnerability to DNA damage.

  7. Effect of passage number on cellular response to DNA-damaging agents: Cell survival and gene expression

    International Nuclear Information System (INIS)

    Chang-Liu, C.M.; Woloschak, G.E.

    1997-01-01

    The effect of different passage numbers on plating efficiency, doubling time, cell growth, and radiation sensitivity was assessed in Syrian hamster embryo (SHE) cells. Changes in gene expression after UV or γ-ray irradiation at different passage numbers were also examined. The SHE cells were maintained in culture medium for up to 64 passages. Cells were exposed to 60 Co γ rays or 254-nm UV radiation. Differential display of cDNAs and northern blots were used for the study of gene expression. With increasing passage number, SHE cells demonstrated decreased doubling time, increased plating efficiency, and a decreased yield in the number of cells per plate. Between passages 41 and 48 a crisis period was evident during which time cell growth in high serum was no longer optimal, and serum concentrations were reduced to maintain cell growth. Sensitivity to ionizing radiation was no different between early- and intermediate-passage cells. However, after UV exposure at low passages (passage 3), confluent cells were more sensitive to the killing effects of UV than were log-phase cells. At intermediate passages (passages 43, 48), confluent cells were slightly more radioresistant than were log-phase cells. By passage 64, however, both confluent and log-phase cells showed similar patterns of UV sensitivity. Expression of γ-actin, PCNA, and p53 transcripts did not change following UV exposure. p53 mRNA was induced following γ-ray exposure of the intermediate (passage 45) epithelial cells. The observed differences in radiation sensitivity associated with increasing passage number may be influenced by radiation-induced gene expression. The authors are conducted experiments to identify these genes

  8. DNA damage-inducible transcripts in mammalian cells

    International Nuclear Information System (INIS)

    Fornace, A.J. Jr.; Alamo, I. Jr.; Hollander, M.C.

    1988-01-01

    Hybridization subtraction at low ratios of RNA to cDNA was used to enrich for the cDNA of transcripts increased in Chinese hamster cells after UV irradiation. Forty-nine different cDNA clones were isolated. Most coded for nonabundant transcripts rapidly induced 2- to 10-fold after UV irradiation. Only 2 of the 20 cDNA clones sequenced matched known sequences (metallothionein I and II). The predicted amino acid sequence of one cDNA had two localized areas of homology with the rat helix-destabilizing protein. These areas of homology were at the two DNA-binding sites of this nucleic acid single-strand-binding protein. The induced transcripts were separated into two general classes. Class I transcripts were induced by UV radiation and not by the alkylating agent methyl methanesulfonate. Class II transcripts were induced by UV radiation and by methyl methanesulfonate. Many class II transcripts were induced also by H2O2 and various alkylating agents but not by heat shock, phorbol 12-tetradecanoate 13-acetate, or DNA-damaging agents which do not produce high levels of base damage. Since many of the cDNA clones coded for transcripts which were induced rapidly and only by certain types of DNA-damaging agents, their induction is likely a specific response to such damage rather than a general response to cell injury

  9. Vorinostat induces reactive oxygen species and DNA damage in acute myeloid leukemia cells.

    Directory of Open Access Journals (Sweden)

    Luca A Petruccelli

    Full Text Available Histone deacetylase inhibitors (HDACi are promising anti-cancer agents, however, their mechanisms of action remain unclear. In acute myeloid leukemia (AML cells, HDACi have been reported to arrest growth and induce apoptosis. In this study, we elucidate details of the DNA damage induced by the HDACi vorinostat in AML cells. At clinically relevant concentrations, vorinostat induces double-strand breaks and oxidative DNA damage in AML cell lines. Additionally, AML patient blasts treated with vorinostat display increased DNA damage, followed by an increase in caspase-3/7 activity and a reduction in cell viability. Vorinostat-induced DNA damage is followed by a G2-M arrest and eventually apoptosis. We found that pre-treatment with the antioxidant N-acetyl cysteine (NAC reduces vorinostat-induced DNA double strand breaks, G2-M arrest and apoptosis. These data implicate DNA damage as an important mechanism in vorinostat-induced growth arrest and apoptosis in both AML cell lines and patient-derived blasts. This supports the continued study and development of vorinostat in AMLs that may be sensitive to DNA-damaging agents and as a combination therapy with ionizing radiation and/or other DNA damaging agents.

  10. Vorinostat Induces Reactive Oxygen Species and DNA Damage in Acute Myeloid Leukemia Cells

    Science.gov (United States)

    Pettersson, Filippa; Retrouvey, Hélène; Skoulikas, Sophia; Miller, Wilson H.

    2011-01-01

    Histone deacetylase inhibitors (HDACi) are promising anti-cancer agents, however, their mechanisms of action remain unclear. In acute myeloid leukemia (AML) cells, HDACi have been reported to arrest growth and induce apoptosis. In this study, we elucidate details of the DNA damage induced by the HDACi vorinostat in AML cells. At clinically relevant concentrations, vorinostat induces double-strand breaks and oxidative DNA damage in AML cell lines. Additionally, AML patient blasts treated with vorinostat display increased DNA damage, followed by an increase in caspase-3/7 activity and a reduction in cell viability. Vorinostat-induced DNA damage is followed by a G2-M arrest and eventually apoptosis. We found that pre-treatment with the antioxidant N-acetyl cysteine (NAC) reduces vorinostat-induced DNA double strand breaks, G2-M arrest and apoptosis. These data implicate DNA damage as an important mechanism in vorinostat-induced growth arrest and apoptosis in both AML cell lines and patient-derived blasts. This supports the continued study and development of vorinostat in AMLs that may be sensitive to DNA-damaging agents and as a combination therapy with ionizing radiation and/or other DNA damaging agents. PMID:21695163

  11. Sunlight-induced DNA damage in human mononuclear cells

    DEFF Research Database (Denmark)

    Møller, Peter; Wallin, Hakan; Holst, Erik

    2002-01-01

    of sunlight was comparable to the interindividual variation, indicating that sunlight exposure and the individual's background were the two most important determinants for the basal level of DNA damage. Influence of other lifestyle factors such as exercise, intake of foods, infections, and age could......In this study of 301 blood samples from 21 subjects, we found markedly higher levels of DNA damage (nonpyrimidine dimer types) in the summer than in the winter detected by single-cell gel electrophoresis. The level of DNA damage was influenced by the average daily influx of sunlight ... to blood sampling. The 3 and 6 day periods before sampling influenced DNA damage the most. The importance of sunlight was further emphasized by a positive association of the DNA damage level to the amount of time the subjects had spent in the sun over a 3 day period prior to the sampling. The effect...

  12. Hypersensitivity to DNA-damaging agents in primary degenerations of excitable tissue

    International Nuclear Information System (INIS)

    Robbins, J.H.

    1983-01-01

    Defects in DNA-repair mechanisms render xeroderma pigmentosum cells hypersensitive to killing by the uv-type of DNA-damaging agent. Some xeroderma pigmentosum patients develop a primary neuronal degeneration, and cell lines from patients with the earliest onset of neurodegeneration are the most sensitive to killing by uv radiation. These findings led to the neuronal DNA integrity theory which holds that when the integrity of neuronal DNA is destroyed by the accumulation of unrepaired DNA damaged spontaneously or by endogenous metabolites, the neurons will undergo a primary degeneration. Cells from patients with Cockayne syndrome, a demyelinating disorder with a primary retinal degeneration, are also hypersensitive to the uv-type of DNA-damaging agent. Cells from patients with the primary neuronal degeneration of ataxia telangiectasia are hypersensitive to the x-ray-type of DNA-damaging agent. Cells from other patients with primary degeneration of excitable tissue also have hypersensitivity to the x-ray-type of DNA-damaging agent. These disorders include (1) primary neuronal degenerations which are either genetic (e.g., Huntington disease, familial dysautonomia, Friedreich ataxia) or sporadic (e.g., Alzheimer disease, Parkinson disease), (2) primary muscle degenerations (e.g., Duchenne muscular dystrophy), and (3) a primary retinal degeneration (Usher syndrome). Death of excitable tissue in vivo in these radiosensitive diseases may result from unrepaired DNA. This hypersensitivity provides the basis for developing suitable presymptomatic and prenatal tests for these diseases, for elucidating their pathogenesis, and for developing future therapies. 119 references, 3 figures, 3 tables

  13. An active site aromatic triad in Escherichia coli DNA Pol IV coordinates cell survival and mutagenesis in different DNA damaging agents.

    Directory of Open Access Journals (Sweden)

    Ryan W Benson

    Full Text Available DinB (DNA Pol IV is a translesion (TLS DNA polymerase, which inserts a nucleotide opposite an otherwise replication-stalling N(2-dG lesion in vitro, and confers resistance to nitrofurazone (NFZ, a compound that forms these lesions in vivo. DinB is also known to be part of the cellular response to alkylation DNA damage. Yet it is not known if DinB active site residues, in addition to aminoacids involved in DNA synthesis, are critical in alkylation lesion bypass. It is also unclear which active site aminoacids, if any, might modulate DinB's bypass fidelity of distinct lesions. Here we report that along with the classical catalytic residues, an active site "aromatic triad", namely residues F12, F13, and Y79, is critical for cell survival in the presence of the alkylating agent methyl methanesulfonate (MMS. Strains expressing dinB alleles with single point mutations in the aromatic triad survive poorly in MMS. Remarkably, these strains show fewer MMS- than NFZ-induced mutants, suggesting that the aromatic triad, in addition to its role in TLS, modulates DinB's accuracy in bypassing distinct lesions. The high bypass fidelity of prevalent alkylation lesions is evident even when the DinB active site performs error-prone NFZ-induced lesion bypass. The analyses carried out with the active site aromatic triad suggest that the DinB active site residues are poised to proficiently bypass distinctive DNA lesions, yet they are also malleable so that the accuracy of the bypass is lesion-dependent.

  14. Sea urchin coelomocytes are resistant to a variety of DNA damaging agents

    Energy Technology Data Exchange (ETDEWEB)

    Loram, Jeannette; Raudonis, Renee; Chapman, Jecar; Lortie, Mae [Bermuda Institute of Ocean Sciences, St. George' s, Bermuda, GE 01 (Bermuda); Bodnar, Andrea, E-mail: andrea.bodnar@bios.edu [Bermuda Institute of Ocean Sciences, St. George' s, Bermuda, GE 01 (Bermuda)

    2012-11-15

    Increasing anthropogenic activities are creating environmental pressures that threaten marine ecosystems. Effective environmental health assessment requires the development of rapid, sensitive, and cost-effective tools to predict negative impacts at the individual and ecosystem levels. To this end, a number of biological assays using a variety of cells and organisms measuring different end points have been developed for biomonitoring programs. The sea urchin fertilization/development test has been useful for evaluating environmental toxicology and it has been proposed that sea urchin coelomocytes represent a novel cellular biosensor of environmental stress. In this study we investigated the sensitivity of coelomocytes from the sea urchin Lytechinus variegatus to a variety of DNA-damaging agents including ultraviolet (UV) radiation, hydrogen peroxide (H{sub 2}O{sub 2}), methylmethane sulfonate (MMS) and benzo[a]pyrene (BaP). LD{sub 50} values determined for coelomocytes after 24 h of exposure to these DNA damaging agents indicated a high level of resistance to all treatments. Significant increases in the formation of apurinic/apyrimidinic (AP or abasic) sites in DNA were only detected using high doses of H{sub 2}O{sub 2}, MMS and UV radiation. Comparison of sea urchin coelomocytes with hemocytes from the gastropod mollusk Aplysia dactylomela and the decapod crustacean Panulirus argus indicated that sensitivity to different DNA damaging agents varies between species. The high level of resistance to genotoxic agents suggests that DNA damage may not be an informative end point for environmental health assessment using sea urchin coelomocytes however, natural resistance to DNA damaging agents may have implications for the occurrence of neoplastic disease in these animals.

  15. Sea urchin coelomocytes are resistant to a variety of DNA damaging agents

    International Nuclear Information System (INIS)

    Loram, Jeannette; Raudonis, Renee; Chapman, Jecar; Lortie, Mae; Bodnar, Andrea

    2012-01-01

    Increasing anthropogenic activities are creating environmental pressures that threaten marine ecosystems. Effective environmental health assessment requires the development of rapid, sensitive, and cost-effective tools to predict negative impacts at the individual and ecosystem levels. To this end, a number of biological assays using a variety of cells and organisms measuring different end points have been developed for biomonitoring programs. The sea urchin fertilization/development test has been useful for evaluating environmental toxicology and it has been proposed that sea urchin coelomocytes represent a novel cellular biosensor of environmental stress. In this study we investigated the sensitivity of coelomocytes from the sea urchin Lytechinus variegatus to a variety of DNA-damaging agents including ultraviolet (UV) radiation, hydrogen peroxide (H 2 O 2 ), methylmethane sulfonate (MMS) and benzo[a]pyrene (BaP). LD 50 values determined for coelomocytes after 24 h of exposure to these DNA damaging agents indicated a high level of resistance to all treatments. Significant increases in the formation of apurinic/apyrimidinic (AP or abasic) sites in DNA were only detected using high doses of H 2 O 2 , MMS and UV radiation. Comparison of sea urchin coelomocytes with hemocytes from the gastropod mollusk Aplysia dactylomela and the decapod crustacean Panulirus argus indicated that sensitivity to different DNA damaging agents varies between species. The high level of resistance to genotoxic agents suggests that DNA damage may not be an informative end point for environmental health assessment using sea urchin coelomocytes however, natural resistance to DNA damaging agents may have implications for the occurrence of neoplastic disease in these animals.

  16. Balancing repair and tolerance of DNA damage caused by alkylating agents.

    Science.gov (United States)

    Fu, Dragony; Calvo, Jennifer A; Samson, Leona D

    2012-01-12

    Alkylating agents constitute a major class of frontline chemotherapeutic drugs that inflict cytotoxic DNA damage as their main mode of action, in addition to collateral mutagenic damage. Numerous cellular pathways, including direct DNA damage reversal, base excision repair (BER) and mismatch repair (MMR), respond to alkylation damage to defend against alkylation-induced cell death or mutation. However, maintaining a proper balance of activity both within and between these pathways is crucial for a favourable response of an organism to alkylating agents. Furthermore, the response of an individual to alkylating agents can vary considerably from tissue to tissue and from person to person, pointing to genetic and epigenetic mechanisms that modulate alkylating agent toxicity.

  17. Alkaline Comet Assay for Assessing DNA Damage in Individual Cells.

    Science.gov (United States)

    Pu, Xinzhu; Wang, Zemin; Klaunig, James E

    2015-08-06

    Single-cell gel electrophoresis, commonly called a comet assay, is a simple and sensitive method for assessing DNA damage at the single-cell level. It is an important technique in genetic toxicological studies. The comet assay performed under alkaline conditions (pH >13) is considered the optimal version for identifying agents with genotoxic activity. The alkaline comet assay is capable of detecting DNA double-strand breaks, single-strand breaks, alkali-labile sites, DNA-DNA/DNA-protein cross-linking, and incomplete excision repair sites. The inclusion of digestion of lesion-specific DNA repair enzymes in the procedure allows the detection of various DNA base alterations, such as oxidative base damage. This unit describes alkaline comet assay procedures for assessing DNA strand breaks and oxidative base alterations. These methods can be applied in a variety of cells from in vitro and in vivo experiments, as well as human studies. Copyright © 2015 John Wiley & Sons, Inc.

  18. Aging of hematopoietic stem cells: DNA damage and mutations?

    Science.gov (United States)

    Moehrle, Bettina M; Geiger, Hartmut

    2016-10-01

    Aging in the hematopoietic system and the stem cell niche contributes to aging-associated phenotypes of hematopoietic stem cells (HSCs), including leukemia and aging-associated immune remodeling. Among others, the DNA damage theory of aging of HSCs is well established, based on the detection of a significantly larger amount of γH2AX foci and a higher tail moment in the comet assay, both initially thought to be associated with DNA damage in aged HSCs compared with young cells, and bone marrow failure in animals devoid of DNA repair factors. Novel data on the increase in and nature of DNA mutations in the hematopoietic system with age, the quality of the DNA damage response in aged HSCs, and the nature of γH2AX foci question a direct link between DNA damage and the DNA damage response and aging of HSCs, and rather favor changes in epigenetics, splicing-factors or three-dimensional architecture of the cell as major cell intrinsic factors of HSCs aging. Aging of HSCs is also driven by a strong contribution of aging of the niche. This review discusses the DNA damage theory of HSC aging in the light of these novel mechanisms of aging of HSCs. Copyright © 2016 ISEH - International Society for Experimental Hematology. Published by Elsevier Inc. All rights reserved.

  19. DNA damage responses in human induced pluripotent stem cells and embryonic stem cells.

    Directory of Open Access Journals (Sweden)

    Olga Momcilovic

    2010-10-01

    Full Text Available Induced pluripotent stem (iPS cells have the capability to undergo self-renewal and differentiation into all somatic cell types. Since they can be produced through somatic cell reprogramming, which uses a defined set of transcription factors, iPS cells represent important sources of patient-specific cells for clinical applications. However, before these cells can be used in therapeutic designs, it is essential to understand their genetic stability.Here, we describe DNA damage responses in human iPS cells. We observe hypersensitivity to DNA damaging agents resulting in rapid induction of apoptosis after γ-irradiation. Expression of pluripotency factors does not appear to be diminished after irradiation in iPS cells. Following irradiation, iPS cells activate checkpoint signaling, evidenced by phosphorylation of ATM, NBS1, CHEK2, and TP53, localization of ATM to the double strand breaks (DSB, and localization of TP53 to the nucleus of NANOG-positive cells. We demonstrate that iPS cells temporary arrest cell cycle progression in the G(2 phase of the cell cycle, displaying a lack of the G(1/S cell cycle arrest similar to human embryonic stem (ES cells. Furthermore, both cell types remove DSB within six hours of γ-irradiation, form RAD51 foci and exhibit sister chromatid exchanges suggesting homologous recombination repair. Finally, we report elevated expression of genes involved in DNA damage signaling, checkpoint function, and repair of various types of DNA lesions in ES and iPS cells relative to their differentiated counterparts.High degrees of similarity in DNA damage responses between ES and iPS cells were found. Even though reprogramming did not alter checkpoint signaling following DNA damage, dramatic changes in cell cycle structure, including a high percentage of cells in the S phase, increased radiosensitivity and loss of DNA damage-induced G(1/S cell cycle arrest, were observed in stem cells generated by induced pluripotency.

  20. Quantification of DNA damage by single-cell electrophoresis

    International Nuclear Information System (INIS)

    Ikushima, Takaji

    1990-01-01

    A simple technique of micro-agarose gel electrophoresis has been developed to quantify DNA damage in individual cells. Cells are embedded in agarose gel on microscope slides, lysed by detergents and then electrophoresed for a short time under neutral or alkaline condition. In irradiated cells, DNA migrates from the nucleus toward the anode, displaying commet-like pattern by staining with DNA-specific fluorescence dye. DNA damage is evaluated by measuring the distance of DNA migration. The technique was applied for measuring DNA damage in single cells exposed to 60 Co γ-rays, or to KUR radiation in the presence or absence of 10 B-enriched boric acid. The enhanced production of double-stranded DNA breaks by 10 B(n,α) 7 Li reaction was demonstrated here. The significant increase in the length of DNA migration was observed in single cells exposed to such a low dose as 20 cGy after alkaline micro electrophoresis. (author)

  1. The intersection between DNA damage response and cell death pathways.

    Science.gov (United States)

    Nowsheen, S; Yang, E S

    2012-10-01

    Apoptosis is a finely regulated process that serves to determine the fate of cells in response to various stresses. One such stress is DNA damage, which not only can signal repair processes but is also intimately involved in regulating cell fate. In this review we examine the relationship between the DNA damage/repair response in cell survival and apoptosis following insults to the DNA. Elucidating these pathways and the crosstalk between them is of great importance, as they eventually contribute to the etiology of human disease such as cancer and may play key roles in determining therapeutic response. This article is part of a Special Issue entitled "Apoptosis: Four Decades Later".

  2. Comparison of checkpoint responses triggered by DNA polymerase inhibition versus DNA damaging agents

    International Nuclear Information System (INIS)

    Liu, J.-S.; Kuo, S.-R.; Melendy, Thomas

    2003-01-01

    To better understand the different cellular responses to replication fork pausing versus blockage, early DNA damage response markers were compared after treatment of cultured mammalian cells with agents that either inhibit DNA polymerase activity (hydroxyurea (HU) or aphidicolin) or selectively induce S-phase DNA damage responses (the DNA alkylating agents, methyl methanesulfonate (MMS) and adozelesin). These agents were compared for their relative abilities to induce phosphorylation of Chk1, H2AX, and replication protein A (RPA), and intra-nuclear focalization of γ-H2AX and RPA. Treatment by aphidicolin and HU resulted in phosphorylation of Chk1, while HU, but not aphidicolin, induced focalization of γ-H2AX and RPA. Surprisingly, pre-treatment with aphidicolin to stop replication fork progression, did not abrogate HU-induced γ-H2AX and RPA focalization. This suggests that HU may act on the replication fork machinery directly, such that fork progression is not required to trigger these responses. The DNA-damaging fork-blocking agents, adozelesin and MMS, both induced phosphorylation and focalization of H2AX and RPA. Unlike adozelesin and HU, the pattern of MMS-induced RPA focalization did not match the BUdR incorporation pattern and was not blocked by aphidicolin, suggesting that MMS-induced damage is not replication fork-dependent. In support of this, MMS was the only reagent used that did not induce phosphorylation of Chk1. These results indicate that induction of DNA damage checkpoint responses due to adozelesin is both replication fork and fork progression dependent, induction by HU is replication fork dependent but progression independent, while induction by MMS is independent of both replication forks and fork progression

  3. Comparison of checkpoint responses triggered by DNA polymerase inhibition versus DNA damaging agents

    Energy Technology Data Exchange (ETDEWEB)

    Liu, J.-S.; Kuo, S.-R.; Melendy, Thomas

    2003-11-27

    To better understand the different cellular responses to replication fork pausing versus blockage, early DNA damage response markers were compared after treatment of cultured mammalian cells with agents that either inhibit DNA polymerase activity (hydroxyurea (HU) or aphidicolin) or selectively induce S-phase DNA damage responses (the DNA alkylating agents, methyl methanesulfonate (MMS) and adozelesin). These agents were compared for their relative abilities to induce phosphorylation of Chk1, H2AX, and replication protein A (RPA), and intra-nuclear focalization of {gamma}-H2AX and RPA. Treatment by aphidicolin and HU resulted in phosphorylation of Chk1, while HU, but not aphidicolin, induced focalization of {gamma}-H2AX and RPA. Surprisingly, pre-treatment with aphidicolin to stop replication fork progression, did not abrogate HU-induced {gamma}-H2AX and RPA focalization. This suggests that HU may act on the replication fork machinery directly, such that fork progression is not required to trigger these responses. The DNA-damaging fork-blocking agents, adozelesin and MMS, both induced phosphorylation and focalization of H2AX and RPA. Unlike adozelesin and HU, the pattern of MMS-induced RPA focalization did not match the BUdR incorporation pattern and was not blocked by aphidicolin, suggesting that MMS-induced damage is not replication fork-dependent. In support of this, MMS was the only reagent used that did not induce phosphorylation of Chk1. These results indicate that induction of DNA damage checkpoint responses due to adozelesin is both replication fork and fork progression dependent, induction by HU is replication fork dependent but progression independent, while induction by MMS is independent of both replication forks and fork progression.

  4. Live cell microscopy of DNA damage response in Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Pinela da Silva, Sonia Cristina; Gallina, Irene; Eckert-Boulet, Nadine Valerie

    2012-01-01

    live cell imaging allows for multiple cellular markers to be monitored over several hours. This chapter reviews useful fluorescent markers and genotoxic agents for studying the DNA damage response in living cells and provides protocols for live cell imaging, time-lapse microscopy, and for induction...

  5. Differences in the regulation by poly(ADP-ribose) of repair of DNA damage from alkylating agents and ultraviolet light according to cell type

    Energy Technology Data Exchange (ETDEWEB)

    Cleaver, J.E.; Bodell, W.J.; Morgan, W.F.; Zelle, B.

    1983-08-10

    Inhibition of poly(ADP-ribose) synthesis by 3-aminobenzamide in various human and hamster cells influenced the responses to DNA damage from methyl methanesulfonate, but not from ultraviolet light. After exposure to methyl methanesulfonate, 3-aminobenzamide increased the strand break frequency in all cell types studied, but only stimulated repair replication in lymphoid and HeLa cells, suggesting these are independent effects. 3-Aminobenzamide also inhibited the pathway for de novo synthesis of DNA purines, suggesting that some of its effects may be due to disturbance of precursor pathways and irrelevant to the role of poly(ADP-ribose) in repair. Previous claims that 3-aminobenzamide stimulates repair synthesis after exposure to UV light are probably artifacts, because the stimulations are only observed in lymphocytes in the presence of a high concentration of hydroxyurea that itself inhibits repair. The initial inhibition of semiconservative DNA synthesis and the excision of the major alkylation products and pyrimidine dimers were unaffected by 3-aminobenzamide. In general poly(ADP-ribose) synthesis appears to be uniquely involved in regulating the ligation stage of repair of alkylation damage but not ultraviolet damage. By regulating the ligation efficiency, poly(ADP-ribosylation) modulates the dynamic balance between incision and ligation, so as to minimize the frequency of DNA breaks. The ligation stage of repair of UV damage appears different and is not regulated by poly(ADP-ribosylation).

  6. In vitro sensitivity of normal and hereditary retinoblastoma fibroblasts to DNA-damaging agents

    International Nuclear Information System (INIS)

    Woods, W.G.; Byrne, T.D.

    1986-01-01

    We investigated the ability of nine fibroblast cell strains from patients with the hereditary form of retinoblastoma (RB) to handle various types of DNA-damaging agents and compared the results with those obtained in nine normal strains. Cell strains were exposed to gamma-radiation, which causes DNA scission; actinomycin D, a DNA-intercalating agent; and mitomycin C, a bifunctional alkylating agent leading to DNA-DNA cross-linking. Cell strains were studied for their ability to survive in a cytotoxicity assay. Nine normal strains exhibited a mean D0 (inverse of the slope of the straight line portion of the survival curve) of 134-178 cGy after radiation exposure, compared to a range of 119-186 cGy in the nine RB strains (P = 0.33). Similarly, exposure to actinomycin D led to D0 values of 0.024-0.069 microgram/ml in the nine normal strains and D0 values of 0.016-0.067 microgram/ml in the RB strains (P = 0.64). The nine RB strains did exhibit a small overall increase in sensitivity after exposure to mitomycin C, with D0 values ranging from 0.14-0.32 microgram/ml versus 0.19-0.66 microgram/ml in the nine normal strains (P = 0.002); however, when the two most resistant normal strains were excluded from analysis, results were similar. Three RB cell strains derived from individuals who had either developed second cancers or who had a family history of additional sarcomas consistently exhibited increases in sensitivity to all three DNA-damaging agents studied compared with other hereditary RB cell strains as well as normal strains. The results suggest that normal human fibroblast cell strains exhibit a wide response to DNA-damaging agents, especially chemical agents. Most hereditary RB strains exhibit sensitivity well within the normal range; however, strains from RB patients predisposed to second cancers exhibit increases in sensitivity to DNA-damaging agents

  7. DNA damage and autophagy

    International Nuclear Information System (INIS)

    Rodriguez-Rocha, Humberto; Garcia-Garcia, Aracely; Panayiotidis, Mihalis I.; Franco, Rodrigo

    2011-01-01

    Both exogenous and endogenous agents are a threat to DNA integrity. Exogenous environmental agents such as ultraviolet (UV) and ionizing radiation, genotoxic chemicals and endogenous byproducts of metabolism including reactive oxygen species can cause alterations in DNA structure (DNA damage). Unrepaired DNA damage has been linked to a variety of human disorders including cancer and neurodegenerative disease. Thus, efficient mechanisms to detect DNA lesions, signal their presence and promote their repair have been evolved in cells. If DNA is effectively repaired, DNA damage response is inactivated and normal cell functioning resumes. In contrast, when DNA lesions cannot be removed, chronic DNA damage triggers specific cell responses such as cell death and senescence. Recently, DNA damage has been shown to induce autophagy, a cellular catabolic process that maintains a balance between synthesis, degradation, and recycling of cellular components. But the exact mechanisms by which DNA damage triggers autophagy are unclear. More importantly, the role of autophagy in the DNA damage response and cellular fate is unknown. In this review we analyze evidence that supports a role for autophagy as an integral part of the DNA damage response.

  8. Sensitization of melanoma cells to alkylating agent-induced DNA damage and cell death via orchestrating oxidative stress and IKK? inhibition

    OpenAIRE

    Tse, Anfernee Kai-Wing; Chen, Ying-Jie; Fu, Xiu-Qiong; Su, Tao; Li, Ting; Guo, Hui; Zhu, Pei-Li; Kwan, Hiu-Yee; Cheng, Brian Chi-Yan; Cao, Hui-Hui; Lee, Sally Kin-Wah; Fong, Wang-Fun; Yu, Zhi-Ling

    2017-01-01

    Nitrosourea represents one of the most active classes of chemotherapeutic alkylating agents for metastatic melanoma. Treatment with nitrosoureas caused severe systemic side effects which hamper its clinical use. Here, we provide pharmacological evidence that reactive oxygen species (ROS) induction and IKKβ inhibition cooperatively enhance nitrosourea-induced cytotoxicity in melanoma cells. We identified SC-514 as a ROS-inducing IKKβ inhibitor which enhanced the function of nitrosoureas. Eleva...

  9. Molecular dosimetry of DNA damage caused by alkylation. I. Single-strand breaks induced by ethylating agents in cultured mammalian cells in relation to survival

    NARCIS (Netherlands)

    Abbondandolo, A.; Dogliotti, E.; Lohman, P.H.M.; Berends, F.

    1982-01-01

    Cultured Chinese hamster ovary cells were treated with ethylating agents. DNA lesions giving rise to single-strand breaks (ssb) or alkali-labile sites were measured by centrifugation in alkaline sucrose gradients after lysis in alkali. 4 agents with different tendencies to ethylate preferentially

  10. DNA damage by carbonyl stress in human skin cells

    International Nuclear Information System (INIS)

    Roberts, Michael J.; Wondrak, Georg T.; Laurean, Daniel Cervantes; Jacobson, Myron K.; Jacobson, Elaine L.

    2003-01-01

    Reactive carbonyl species (RCS) are potent mediators of cellular carbonyl stress originating from endogenous chemical processes such as lipid peroxidation and glycation. Skin deterioration as observed in photoaging and diabetes has been linked to accumulative protein damage from glycation, but the effects of carbonyl stress on skin cell genomic integrity are ill defined. In this study, the genotoxic effects of acute carbonyl stress on HaCaT keratinocytes and CF3 fibroblasts were assessed. Administration of the α-dicarbonyl compounds glyoxal and methylglyoxal as physiologically relevant RCS inhibited skin cell proliferation, led to intra-cellular protein glycation as evidenced by the accumulation of N ε -(carboxymethyl)-L-lysine (CML) in histones, and caused extensive DNA strand cleavage as assessed by the comet assay. These effects were prevented by treatment with the carbonyl scavenger D-penicillamine. Both glyoxal and methylglyoxal damaged DNA in intact cells. Glyoxal caused DNA strand breaks while methylglyoxal produced extensive DNA-protein cross-linking as evidenced by pronounced nuclear condensation and total suppression of comet formation. Glycation by glyoxal and methylglyoxal resulted in histone cross-linking in vitro and induced oxygen-dependent cleavage of plasmid DNA, which was partly suppressed by the hydroxyl scavenger mannitol. We suggest that a chemical mechanism of cellular DNA damage by carbonyl stress occurs in which histone glycoxidation is followed by reactive oxygen induced DNA stand breaks. The genotoxic potential of RCS in cultured skin cells and its suppression by a carbonyl scavenger as described in this study have implications for skin damage and carcinogenesis and its prevention by agents selective for carbonyl stress

  11. DNA damage by carbonyl stress in human skin cells

    Energy Technology Data Exchange (ETDEWEB)

    Roberts, Michael J.; Wondrak, Georg T.; Laurean, Daniel Cervantes; Jacobson, Myron K.; Jacobson, Elaine L

    2003-01-28

    Reactive carbonyl species (RCS) are potent mediators of cellular carbonyl stress originating from endogenous chemical processes such as lipid peroxidation and glycation. Skin deterioration as observed in photoaging and diabetes has been linked to accumulative protein damage from glycation, but the effects of carbonyl stress on skin cell genomic integrity are ill defined. In this study, the genotoxic effects of acute carbonyl stress on HaCaT keratinocytes and CF3 fibroblasts were assessed. Administration of the {alpha}-dicarbonyl compounds glyoxal and methylglyoxal as physiologically relevant RCS inhibited skin cell proliferation, led to intra-cellular protein glycation as evidenced by the accumulation of N{sup {epsilon}}-(carboxymethyl)-L-lysine (CML) in histones, and caused extensive DNA strand cleavage as assessed by the comet assay. These effects were prevented by treatment with the carbonyl scavenger D-penicillamine. Both glyoxal and methylglyoxal damaged DNA in intact cells. Glyoxal caused DNA strand breaks while methylglyoxal produced extensive DNA-protein cross-linking as evidenced by pronounced nuclear condensation and total suppression of comet formation. Glycation by glyoxal and methylglyoxal resulted in histone cross-linking in vitro and induced oxygen-dependent cleavage of plasmid DNA, which was partly suppressed by the hydroxyl scavenger mannitol. We suggest that a chemical mechanism of cellular DNA damage by carbonyl stress occurs in which histone glycoxidation is followed by reactive oxygen induced DNA stand breaks. The genotoxic potential of RCS in cultured skin cells and its suppression by a carbonyl scavenger as described in this study have implications for skin damage and carcinogenesis and its prevention by agents selective for carbonyl stress.

  12. Harnessing the p53-PUMA Axis to Overcome DNA Damage Resistance in Renal Cell Carcinoma

    Directory of Open Access Journals (Sweden)

    Xiaoguang Zhou

    2014-12-01

    Full Text Available Resistance to DNA damage–induced apoptosis is a hallmark of cancer and a major cause of treatment failure and lethal disease outcome. A tumor entity that is largely resistant to DNA-damaging therapies including chemo- or radiotherapy is renal cell carcinoma (RCC. This study was designed to explore the underlying molecular mechanisms of DNA damage resistance in RCC to develop strategies to resensitize tumor cells to DNA damage–induced apoptosis. Here, we show that apoptosis-resistant RCC cells have a disconnect between activation of p53 and upregulation of the downstream proapoptotic protein p53 upregulated modulator of apoptosis (PUMA. We demonstrate that this disconnect is not caused by gene-specific repression through CCCTC-binding factor (CTCF but instead by aberrant chromatin compaction. Treatment with an HDAC inhibitor was found to effectively reactivate PUMA expression on the mRNA and protein level and to revert resistance to DNA damage–induced cell death. Ectopic expression of PUMA was found to resensitize a panel of RCC cell lines to four different DNA-damaging agents tested. Remarkably, all RCC cell lines analyzed were wild-type for p53, and a knockdown was likewise able to sensitize RCC cells to acute genotoxic stress. Taken together, our results indicate that DNA damage resistance in RCC is reversible, involves the p53-PUMA axis, and is potentially targetable to improve the oncological outcomes of RCC patients.

  13. Hydroxytyrosol Protects against Oxidative DNA Damage in Human Breast Cells

    Directory of Open Access Journals (Sweden)

    José J. Gaforio

    2011-10-01

    Full Text Available Over recent years, several studies have related olive oil ingestion to a low incidence of several diseases, including breast cancer. Hydroxytyrosol and tyrosol are two of the major phenols present in virgin olive oils. Despite the fact that they have been linked to cancer prevention, there is no evidence that clarifies their effect in human breast tumor and non-tumor cells. In the present work, we present hydroxytyrosol and tyrosol’s effects in human breast cell lines. Our results show that hydroxytyrosol acts as a more efficient free radical scavenger than tyrosol, but both fail to affect cell proliferation rates, cell cycle profile or cell apoptosis in human mammary epithelial cells (MCF10A or breast cancer cells (MDA-MB-231 and MCF7. We found that hydroxytyrosol decreases the intracellular reactive oxygen species (ROS level in MCF10A cells but not in MCF7 or MDA-MB-231 cells while very high amounts of tyrosol is needed to decrease the ROS level in MCF10A cells. Interestingly, hydroxytyrosol prevents oxidative DNA damage in the three breast cell lines. Therefore, our data suggest that simple phenol hydroxytyrosol could contribute to a lower incidence of breast cancer in populations that consume virgin olive oil due to its antioxidant activity and its protection against oxidative DNA damage in mammary cells.

  14. Moderate variations in CDC25B protein levels modulate the response to DNA damaging agents

    International Nuclear Information System (INIS)

    Aressy, B.; Bugler, B.; Valette, A.; Ducommun, B.; Biard, D.

    2008-01-01

    CDC25B, one of the three members of the CDC25 dual-specificity phosphatase family, plays a critical role in the control of the cell cycle and in the checkpoint response to DNA damage. CDC25B is responsible for the initial dephosphorylation and activation of the cyclin-dependent kinases, thus initiating the train of events leading to entry into mitosis. The critical role played by CDC25B is illustrated by the fact that it is specifically required for checkpoint recovery and that unscheduled accumulation of CDC25B is responsible for illegitimate entry into mitosis. Here, we report that in p53 colon carcinoma cells, a moderate increase in the CDC25B level is sufficient to impair the DNA damage checkpoint, to increase spontaneous mutagenesis, and to sensitize cells to ionising radiation and genotoxic agents. Using a tumour cell spheroid assay as an alternative to animal studies, we demonstrate that the level of CDC25B expression modulates growth inhibition and apoptotic death. Since CDC25B overexpression has been observed in a significant number of human cancers, including colon carcinoma, and is often associated with high grade tumours and poor prognosis, our work suggests that the expression level of CDC25B might be a potential key parameter of the cellular response to cancer therapy. (authors)

  15. Inhibition of exportin-1 function results in rapid cell cycle-associated DNA damage in cancer cells.

    Science.gov (United States)

    Burke, Russell T; Marcus, Joshua M; Orth, James D

    2017-06-13

    Selective inhibitors of nuclear export (SINE) are small molecules in development as anti-cancer agents. The first-in-class SINE, selinexor, is in clinical trials for blood and solid cancers. Selinexor forms a covalent bond with exportin-1 at cysteine-528, and blocks its ability to export cargos. Previous work has shown strong cell cycle effects and drug-induced cell death across many different cancer-derived cell lines. Here, we report strong cell cycle-associated DNA double-stranded break formation upon the treatment of cancer cells with SINE. In multiple cell models, selinexor treatment results in the formation of clustered DNA damage foci in 30-40% of cells within 8 hours that is dependent upon cysteine-528. DNA damage strongly correlates with G1/S-phase and decreased DNA replication. Live cell microscopy reveals an association between DNA damage and cell fate. Cells that form damage in G1-phase more often die or arrest, while those damaged in S/G2-phase frequently progress to cell division. Up to half of all treated cells form damage foci, and most cells that die after being damaged, were damaged in G1-phase. By comparison, non-transformed cell lines show strong cell cycle effects but little DNA damage and less death than cancer cells. Significant drug combination effects occur when selinexor is paired with different classes of agents that either cause DNA damage or that diminish DNA damage repair. These data present a novel effect of exportin-1 inhibition and provide a strong rationale for multiple combination treatments of selinexor with agents that are currently in use for the treatment of different solid cancers.

  16. DNA Damage by Radiation in Tradescantia Leaf Cells

    International Nuclear Information System (INIS)

    Han, Min; Hyun, Kyung Man; Ryu, Tae Ho; Kim, Jin Kyu; Nili, Mohammad

    2010-01-01

    The comet assay is currently used in different areas of biological sciences to detect DNA damage. The comet assay, due to its simplicity, sensitivity and need of a few cells, is ideal as a short-term genotoxicity test. The comet assay can theoretically be applied to every type of eukaryotic cell, including plant cells. Plants are very useful as monitors of genetic effects caused by pollution in the atmosphere, water and soil. Tradescantia tests are very useful tools for screening the mutagenic potential in the environment. Experiments were conducted to study the genotoxic effects of ionizing radiations on the genome integrity, particularly of Tradescantia. The increasingly frequent use of Tradescantia as a sensitive environmental bioindicator of genotoxic effects. This study was designed to assess the genotoxicity of ionizing radiation using Tradescnatia-comet assay. The development of comet assay has enabled investigators to detect DNA damage at the levels of cells. To adapt this assay to plant cells, nuclei were directly obtained from Tradescantia leaf samples. A significant dose-dependent increase in the average tail moment values over the negative control was observed. Recently the adaptation of this technique to plant cells opens new possibilities for studies in variety area. The future applications of the comet assay could impact some other important areas, certainly, one of the limiting factors to its utility is the imagination of the investigator.

  17. DNA Damage by Radiation in Tradescantia Leaf Cells

    Energy Technology Data Exchange (ETDEWEB)

    Han, Min; Hyun, Kyung Man; Ryu, Tae Ho; Kim, Jin Kyu [Korea Atomic Energy Research Institute, Advanced Radiation Technology Institute, Jeongeup (Korea, Republic of); Nili, Mohammad [Dawnesh Radiation Research Institute, Barcelona (Spain)

    2010-04-15

    The comet assay is currently used in different areas of biological sciences to detect DNA damage. The comet assay, due to its simplicity, sensitivity and need of a few cells, is ideal as a short-term genotoxicity test. The comet assay can theoretically be applied to every type of eukaryotic cell, including plant cells. Plants are very useful as monitors of genetic effects caused by pollution in the atmosphere, water and soil. Tradescantia tests are very useful tools for screening the mutagenic potential in the environment. Experiments were conducted to study the genotoxic effects of ionizing radiations on the genome integrity, particularly of Tradescantia. The increasingly frequent use of Tradescantia as a sensitive environmental bioindicator of genotoxic effects. This study was designed to assess the genotoxicity of ionizing radiation using Tradescnatia-comet assay. The development of comet assay has enabled investigators to detect DNA damage at the levels of cells. To adapt this assay to plant cells, nuclei were directly obtained from Tradescantia leaf samples. A significant dose-dependent increase in the average tail moment values over the negative control was observed. Recently the adaptation of this technique to plant cells opens new possibilities for studies in variety area. The future applications of the comet assay could impact some other important areas, certainly, one of the limiting factors to its utility is the imagination of the investigator.

  18. Targeting Ongoing DNA Damage in Multiple Myeloma: Effects of DNA Damage Response Inhibitors on Plasma Cell Survival

    Directory of Open Access Journals (Sweden)

    Ana Belén Herrero

    2017-05-01

    Full Text Available Human myeloma cell lines (HMCLs and a subset of myeloma patients with poor prognosis exhibit high levels of replication stress (RS, leading to DNA damage. In this study, we confirmed the presence of DNA double-strand breaks (DSBs in several HMCLs by measuring γH2AX and RAD51 foci and analyzed the effect of various inhibitors of the DNA damage response on MM cell survival. Inhibition of ataxia telangiectasia and Rad3-related protein (ATR, the main kinase mediating the response to RS, using the specific inhibitor VE-821 induced more cell death in HMCLs than in control lymphoblastoid cells and U266, an HMCL with a low level of DNA damage. The absence of ATR was partially compensated by ataxia telangiectasia-mutated protein (ATM, since chemical inhibition of both kinases using VE-821 and KU-55933 significantly increased the death of MM cells with DNA damage. We found that ATM and ATR are involved in DSB repair by homologous recombination (HR in MM. Inhibition of both kinases resulted in a stronger inhibition that may underlie cell death induction, since abolition of HR using two different inhibitors severely reduced survival of HMCLs that exhibit DNA damage. On the other hand, inhibition of the other route involved in DSB repair, non-homologous end joining (NHEJ, using the DNA-PK inhibitor NU7441, did not affect MM cell viability. Interestingly, we found that NHEJ inhibition did not increase cell death when HR was simultaneously inhibited with the RAD51 inhibitor B02, but it clearly increased the level of cell death when HR was inhibited with the MRE11 inhibitor mirin, which interferes with recombination before DNA resection takes place. Taken together, our results demonstrate for the first time that MM cells with ongoing DNA damage rely on an intact HR pathway, which thereby suggests therapeutic opportunities. We also show that inhibition of HR after the initial step of end resection might be more appropriate for inducing MM cell death, since it

  19. Effects of chemical-induced DNA damage on male germ cells

    Energy Technology Data Exchange (ETDEWEB)

    Holme, J.A.; Bjoerge, C.; Trbojevic, M.; Olsen, A.K.; Brunborg, G.; Soederlund, E.J. [National Inst. of Public Health, Oslo (Norway). Dept. of Environmental Medicine; Bjoeras, M.; Seeberg, E. [National Hospital, Oslo (Norway). Dept. of Microbiology; Scholz, T.; Dybing, E.; Wiger, R. [National Hospital, Oslo (Norway). Inst. for Surgical Research and Surgical Dept. B

    1998-12-31

    Several recent studies indicate declines in sperm production, as well as increases in the incidence of genitourinary abnormalities such as testicular cancer, cryptorchidism and hypospadias. It is not known if these effects are due to exposure to chemical pollutants or if other ethiological factors are involved. Animal studies indicate that chemicals will induce such effects by various genetic, epigenetic or non-genetic mechanisms. Recently, much attention has been focused on embryonic/fetal exposure to oestrogen-mimicking chemicals (Toppari et al., 1996). However, the possibility that chemicals may cause reproductive toxicity by other mechanisms such as interactions with DNA, should not be ignored. DNA damage in germ cells may lead to the production of mutated spermatozoa, which in turn may result in spontaneous abortions, malformations and/or genetic defects in the offspring. Regarding the consequences of DNA alterations for carcinogenesis it is possible that genetic damage may occur germ cells, but the consequences are not expressed until certain genetic events occur in postnatal life. Transmission of genetic risk is best demonstrated by cancer-prone disorders such as hereditary retinoblastoma and the Li-Fraumeni syndrome. A number of experiments indicate that germ cells and proliferating cells may be particularly sensitive to DNA damaging agents compared to other cells. Furthermore, several lines of evidence have indicated that one of the best documented male reproductive toxicants, 1,2-dibrome-3-chloropropane (DBCP), causes testicular toxicity through DNA damage. It is possible that testicular cells at certain maturational stages are more subject to DNA damage, have less efficient DNA repair, or have different thresholds for initiating apoptosis following DNA damage than other cell types. (orig.)

  20. Genome-Wide Requirements for Resistance to Functionally Distinct DNA-Damaging Agents.

    Directory of Open Access Journals (Sweden)

    2005-08-01

    Full Text Available The mechanistic and therapeutic differences in the cellular response to DNA-damaging compounds are not completely understood, despite intense study. To expand our knowledge of DNA damage, we assayed the effects of 12 closely related DNA-damaging agents on the complete pool of ~4,700 barcoded homozygous deletion strains of Saccharomyces cerevisiae. In our protocol, deletion strains are pooled together and grown competitively in the presence of compound. Relative strain sensitivity is determined by hybridization of PCR-amplified barcodes to an oligonucleotide array carrying the barcode complements. These screens identified genes in well-characterized DNA-damage-response pathways as well as genes whose role in the DNA-damage response had not been previously established. High-throughput individual growth analysis was used to independently confirm microarray results. Each compound produced a unique genome-wide profile. Analysis of these data allowed us to determine the relative importance of DNA-repair modules for resistance to each of the 12 profiled compounds. Clustering the data for 12 distinct compounds uncovered both known and novel functional interactions that comprise the DNA-damage response and allowed us to define the genetic determinants required for repair of interstrand cross-links. Further genetic analysis allowed determination of epistasis for one of these functional groups.

  1. Nek1 silencing slows down DNA repair and blocks DNA damage-induced cell cycle arrest.

    Science.gov (United States)

    Pelegrini, Alessandra Luíza; Moura, Dinara Jaqueline; Brenner, Bethânia Luise; Ledur, Pitia Flores; Maques, Gabriela Porto; Henriques, João Antônio Pegas; Saffi, Jenifer; Lenz, Guido

    2010-09-01

    Never in mitosis A (NIMA)-related kinases (Nek) are evolutionarily conserved proteins structurally related to the Aspergillus nidulans mitotic regulator NIMA. Nek1 is one of the 11 isoforms of the Neks identified in mammals. Different lines of evidence suggest the participation of Nek1 in response to DNA damage, which is also supported by the interaction of this kinase with proteins involved in DNA repair pathways and cell cycle regulation. In this report, we show that cells with Nek1 knockdown (KD) through stable RNA interference present a delay in DNA repair when treated with methyl-methanesulfonate (MMS), hydrogen peroxide (H(2)O(2)) and cisplatin (CPT). In particular, interstrand cross links induced by CPT take much longer to be resolved in Nek1 KD cells when compared to wild-type (WT) cells. In KD cells, phosphorylation of Chk1 in response to CPT was strongly reduced. While WT cells accumulate in G(2)/M after DNA damage with MMS and H(2)O(2), Nek1 KD cells do not arrest, suggesting that G(2)/M arrest induced by the DNA damage requires Nek1. Surprisingly, CPT-treated Nek1 KD cells arrest with a 4N DNA content similar to WT cells. This deregulation in cell cycle control in Nek1 KD cells leads to an increased sensitivity to genotoxic agents when compared to WT cells. These results suggest that Nek1 is involved in the beginning of the cellular response to genotoxic stress and plays an important role in preventing cell death induced by DNA damage.

  2. DNA Damage Induced by Alkylating Agents and Repair Pathways

    OpenAIRE

    Natsuko Kondo; Akihisa Takahashi; Koji Ono; Takeo Ohnishi

    2010-01-01

    The cytotoxic effects of alkylating agents are strongly attenuated by cellular DNA repair processes, necessitating a clear understanding of the repair mechanisms. Simple methylating agents form adducts at N- and O-atoms. N-methylations are removed by base excision repair, AlkB homologues, or nucleotide excision repair (NER). O 6-methylguanine (MeG), which can eventually become cytotoxic and mutagenic, is repaired by O 6-methylguanine-DNA methyltransferase, and O 6MeG:T mispairs are recognized...

  3. [Sea urchin embryo, DNA-damaged cell cycle checkpoint and the mechanisms initiating cancer development].

    Science.gov (United States)

    Bellé, Robert; Le Bouffant, Ronan; Morales, Julia; Cosson, Bertrand; Cormier, Patrick; Mulner-Lorillon, Odile

    2007-01-01

    Cell division is an essential process for heredity, maintenance and evolution of the whole living kingdom. Sea urchin early development represents an excellent experimental model for the analysis of cell cycle checkpoint mechanisms since embryonic cells contain a functional DNA-damage checkpoint and since the whole sea urchin genome is sequenced. The DNA-damaged checkpoint is responsible for an arrest in the cell cycle when DNA is damaged or incorrectly replicated, for activation of the DNA repair mechanism, and for commitment to cell death by apoptosis in the case of failure to repair. New insights in cancer biology lead to two fundamental concepts about the very first origin of cancerogenesis. Cancers result from dysfunction of DNA-damaged checkpoints and cancers appear as a result of normal stem cell (NCS) transformation into a cancer stem cell (CSC). The second aspect suggests a new definition of "cancer", since CSC can be detected well before any clinical evidence. Since early development starts from the zygote, which is a primary stem cell, sea urchin early development allows analysis of the early steps of the cancerization process. Although sea urchins do not develop cancers, the model is alternative and complementary to stem cells which are not easy to isolate, do not divide in a short time and do not divide synchronously. In the field of toxicology and incidence on human health, the sea urchin experimental model allows assessment of cancer risk from single or combined molecules long before any epidemiologic evidence is available. Sea urchin embryos were used to test the worldwide used pesticide Roundup that contains glyphosate as the active herbicide agent; it was shown to activate the DNA-damage checkpoint of the first cell cycle of development. The model therefore allows considerable increase in risk evaluation of new products in the field of cancer and offers a tool for the discovery of molecular markers for early diagnostic in cancer biology

  4. DNA damage and polyploidization.

    Science.gov (United States)

    Chow, Jeremy; Poon, Randy Y C

    2010-01-01

    A growing body of evidence indicates that polyploidization triggers chromosomal instability and contributes to tumorigenesis. DNA damage is increasingly being recognized for its roles in promoting polyploidization. Although elegant mechanisms known as the DNA damage checkpoints are responsible for halting the cell cycle after DNA damage, agents that uncouple the checkpoints can induce unscheduled entry into mitosis. Likewise, defects of the checkpoints in several disorders permit mitotic entry even in the presence of DNA damage. Forcing cells with damaged DNA into mitosis causes severe chromosome segregation defects, including lagging chromosomes, chromosomal fragments and chromosomal bridges. The presence of these lesions in the cleavage plane is believed to abort cytokinesis. It is postulated that if cytokinesis failure is coupled with defects of the p53-dependent postmitotic checkpoint pathway, cells can enter S phase and become polyploids. Progress in the past several years has unraveled some of the underlying principles of these pathways and underscored the important role of DNA damage in polyploidization. Furthermore, polyploidization per se may also be an important determinant of sensitivity to DNA damage, thereby may offer an opportunity for novel therapies.

  5. Endogenous DNA Damage and Risk of Testicular Germ Cell Tumors

    Energy Technology Data Exchange (ETDEWEB)

    Cook, M B; Sigurdson, A J; Jones, I M; Thomas, C B; Graubard, B I; Korde, L; Greene, M H; McGlynn, K A

    2008-01-18

    Testicular germ cell tumors (TGCT) are comprised of two histologic groups, seminomas and nonseminomas. We postulated that the possible divergent pathogeneses of these histologies may be partially explained by variable endogenous DNA damage. To assess our hypothesis, we conducted a case-case analysis of seminomas and nonseminomas using the alkaline comet assay to quantify single-strand DNA breaks and alkali-labile sites. The Familial Testicular Cancer study and the U.S. Radiologic Technologists cohort provided 112 TGCT cases (51 seminomas & 61 nonseminomas). A lymphoblastoid cell line was cultured for each patient and the alkaline comet assay was used to determine four parameters: tail DNA, tail length, comet distributed moment (CDM) and Olive tail moment (OTM). Odds ratios (OR) and 95% confidence intervals (95%CI) were estimated using logistic regression. Values for tail length, tail DNA, CDM and OTM were modeled as categorical variables using the 50th and 75th percentiles of the seminoma group. Tail DNA was significantly associated with nonseminoma compared to seminoma (OR{sub 50th percentile} = 3.31, 95%CI: 1.00, 10.98; OR{sub 75th percentile} = 3.71, 95%CI: 1.04, 13.20; p for trend=0.039). OTM exhibited similar, albeit statistically non-significant, risk estimates (OR{sub 50th percentile} = 2.27, 95%CI: 0.75, 6.87; OR{sub 75th percentile} = 2.40, 95%CI: 0.75, 7.71; p for trend=0.12) whereas tail length and CDM showed no association. In conclusion, the results for tail DNA and OTM indicate that endogenous DNA damage levels are higher in patients who develop nonseminoma compared with seminoma. This may partly explain the more aggressive biology and younger age-of-onset of this histologic subgroup compared with the relatively less aggressive, later-onset seminoma.

  6. Melanin photosensitizes ultraviolet light (UVC) DNA damage in pigmented cells

    International Nuclear Information System (INIS)

    Huselton, C.A.; Hill, H.Z.

    1990-01-01

    Melanins, pigments of photoprotection and camouflage, are very photoreactive and can both absorb and emit active oxygen species. Nevertheless, black skinned individuals rarely develop skin cancer and melanin is assumed to act as a solar screen. Since DNA is the target for solar carcinogenesis, the effect of melanin on Ultraviolet (UV)-induced thymine lesions was examined in mouse melanoma and carcinoma cells that varied in melanin content. Cells prelabeled with 14C-dThd were irradiated with UVC; DNA was isolated, purified, degraded to bases by acid hydrolysis and analyzed by HPLC. Thymine dimers were detected in all of the extracts of irradiated cells. Melanotic and hypomelanotic but not mammary carcinoma cell DNA from irradiated cells contained hydrophilic thymine derivatives. The quantity of these damaged bases was a function of both the UVC dose and the cellular melanin content. One such derivative was identified by gas chromatography-mass spectroscopy as thymine glycol. The other appears to be derived from thymine glycol by further oxidation during acid hydrolysis of the DNA. The finding of oxidative DNA damage in melanin-containing cells suggests that melanin may be implicated in the etiology of caucasian skin cancer, particularly melanoma. Furthermore, the projected decrease in stratospheric ozone could impact in an unanticipated deleterious manner on dark-skinned individuals

  7. Synergy of irofulven in combination with other DNA damaging agents: synergistic interaction with altretamine, alkylating, and platinum-derived agents in the MV522 lung tumor model.

    Science.gov (United States)

    Kelner, Michael J; McMorris, Trevor C; Rojas, Rafael J; Estes, Leita A; Suthipinijtham, Pharnuk

    2008-12-01

    Irofulven (MGI 114, NSC 683863) is a semisynthetic derivative of illudin S, a natural product present in the Omphalotus illudins (Jack O'Lantern) mushroom. This novel agent produces DNA damage, that in contrast to other agents, is predominately ignored by the global genome repair pathway of the nucleotide excision repair (NER)(2) system. The aim of this study was to determine the antitumor activity of irofulven when administered in combination with 44 different DNA damaging agents, whose damage is in general detected and repaired by the genome repair pathway. The human lung carcinoma MV522 cell line and its corresponding xenograft model were used to evaluate the activity of irofulven in combination with different DNA damaging agents. Two main classes of DNA damaging agents, platinum-derived agents, and select bifunctional alkylating agents, demonstrated in vivo synergistic or super-additive interaction with irofulven. DNA helicase inhibiting agents also demonstrated synergy in vitro, but an enhanced interaction with irofulven could not be demonstrated in vivo. There was no detectable synergistic activity between irofulven and agents capable of inducing DNA cleavage or intercalating into DNA. These results indicate that the antitumor activity of irofulven is enhanced when combined with platinum-derived agents, altretamine, and select alkylating agents such as melphalan or chlorambucil. A common factor between these agents appears to be the production of intrastrand DNA crosslinks. The synergistic interaction between irofulven and other agents may stem from the nucleotide excision repair system being selectively overwhelmed at two distinct points in the pathway, resulting in prolonged stalling of transcription forks, and subsequent initiation of apoptosis.

  8. Orchestration of DNA Damage Checkpoint Dynamics across the Human Cell Cycle.

    Science.gov (United States)

    Chao, Hui Xiao; Poovey, Cere E; Privette, Ashley A; Grant, Gavin D; Chao, Hui Yan; Cook, Jeanette G; Purvis, Jeremy E

    2017-11-22

    Although molecular mechanisms that prompt cell-cycle arrest in response to DNA damage have been elucidated, the systems-level properties of DNA damage checkpoints are not understood. Here, using time-lapse microscopy and simulations that model the cell cycle as a series of Poisson processes, we characterize DNA damage checkpoints in individual, asynchronously proliferating cells. We demonstrate that, within early G1 and G2, checkpoints are stringent: DNA damage triggers an abrupt, all-or-none cell-cycle arrest. The duration of this arrest correlates with the severity of DNA damage. After the cell passes commitment points within G1 and G2, checkpoint stringency is relaxed. By contrast, all of S phase is comparatively insensitive to DNA damage. This checkpoint is graded: instead of halting the cell cycle, increasing DNA damage leads to slower S phase progression. In sum, we show that a cell's response to DNA damage depends on its exact cell-cycle position and that checkpoints are phase-dependent, stringent or relaxed, and graded or all-or-none. Copyright © 2017 Elsevier Inc. All rights reserved.

  9. Un-repairable DNA damage in cell due to irradiation

    International Nuclear Information System (INIS)

    Yoshii, Giichi

    1992-01-01

    Radiation-induced cell reproductive deactivation is caused by damage to DNA. In a cell, cellular DNA radical reacts with diffusion controlled rate and generates DNA peroxide radical. The chemical repair of DNA radical with hydrogen donation by thiol competes with the reaction of oxygen with same radicals in the DNA molecules. From the point reaction rates, the prolongation of radical life time is not as great as expected from the reduction in the glutathione content of the cell. This indicates that further reducting compounds (protein bound thiol) are present in the cell. The residual radicals are altered to strand breaks, base damages and so on. The effective lesions for a number of endpoints is un-repaired double strand break, which has been discovered in a cluster. This event gives risk to high LET radiation or to a track end of X-rays. For X- or electron irradiations the strand breaks are frequently induced by the interactions between sublesions on two strands in DNA. A single strand break followed by radical action may be unstable excited state, because of remaining sugar radical action and of having negative charged phosphates, in which strands breaks will be rejoined in a short time to stable state. On the same time, a break in the double helix will be immediately produced if two breaks are on either or approximately opposite locations. The formation of a double strand break in the helix depends on the ion strength of the cell. The potassium ions are largely released from polyanionic strand during irradiation, which results in the induction of denatured region. Double strand break with the denatured region seems to be un-repairable DNA damage. (author)

  10. Repair of DNA damage induced by ionizing radiation and benzo[a]pyrene in mammalian cells

    International Nuclear Information System (INIS)

    Cerutti, P.; Shinohara, K.; Remsen, J.

    1977-01-01

    The biological effects of DNA-damaging agents are codetermined by the structural characteristics of the lesions, the quality and extent of the local distortion of DNA and chromatin structure, and the mode(s) of damage processing used by a given type of cell. Persistent damage (i.e., damage that is not removed before it is reached by DNA replication) may be mostly responsible for mutagenesis and carcinogenesis. To understand the effects of environmental physical and chemical DNA-damaging agents on human health, the mechanisms of damage processing used by human cells have to be elucidated. We report our studies of the excision of gamma-ray products of the 5,6-dihydroxydihydrothymine type (t 0 /sub 2//sup γ/) in normal human fibroblasts and in fibroblasts from patients with the hereditary diseases Fanconi's anemia (FA) and ataxia telangiectasia (AT). Both diseases are characterized by chromosomal instability and increased susceptibility for the development of cancer. Formation and repair of DNA-benzo[a]pyrene adducts were studied in baby hamster kidney cells, secondary mouse embryo cells, and human lymphoma. The relative persistence of DNA-B[a]P may explain the high mutagenicity of the 7,8-dihydroxy-9,10-epoxy-tetrahydrobenzo[a]pyrene metabolites in rodent cells that has been observed by several investigators

  11. Bisphenol a promotes cell survival following oxidative DNA damage in mouse fibroblasts.

    Directory of Open Access Journals (Sweden)

    Natalie R Gassman

    Full Text Available Bisphenol A (BPA is a biologically active industrial chemical used in production of consumer products. BPA has become a target of intense public scrutiny following concerns about its association with human diseases such as obesity, diabetes, reproductive disorders, and cancer. Recent studies link BPA with the generation of reactive oxygen species, and base excision repair (BER is responsible for removing oxidatively induced DNA lesions. Yet, the relationship between BPA and BER has yet to be examined. Further, the ubiquitous nature of BPA allows continuous exposure of the human genome concurrent with the normal endogenous and exogenous insults to the genome, and this co-exposure may impact the DNA damage response and repair. To determine the effect of BPA exposure on base excision repair of oxidatively induced DNA damage, cells compromised in double-strand break repair were treated with BPA alone or co-exposed with either potassium bromate (KBrO3 or laser irradiation as oxidative damaging agents. In experiments with KBrO3, co-treatment with BPA partially reversed the KBrO3-induced cytotoxicity observed in these cells, and this was coincident with an increase in guanine base lesions in genomic DNA. The improvement in cell survival and the increase in oxidatively induced DNA base lesions were reminiscent of previous results with alkyl adenine DNA glycosylase-deficient cells, suggesting that BPA may prevent initiation of repair of oxidized base lesions. With laser irradiation-induced DNA damage, treatment with BPA suppressed DNA repair as revealed by several indicators. These results are consistent with the hypothesis that BPA can induce a suppression of oxidized base lesion DNA repair by the base excision repair pathway.

  12. Polyphosphate is a key factor for cell survival after DNA damage in eukaryotic cells.

    Science.gov (United States)

    Bru, Samuel; Samper-Martín, Bàrbara; Quandt, Eva; Hernández-Ortega, Sara; Martínez-Laínez, Joan M; Garí, Eloi; Rafel, Marta; Torres-Torronteras, Javier; Martí, Ramón; Ribeiro, Mariana P C; Jiménez, Javier; Clotet, Josep

    2017-09-01

    Cells require extra amounts of dNTPs to repair DNA after damage. Polyphosphate (polyP) is an evolutionary conserved linear polymer of up to several hundred inorganic phosphate (Pi) residues that is involved in many functions, including Pi storage. In the present article, we report on findings demonstrating that polyP functions as a source of Pi when required to sustain the dNTP increment essential for DNA repair after damage. We show that mutant yeast cells without polyP produce less dNTPs upon DNA damage and that their survival is compromised. In contrast, when polyP levels are ectopically increased, yeast cells become more resistant to DNA damage. More importantly, we show that when polyP is reduced in HEK293 mammalian cell line cells and in human dermal primary fibroblasts (HDFa), these cells become more sensitive to DNA damage, suggesting that the protective role of polyP against DNA damage is evolutionary conserved. In conclusion, we present polyP as a molecule involved in resistance to DNA damage and suggest that polyP may be a putative target for new approaches in cancer treatment or prevention. Copyright © 2017 Elsevier B.V. All rights reserved.

  13. Link between DNA damage and centriole disengagement/reduplication in untransformed human cells.

    Science.gov (United States)

    Douthwright, Stephen; Sluder, Greenfield

    2014-10-01

    The radiation and radiomimetic drugs used to treat human tumors damage DNA in both cancer cells and normal proliferating cells. Centrosome amplification after DNA damage is well established for transformed cell types but is sparsely reported and not fully understood in untransformed cells. We characterize centriole behavior after DNA damage in synchronized untransformed human cells. One hour treatment of S phase cells with the radiomimetic drug, Doxorubicin, prolongs G2 by at least 72 h, though 14% of the cells eventually go through mitosis in that time. By 72 h after DNA damage we observe a 52% incidence of centriole disengagement plus a 10% incidence of extra centrioles. We find that either APC/C or Plk activities can disengage centrioles after DNA damage, though they normally work in concert. All disengaged centrioles are associated with γ-tubulin and maturation markers and thus, should in principle be capable of reduplicating and organizing spindle poles. The low incidence of reduplication of disengaged centrioles during G2 is due to the p53-dependent expression of p21 and the consequent loss of Cdk2 activity. We find that 26% of the cells going through mitosis after DNA damage contain disengaged or extra centrioles. This could produce genomic instability through transient or persistent spindle multipolarity. Thus, for cancer patients the use of DNA damaging therapies raises the chances of genomic instability and evolution of transformed characteristics in proliferating normal cell populations. © 2014 Wiley Periodicals, Inc.

  14. DNA damage in male gonad cells of Green mussel (Perna viridis) upon exposure to tobacco products

    Digital Repository Service at National Institute of Oceanography (India)

    Nagarajappa; Ganguly, A.; Goswami, U.

    DNA damage (determined by the Comet Assay) and the occurrence of deformed nuclei were measured as endpoints of genotoxicity in male gonad cells of the marine mussel (Perna viridis). Upon exposure of the organism to varying concentrations...

  15. PKC 412 sensitizes U1810 non-small cell lung cancer cells to DNA damage

    International Nuclear Information System (INIS)

    Hemstroem, Therese H.; Joseph, Bertrand; Schulte, Gunnar; Lewensohn, Rolf; Zhivotovsky, Boris

    2005-01-01

    Non-small cell lung carcinoma (NSCLC) is characterized by resistance to drug-induced apoptosis, which might explain the survival of lung cancer cells following treatment. Recently we have shown that the broad-range kinase inhibitor staurosporine (STS) reactivates the apoptotic machinery in U1810 NSCLC cells [Joseph et al., Oncogene 21 (2002) 65]. Lately, several STS analogs that are more specific in kinase inhibition have been suggested for tumor treatment. In this study the apoptosis-inducing ability of the STS analogs PKC 412 and Ro 31-8220 used alone or in combination with DNA-damaging agents in U1810 cells was investigated. In these cells Ro 31-8220 neither induced apoptosis when used alone, nor sensitized cells to etoposide treatment. PKC 412 as a single agent induced death of a small number of U1810 cells, whereas it efficiently triggered a dose- and time-dependent apoptosis in U1285 small cell lung carcinoma cells. In both cell types PKC 412 triggered release of mitochondrial proteins followed by caspase activation. However, concomitant activation of a caspase-independent pathway was essential to kill NSCLC cells. Importantly, PKC 412 was able to sensitize etoposide- and radiation-induced death of U1810 cells. The best sensitization was achieved when PKC 412 was administered 24 h after treatments. In U1810 cells, Ro 31-8220 decreased PMA-induced ERK phosphorylation as efficiently as PKC 412, indicating that the failure of Ro 31-8220 to induce apoptosis was not due to weaker inhibition of conventional and novel PKC isoforms. However, Ro 31-8220 increased the basal level of ERK and Akt phosphorylation in both cell lines, whereas Akt phosphorylation was suppressed in the U1810 cells, which might influence apoptosis. These results suggest that PKC 412 could be a useful tool in increasing the efficiency of therapy of NSCLC

  16. Processing of radiation-induced clustered DNA damage generates DSB in mammalian cells

    International Nuclear Information System (INIS)

    Gulston, M.K.; De Lara, C.M.; Davis, E.L.; Jenner, T.J.; O'Neill, P.

    2003-01-01

    Full text: Clustered DNA damage sites, in which two or more lesions are formed within a few helical turns of the DNA after passage of a single radiation track, are signatures of DNA modifications induced by ionizing radiation in mammalian cell. With 60 Co-radiation, the abundance of clustered DNA damage induced in CHO cells is ∼4x that of prompt double strand breaks (DSB) determined by PFGE. Less is known about the processing of non-DSB clustered DNA damage induced in cells. To optimize observation of any additional DSB formed during processing of DNA damage at 37 deg C, xrs-5 cells deficient in non-homologous end joining were used. Surprisingly, ∼30% of the DSB induced by irradiation at 37 deg C are rejoined within 4 minutes in both mutant and wild type cells. No significant mis-repair of these apparent DSB was observed. It is suggested that a class of non-DSB clustered DNA damage is formed which repair correctly within 4 min but, if 'trapped' prior to repair, are converted into DSB during the lysis procedure of PFGE. However at longer times, a proportion of non-DSB clustered DNA damage sites induced by γ-radiation are converted into DSB within ∼30 min following post-irradiation incubation at 37 deg C. The corresponding formation of additional DSB was not apparent in wild type CHO cells. From these observations, it is estimated that only ∼10% of the total yield of non DSB clustered DNA damage sites are converted into DSB through cellular processing. The biological consequences that the majority of non-DSB clustered DNA damage sites are not converted into DSBs may be significant even at low doses, since a finite chance exists of these clusters being formed in a cell by a single radiation track

  17. DNA-damaging agents stimulate gene expression at specific loci in Escherichia coli

    Energy Technology Data Exchange (ETDEWEB)

    Kenyon, C.J.; Walker, G.C.

    1988-05-01

    Operon fusions in Escherichia coli were obtained that showed increased beta-galactosidase expression in response to treatment with the DNA-damaging agent mitomycin C. These fusions were generated by using the Mud(ApR, lac) vector to insert the lactose structural genes randomly into the bacterial chromosome. Induction of beta-galactosidase in these strains, which carried fusions of lac to these din (damage-inducible) loci, was (i) triggered by UV light as well as by mitomycin C and (ii) abolished by either a recA- or a lexA- mutation. Similar characteristics of induction were observed when the lactose genes were fused to a prophage lambda promoter by using Mud(ApR, lac). These results indicate that E. coli contains a set of genes that, like prophage lambda genes, are expressed in response to DNA-damaging agents and regulated by the recA and lexA gene products. These din genes map at five bacterial loci. One din::Mud(ApR, lac) insertion results in a UV-sensitive phenotype and may be within the uvrA transcriptional unit.

  18. Increased DNA damage in blood cells of rat treated with lead as assessed by comet assay

    Directory of Open Access Journals (Sweden)

    Mohammad Arif

    2008-06-01

    Full Text Available A growing body of evidence suggests that oxidative stress is the key player in the pathogenesis of lead-induced toxicity. The present study investigated lead induced oxidative DNA damage, if any in rat blood cells by alkaline comet assay. Lead was administered intraperitoneally to rats at doses of 25, 50 and 100 mg/kg body weight for 5 days consecutively. Blood collected on day six from sacrificed lead-treated rats was used to assess the extent of DNA damage by comet assay which entailed measurement of comet length, olive tail moment, tail DNA (% and tail length. The results showed that treatment with lead significantly increased DNA damage in a dose-dependent manner. Therefore, our data suggests that lead treatment is associated with oxidative stress-induced DNA damage in rat blood cells which could be used as an early bio-marker of lead-toxicity.

  19. ATM-activated autotaxin (ATX) propagates inflammation and DNA damage in lung epithelial cells: a new mode of action for silica-induced DNA damage?

    Science.gov (United States)

    Zheng, Huiyuan; Högberg, Johan; Stenius, Ulla

    2017-12-07

    Silica exposure is a common risk factor for lung cancer. It has been claimed that key elements in cancer development are activation of inflammatory cells that indirectly induce DNA damage and proliferative stimuli in respiratory epithelial cells. We studied DNA damage induced by silica particles in respiratory epithelial cells and focused the role of the signaling enzyme autotaxin (ATX). A549 and 16 bronchial epithelial cells (16HBE) lung epithelial cells were exposed to silica particles. Reactive oxygen species (ROS), NOD-like receptor family pyrin domain containing-3 (NLRP3) inflammasome activation, ATX, ataxia telangiectasia mutated (ATM), and DNA damage (γH2AX, pCHK1, pCHK2, comet assay) were end points. Low doses of silica induced NLRP3 activation, DNA damage accumulation, and ATM phosphorylation. A novel finding was that ATM induced ATX generation and secretion. Not only silica but also rotenone, camptothecin and H2O2 activated ATX via ATM, suggesting that ATX is part of a generalized ATM response to double-strand breaks (DSBs). Surprisingly, ATX inhibition mitigated DNA damage accumulation at later time points (6-16 h), and ATX transfection caused NLRP3 activation and DNA damage. Furthermore, the product of ATX enzymatic activity, lysophosphatidic acid, recapitulated the effects of ATX transfection. These data indicate an ATM-ATX-dependent loop that propagates inflammation and DSB accumulation, making low doses of silica effective inducers of DSBs in epithelial cells. We conclude that an ATM-ATX axis interconnects DSBs with silica-induced inflammation and propagates these effects in epithelial cells. Further studies of this adverse outcome pathway may give an accurate assessment of the lowest doses of silica that causes cancer. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  20. A germline FANCA alteration that is associated with increased sensitivity to DNA damaging agents.

    Science.gov (United States)

    Wilkes, David C; Sailer, Verena; Xue, Hui; Cheng, Hongwei; Collins, Colin C; Gleave, Martin; Wang, Yuzhuo; Demichelis, Francesca; Beltran, Himisha; Rubin, Mark A; Rickman, David S

    2017-09-01

    Defects in genes involved in DNA damage repair (DDR) pathway are emerging as novel biomarkers and targets for new prostate cancer drug therapies. A previous report revealed an association between an exceptional response to cisplatin treatment and a somatic loss of heterozygosity (LOH) of FANCA in a patient with metastatic prostate cancer who also harbored a germline FANCA variant (S1088F). Although germline FANCA mutations are the most frequent alterations in patients with Fanconi anemia, germline alterations are less common in prostate cancer. We hypothesized that the germline S1088F FANCA variant in combination with FANCA LOH was deleterious for FANCA function and contributed to the patient's exceptional response to cisplatin. We show that although it properly localizes to the nucleus, the S1088F FANCA mutant protein disrupts the FANC protein complex resulting in increased sensitivity to DNA damaging agents. Because molecular stratification is emerging as a strategy for treating men with metastatic, castrate-resistant prostate cancer harboring specific DDR gene defects, our findings suggest that more biomarker studies are needed to better define clinically relevant germline and somatic alterations. © 2017 Wilkes et al.; Published by Cold Spring Harbor Laboratory Press.

  1. Ginsenoside Rg3 induces DNA damage in human osteosarcoma cells and reduces MNNG-induced DNA damage and apoptosis in normal human cells.

    Science.gov (United States)

    Zhang, Yue-Hui; Li, Hai-Dong; Li, Bo; Jiang, Sheng-Dan; Jiang, Lei-Sheng

    2014-02-01

    Panax ginseng is a Chinese medicinal herb. Ginsenosides are the main bioactive components of P. ginseng, and ginsenoside Rg3 is the primary ginsenoside. Ginsenosides can potently kill various types of cancer cells. The present study was designed to evaluate the potential genotoxicity of ginsenoside Rg3 in human osteosarcoma cells and the protective effect of ginsenoside Rg3 with respect to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-induced DNA damage and apoptosis in a normal human cell line (human fibroblasts). Four human osteosarcoma cell lines (MG-63, OS732, U-2OS and HOS cells) and a normal human cell line (human fibroblasts) were employed to investigate the cytotoxicity of ginsenosides Rg3 by MTT assay. Alkaline comet assay and γH2AX focus staining were used to detect the DNA damage in MG-63 and U-2OS cells. The extent of cell apoptosis was determined by flow cytometry and a DNA ladder assay. Our results demonstrated that the cytotoxicity of ginsenoside Rg3 was dose-dependent in the human osteosarcoma cell lines, and MG-63 and U-2OS cells were the most sensitive to ginsenoside Rg3. As expected, compared to the negative control, ginsenoside Rg3 significantly increased DNA damage in a concentration-dependent manner. In agreement with the comet assay data, the percentage of γH2AX-positive MG-63 and U-2OS cells indicated that ginsenoside Rg3 induced DNA double-strand breaks in a concentration-dependent manner. The results also suggest that ginsenoside Rg3 reduces the extent of MNNG-induced DNA damage and apoptosis in human fibroblasts.

  2. PRAP1 is a novel executor of p53-dependent mechanisms in cell survival after DNA damage.

    Science.gov (United States)

    Huang, B H; Zhuo, J L; Leung, C H W; Lu, G D; Liu, J J; Yap, C T; Hooi, S C

    2012-12-13

    p53 has a crucial role in governing cellular mechanisms in response to a broad range of genotoxic stresses. During DNA damage, p53 can either promote cell survival by activating senescence or cell-cycle arrest and DNA repair to maintain genomic integrity for cell survival or direct cells to undergo apoptosis to eliminate extensively damaged cells. The ability of p53 to execute these two opposing cell fates depends on distinct signaling pathways downstream of p53. In this study, we showed that under DNA damage conditions induced by chemotherapeutic drugs, gamma irradiation and hydrogen peroxide, p53 upregulates a novel protein, proline-rich acidic protein 1 (PRAP1). We identified functional p53-response elements within intron 1 of PRAP1 gene and showed that these regions interact directly with p53 using ChIP assays, indicating that PRAP1 is a novel p53 target gene. The induction of PRAP1 expression by p53 may promote resistance of cancer cells to chemotherapeutic drugs such as 5-fluorouracil (5-FU), as knockdown of PRAP1 increases apoptosis in cancer cells after 5-FU treatment. PRAP1 appears to protect cells from apoptosis by inducing cell-cycle arrest, suggesting that the induction of PRAP1 expression by p53 in response to DNA-damaging agents contributes to cancer cell survival. Our findings provide a greater insight into the mechanisms underlying the pro-survival role of p53 in response to cytotoxic treatments.

  3. Persistence and dynamics of DNA damage signal amplification determined by microcolony formation and live-cell imaging

    International Nuclear Information System (INIS)

    Oka, Yasuyoshi; Yamauchi, Motohiro; Suzuki, Masatoshi; Yamashita, Shunichi; Suzuki, Keiji

    2011-01-01

    Cell cycle checkpoints are essential cellular process protecting the integrity of the genome from DNA damaging agents. In the present study, we developed a microcolony assay, in which normal human diploid fibroblast-like cells exposed to ionizing radiation, were plated onto coverslips at very low density (3 cells/cm 2 ). Cells were grown for up to 3 days, and phosphorylated ataxia-telangiectasia mutated (ATM) at Ser1981 and 53BP1 foci were analyzed as the markers for an amplified DNA damage signal. We observed a dose-dependent increase in the fraction of non-dividing cells, whose increase was compromised by knocking down p53 expression. While large persistent foci were predominantly formed in non-dividing cells, we observed some growing colonies that contained cells with large foci. As each microcolony was derived from a single cell, it appeared that some cells could proliferate with large foci. A live-imaging analysis using hTERT-immortalized normal human diploid cells transfected with the EGFP-tagged 53BP1 gene revealed that the formation of persistent large foci was highly dynamic. Delayed appearance and disappearance of large foci were frequently observed in exposed cells visualized 12-72 hours after X-irradiation. Thus, our results indicate that amplified DNA damage signal could be ignored, which may be explained in part by the dynamic nature of the amplification process. (author)

  4. Phosphoramide mustard exposure induces DNA adduct formation and the DNA damage repair response in rat ovarian granulosa cells

    Energy Technology Data Exchange (ETDEWEB)

    Ganesan, Shanthi, E-mail: shanthig@iastate.edu; Keating, Aileen F., E-mail: akeating@iastate.edu

    2015-02-01

    Phosphoramide mustard (PM), the ovotoxic metabolite of the anti-cancer agent cyclophosphamide (CPA), destroys rapidly dividing cells by forming NOR-G-OH, NOR-G and G-NOR-G adducts with DNA, potentially leading to DNA damage. A previous study demonstrated that PM induces ovarian DNA damage in rat ovaries. To investigate whether PM induces DNA adduct formation, DNA damage and induction of the DNA repair response, rat spontaneously immortalized granulosa cells (SIGCs) were treated with vehicle control (1% DMSO) or PM (3 or 6 μM) for 24 or 48 h. Cell viability was reduced (P < 0.05) after 48 h of exposure to 3 or 6 μM PM. The NOR-G-OH DNA adduct was detected after 24 h of 6 μM PM exposure, while the more cytotoxic G-NOR-G DNA adduct was formed after 48 h by exposure to both PM concentrations. Phosphorylated H2AX (γH2AX), a marker of DNA double stranded break occurrence, was also increased by PM exposure, coincident with DNA adduct formation. Additionally, induction of genes (Atm, Parp1, Prkdc, Xrcc6, and Brca1) and proteins (ATM, γH2AX, PARP-1, PRKDC, XRCC6, and BRCA1) involved in DNA repair were observed in both a time- and dose-dependent manner. These data support that PM induces DNA adduct formation in ovarian granulosa cells, induces DNA damage and elicits the ovarian DNA repair response. - Highlights: • PM forms ovarian DNA adducts. • DNA damage marker γH2AX increased by PM exposure. • PM induces ovarian DNA double strand break repair.

  5. Mechanisms of DNA damage repair in adult stem cells and implications for cancer formation.

    Science.gov (United States)

    Weeden, Clare E; Asselin-Labat, Marie-Liesse

    2018-01-01

    Maintenance of genomic integrity in tissue-specific stem cells is critical for tissue homeostasis and the prevention of deleterious diseases such as cancer. Stem cells are subject to DNA damage induced by endogenous replication mishaps or exposure to exogenous agents. The type of DNA lesion and the cell cycle stage will invoke different DNA repair mechanisms depending on the intrinsic DNA repair machinery of a cell. Inappropriate DNA repair in stem cells can lead to cell death, or to the formation and accumulation of genetic alterations that can be transmitted to daughter cells and so is linked to cancer formation. DNA mutational signatures that are associated with DNA repair deficiencies or exposure to carcinogenic agents have been described in cancer. Here we review the most recent findings on DNA repair pathways activated in epithelial tissue stem and progenitor cells and their implications for cancer mutational signatures. We discuss how deep knowledge of early molecular events leading to carcinogenesis provides insights into DNA repair mechanisms operating in tumours and how these could be exploited therapeutically. Copyright © 2017 Elsevier B.V. All rights reserved.

  6. Apple Flavonoids Suppress Carcinogen-Induced DNA Damage in Normal Human Bronchial Epithelial Cells

    Directory of Open Access Journals (Sweden)

    Vazhappilly Cijo George

    2017-01-01

    Full Text Available Scope. Human neoplastic transformation due to DNA damage poses an increasing global healthcare concern. Maintaining genomic integrity is crucial for avoiding tumor initiation and progression. The present study aimed to investigate the efficacy of an apple flavonoid fraction (AF4 against various carcinogen-induced toxicity in normal human bronchial epithelial cells and its mechanism of DNA damage response and repair processes. Methods and Results. AF4-pretreated cells were exposed to nicotine-derived nitrosamine ketones (NNK, NNK acetate (NNK-Ae, methotrexate (MTX, and cisplatin to validate cytotoxicity, total reactive oxygen species, intracellular antioxidants, DNA fragmentation, and DNA tail damage. Furthermore, phosphorylated histone (γ-H2AX and proteins involved in DNA damage (ATM/ATR, Chk1, Chk2, and p53 and repair (DNA-PKcs and Ku80 mechanisms were evaluated by immunofluorescence and western blotting, respectively. The results revealed that AF4-pretreated cells showed lower cytotoxicity, total ROS generation, and DNA fragmentation along with consequent inhibition of DNA tail moment. An increased level of γ-H2AX and DNA damage proteins was observed in carcinogen-treated cells and that was significantly (p≤0.05 inhibited in AF4-pretreated cells, in an ATR-dependent manner. AF4 pretreatment also facilitated the phosphorylation of DNA-PKcs and thus initiation of repair mechanisms. Conclusion. Apple flavonoids can protect in vitro oxidative DNA damage and facilitate repair mechanisms.

  7. Persistence of DNA damage following exposure of human bladder cells to chronic monomethylarsonous acid

    International Nuclear Information System (INIS)

    Wnek, S.M.; Medeiros, M.K.; Eblin, K.E.; Gandolfi, A.J.

    2009-01-01

    Malignant transformation was demonstrated in UROtsa cells following 52-weeks of exposure to 50 nM monomethylarsonous acid (MMA III ); the result was the malignantly transformed cell line, URO-MSC. URO-MSC cells were used to study the induction of DNA damage and the alteration of DNA repair enzymes in both the presence of MMA III [URO-MSC(+)] and after subsequent removal of MMA III [URO-MSC(-)] following chronic, low-level exposure. In the presence of MMA III , URO-MSC(+) cells demonstrated a sustained increase in DNA damage following 12-weeks of exposure; in particular, a significant increase in DNA single-strand breaks at 12-weeks of exposure consistently elevated through 52 weeks. The persistence of DNA damage in URO-MSC cells was assessed after a 2-week removal of MMA III . URO-MSC(-) cells demonstrated a decrease in DNA damage compared to URO-MSC(+); however, DNA damage in URO-MSC(-) remained significantly elevated when compared to untreated UROtsa and increased in a time-dependent manner. Reactive oxygen species (ROS) were demonstrated to be a critical component in the generation of DNA damage determined through the incubation of ROS scavengers with URO-MSC cells. Poly (ADP-ribose) polymerase (PARP) is a key repair enzyme in DNA single-strand break repair. URO-MSC(+) resulted in a slight increase in PARP activity after 36-weeks of MMA III exposure, suggesting the presence of MMA III is inhibiting the increase in PARP activity. In support, PARP activity in URO-MSC(-) increased significantly, coinciding with a subsequent decrease in DNA damage demonstrated in URO-MSC(-) compared to URO-MSC(+). These data demonstrate that chronic, low-level exposure of UROtsa cells to 50 nM MMA III results in: the induction of DNA damage that remains elevated upon removal of MMA III ; increased levels of ROS that play a role in MMA III induced-DNA damage; and decreased PARP activity in the presence of MMA III .

  8. Human embryonic stem cells have enhanced repair of multiple forms of DNA damage

    DEFF Research Database (Denmark)

    Maynard, Scott; Swistowska, Anna Maria; Lee, Jae Wan

    2008-01-01

    cells compared with various differentiated murine cells. Using single-cell gel electrophoresis (comet assay) we found that human embryonic stem cells (BG01, I6) have more efficient repair of different types of DNA damage (generated from H2O2, UV-C, ionizing radiation, or psoralen) than human primary...

  9. Non-homologous end joining pathway is the major route of protection against 4β-hydroxywithanolide E-induced DNA damage in MCF-7 cells.

    Science.gov (United States)

    You, B-J; Wu, Y-C; Lee, C-L; Lee, H-Z

    2014-03-01

    4β-Hydroxywithanolide E is a bioactive withanolide extracted from Physalis peruviana. 4β-Hydroxywithanolide E caused reactive oxygen species production and cell apoptosis in human breast cancer MCF-7 cells. We further found that 4β-hydroxywithanolide E induced DNA damage and regulated the DNA damage signaling in MCF-7 cells. The DNA damage sensors and repair proteins act promptly to remove DNA lesions by 4β-hydroxywithanolide E. The ataxia-telangiectasia mutated protein (ATM)-dependent DNA damage signaling pathway is involved in 4β-hydroxywithanolide E-induced apoptosis of MCF-7 cells. Non-homologous end joining pathway, but not homologous recombination, is the major route of protection of MCF-7 cells against 4β-hydroxywithanolide E-induced DNA damage. 4β-Hydroxywithanolide E had no significant impact on the base excision repair pathway. In this study, we examined the 4β-hydroxywithanolide E-induced DNA damage as a research tool in project investigating the DNA repair signaling in breast cancer cells. We also suggest that 4β-hydroxywithanolide E assert its anti-tumor activity in carcinogenic progression and develop into a dietary chemopreventive agent. Copyright © 2014 Elsevier Ltd. All rights reserved.

  10. AtMMS21, an SMC5/6 complex subunit, is involved in stem cell niche maintenance and DNA damage responses in Arabidopsis roots.

    Science.gov (United States)

    Xu, Panglian; Yuan, Dongke; Liu, Ming; Li, Chunxin; Liu, Yiyang; Zhang, Shengchun; Yao, Nan; Yang, Chengwei

    2013-04-01

    Plants maintain stem cells in meristems to sustain lifelong growth; these stem cells must have effective DNA damage responses to prevent mutations that can propagate to large parts of the plant. However, the molecular links between stem cell functions and DNA damage responses remain largely unexplored. Here, we report that the small ubiquitin-related modifier E3 ligase AtMMS21 (for methyl methanesulfonate sensitivity gene21) acts to maintain the root stem cell niche by mediating DNA damage responses in Arabidopsis (Arabidopsis thaliana). Mutation of AtMMS21 causes defects in the root stem cell niche during embryogenesis and postembryonic stages. AtMMS21 is essential for the proper expression of stem cell niche-defining transcription factors. Moreover, mms21-1 mutants are hypersensitive to DNA-damaging agents, have a constitutively increased DNA damage response, and have more DNA double-strand breaks (DSBs) in the roots. Also, mms21-1 mutants exhibit spontaneous cell death within the root stem cell niche, and treatment with DSB-inducing agents increases this cell death, suggesting that AtMMS21 is required to prevent DSB-induced stem cell death. We further show that AtMMS21 functions as a subunit of the STRUCTURAL MAINTENANCE OF CHROMOSOMES5/6 complex, an evolutionarily conserved chromosomal ATPase required for DNA repair. These data reveal that AtMMS21 acts in DSB amelioration and stem cell niche maintenance during Arabidopsis root development.

  11. Poly(ADP-ribose) synthesis following DNA damage in cells heterozygous or homozygous for the xeroderma pigmentosum genotype

    International Nuclear Information System (INIS)

    McCurry, L.S.; Jacobson, M.K.

    1981-01-01

    Treatment of normal human cells with DNA-damaging agents such as uv light or N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) stimulates the conversion of NAD to the chromosomal polymer poly(ADP-ribose) which in turn results in a rapid depletion of the cellular NAD pool. The effect of uv light or MNNG on the NAD pools of seven cell lines of human fibroblasts either homozygous or heterozygous for the xeroderma pigmentosum genotype has been studied. Xeroderma pigmentosum cells of genetic complementation groups A, C, and D are deficient in the excision repair of DNA damage caused by uv light. Following uv treatment, the NAD content of these cells was unchanged or only slightly reduced. All of the cell lines are able to excise DNA damage caused by MNNG and all of the cell lines had a greatly reduced content of NAD following MNNG treatment. The results demonstrate a close relationship between the conversion of NAD to poly(ADP-ribose) and DNA excision repair in human cells

  12. Differentiation of Human Induced Pluripotent or Embryonic Stem Cells Decreases the DNA Damage Repair by Homologous Recombination

    Directory of Open Access Journals (Sweden)

    Kalpana Mujoo

    2017-11-01

    Full Text Available The nitric oxide (NO-cyclic GMP pathway contributes to human stem cell differentiation, but NO free radical production can also damage DNA, necessitating a robust DNA damage response (DDR to ensure cell survival. How the DDR is affected by differentiation is unclear. Differentiation of stem cells, either inducible pluripotent or embryonic derived, increased residual DNA damage as determined by γ-H2AX and 53BP1 foci, with increased S-phase-specific chromosomal aberration after exposure to DNA-damaging agents, suggesting reduced homologous recombination (HR repair as supported by the observation of decreased HR-related repair factor foci formation (RAD51 and BRCA1. Differentiated cells also had relatively increased fork stalling and R-loop formation after DNA replication stress. Treatment with NO donor (NOC-18, which causes stem cell differentiation has no effect on double-strand break (DSB repair by non-homologous end-joining but reduced DSB repair by HR. Present studies suggest that DNA repair by HR is impaired in differentiated cells.

  13. Inhibition of HAS2 induction enhances the radiosensitivity of cancer cells via persistent DNA damage

    International Nuclear Information System (INIS)

    Shen, Yan Nan; Shin, Hyun-Jin; Joo, Hyun-Yoo; Park, Eun-Ran; Kim, Su-Hyeon; Hwang, Sang-Gu; Park, Sang Jun; Kim, Chun-Ho; Lee, Kee-Ho

    2014-01-01

    Highlights: •HAS2 may be a promising target for the radiosensitization of human cancer. •HAS2 is elevated (up to ∼10-fold) in irradiated radioresistant and -sensitive cancer cells. •HAS2 knockdown sensitizes cancer cells to radiation. •HAS2 knockdown potentiates irradiation-induced DNA damage and apoptotic death. •Thus, the irradiation-induced up-regulation of HAS2 contributes to the radioresistance of cancer cells. -- Abstract: Hyaluronan synthase 2 (HAS2), a synthetic enzyme for hyaluronan, regulates various aspects of cancer progression, including migration, invasion and angiogenesis. However, the possible association of HAS2 with the response of cancer cells to anticancer radiotherapy, has not yet been elucidated. Here, we show that HAS2 knockdown potentiates irradiation-induced DNA damage and apoptosis in cancer cells. Upon exposure to radiation, all of the tested human cancer cell lines exhibited marked (up to 10-fold) up-regulation of HAS2 within 24 h. Inhibition of HAS2 induction significantly reduced the survival of irradiated radioresistant and -sensitive cells. Interestingly, HAS2 depletion rendered the cells to sustain irradiation-induced DNA damage, thereby leading to an increase of apoptotic death. These findings indicate that HAS2 knockdown sensitizes cancer cells to radiation via persistent DNA damage, further suggesting that the irradiation-induced up-regulation of HAS2 contributes to the radioresistance of cancer cells. Thus, HAS2 could potentially be targeted for therapeutic interventions aimed at radiosensitizing cancer cells

  14. Cellular Response to Bleomycin-Induced DNA Damage in Human Fibroblast Cells in Space

    Science.gov (United States)

    Lu, Tao; Zhang, Ye; Wong, Michael; Stodieck, Louis; Karouia, Fathi; Wu, Honglu

    2015-01-01

    Outside the protection of the geomagnetic field, astronauts and other living organisms are constantly exposed to space radiation that consists of energetic protons and other heavier charged particles. Whether spaceflight factors, microgravity in particular, have effects on cellular responses to DNA damage induced by exposure to radiation or cytotoxic chemicals is still unknown, as is their impact on the radiation risks for astronauts and on the mutation rate in microorganisms. Although possible synergistic effects of space radiation and other spaceflight factors have been investigated since the early days of the human space program, the published results were mostly conflicting and inconsistent. To investigate effects of spaceflight on cellular responses to DNA damages, human fibroblast cells flown to the International Space Station (ISS) were treated with bleomycin for three hours in the true microgravity environment, which induced DNA damages including double-strand breaks (DSB) similar to the ionizing radiation. Damages in the DNA were measured by the phosphorylation of a histone protein H2AX (g-H2AX), which showed slightly more foci in the cells on ISS than in the ground control. The expression of genes involved in DNA damage response was also analyzed using the PCR array. Although a number of the genes, including CDKN1A and PCNA, were significantly altered in the cells after bleomycin treatment, no significant difference in the expression profile of DNA damage response genes was found between the flight and ground samples. At the time of the bleomycin treatment, the cells on the ISS were found to be proliferating faster than the ground control as measured by the percentage of cells containing positive Ki-67 signals. Our results suggested that the difference in g-H2AX focus counts between flight and ground was due to the faster growth rate of the cells in space, but spaceflight did not affect initial transcriptional responses of the DNA damage response genes to

  15. Cells Lacking mtDNA Display Increased dNTP Pools upon DNA Damage

    DEFF Research Database (Denmark)

    Skovgaard, Tine; Rasmussen, Lene Juel; Munch-Petersen, Birgitte

    Imbalanced dNTP pools are highly mutagenic due to a deleterious effect on DNA polymerase fidelity. Mitochondrial DNA defects, including mutations and deletions, are commonly found in a wide variety of different cancer types. In order to further study the interconnection between dNTP pools...... and mitochondrial function we have examined the effect of DNA damage on dNTP pools in cells deficient of mtDNA. We show that DNA damage induced by UV irradiation, in a dose corresponding to LD50, induces an S phase delay in different human osteosarcoma cell lines. The UV pulse also has a destabilizing effect...... shows that normal mitochondrial function is prerequisite for retaining stable dNTP pools upon DNA damage. Therefore it is likely that mitochondrial deficiency defects may cause an increase in DNA mutations by disrupting dNTP pool balance....

  16. Assessment of DNA damage in radiation workers by using single cell gel electrophoresis

    International Nuclear Information System (INIS)

    Jia Lili; Zhang Tao; Yang Yonghua; Wang Yan; Du Liqing; Cao Jia; Wang Hong; Liu Qiang; Fan Feiyue

    2010-01-01

    Objective: To assess the DNA damage of radiation workers in different grade hospitals, and to explore the correlation between the types of work or work time and the levels of DNA damage. Methods: DNA single strand break were detected by using alkaline single cell gel electrophoresis (SCGE), and the comet was analyzed with CASP (Comet Assay Software Project). TDNA%, TL, TM and OTM were calculated. Results: The parameters of SCGE in the radiation group were higher than those of control group (F=3.93, P<0.01). The significant difference was found not only among the different types of work or different work time, but also among the different grade hospitals (F=1.83, 1.91, P<0.05). Conclusions: Various levels of DNA damage could be detected in the radiation workers of the two hospitals. DNA damage of radiation workers is less serious in the higher-grade hospital than the lower grade one. Different types of work or work time might affect the DNA damage level. (authors)

  17. Essential Function of Dicer in Resolving DNA Damage in the Rapidly Dividing Cells of the Developing and Malignant Cerebellum

    Directory of Open Access Journals (Sweden)

    Vijay Swahari

    2016-01-01

    Full Text Available Maintenance of genomic integrity is critical during neurodevelopment, particularly in rapidly dividing cerebellar granule neuronal precursors that experience constitutive replication-associated DNA damage. As Dicer was recently recognized to have an unexpected function in the DNA damage response, we examined whether Dicer was important for preserving genomic integrity in the developing brain. We report that deletion of Dicer in the developing mouse cerebellum resulted in the accumulation of DNA damage leading to cerebellar progenitor degeneration, which was rescued with p53 deficiency; deletion of DGCR8 also resulted in similar DNA damage and cerebellar degeneration. Dicer deficiency also resulted in DNA damage and death in other rapidly dividing cells including embryonic stem cells and the malignant cerebellar progenitors in a mouse model of medulloblastoma. Together, these results identify an essential function of Dicer in resolving the spontaneous DNA damage that occurs during the rapid proliferation of developmental progenitors and malignant cells.

  18. Resistance to DNA Damaging Agents Produced Invasive Phenotype of Rat Glioma Cells—Characterization of a New in Vivo Model

    Directory of Open Access Journals (Sweden)

    Sonja Stojković

    2016-06-01

    Full Text Available Chemoresistance and invasion properties are severe limitations to efficient glioma therapy. Therefore, development of glioma in vivo models that more accurately resemble the situation observed in patients emerges. Previously, we established RC6 rat glioma cell line resistant to DNA damaging agents including antiglioma approved therapies such as 3-bis(2-chloroethyl-1-nitrosourea (BCNU and temozolomide (TMZ. Herein, we evaluated the invasiveness of RC6 cells in vitro and in a new orthotopic animal model. For comparison, we used C6 cells from which RC6 cells originated. Differences in cell growth properties were assessed by real-time cell analyzer. Cells’ invasive potential in vitro was studied in fluorescently labeled gelatin and by formation of multicellular spheroids in hydrogel. For animal studies, fluorescently labeled cells were inoculated into adult male Wistar rat brains. Consecutive coronal and sagittal brain sections were analyzed 10 and 25 days post-inoculation, while rats’ behavior was recorded during three days in the open field test starting from 25th day post-inoculation. We demonstrated that development of chemoresistance induced invasive phenotype of RC6 cells with significant behavioral impediments implying usefulness of orthotopic RC6 glioma allograft in preclinical studies for the examination of new approaches to counteract both chemoresistance and invasion of glioma cells.

  19. Xeroderma Pigmentosum Group C Deficiency Alters Cigarette Smoke DNA Damage Cell Fate and Accelerates Emphysema Development.

    Science.gov (United States)

    Sears, Catherine R; Zhou, Huaxin; Justice, Matthew J; Fisher, Amanda J; Saliba, Jacob; Lamb, Isaac; Wicker, Jessica; Schweitzer, Kelly S; Petrache, Irina

    2018-03-01

    Cigarette smoke (CS) exposure is a major risk factor for the development of emphysema, a common disease characterized by loss of cells comprising the lung parenchyma. The mechanisms of cell injury leading to emphysema are not completely understood but are thought to involve persistent cytotoxic or mutagenic DNA damage induced by CS. Using complementary cell culture and mouse models of CS exposure, we investigated the role of the DNA repair protein, xeroderma pigmentosum group C (XPC), on CS-induced DNA damage repair and emphysema. Expression of XPC was decreased in mouse lungs after chronic CS exposure and XPC knockdown in cultured human lung epithelial cells decreased their survival after CS exposure due to activation of the intrinsic apoptosis pathway. Similarly, cell autophagy and apoptosis were increased in XPC-deficient mouse lungs and were further increased by CS exposure. XPC deficiency was associated with structural and functional changes characteristic of emphysema, which were worsened by age, similar to levels observed with chronic CS exposure. Taken together, these findings suggest that repair of DNA damage by XPC plays an important and previously unrecognized role in the maintenance of alveolar structures. These findings support that loss of XPC, possibly due to chronic CS exposure, promotes emphysema development and further supports a link between DNA damage, impaired DNA repair, and development of emphysema.

  20. Oxidative DNA damage and mammary cell proliferation by alcohol-derived salsolinol.

    Science.gov (United States)

    Murata, Mariko; Midorikawa, Kaoru; Kawanishi, Shosuke

    2013-10-21

    Drinking alcohol is a risk factor for breast cancer. Salsolinol (SAL) is endogenously formed by a condensation reaction of dopamine with acetaldehyde, a major ethanol metabolite, and SAL is detected in blood and urine after alcohol intake. We investigated the possibility that SAL can participate in tumor initiation and promotion by causing DNA damage and cell proliferation, leading to alcohol-associated mammary carcinogenesis. SAL caused oxidative DNA damage including 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), in the presence of transition metal ions, such as Cu(II) and Fe(III)EDTA. Inhibitory effects of scavengers on SAL-induced DNA damage and the electron spin resonance study indicated the involvement of H₂O₂, which is generated via the SAL radical. Experiments on scavengers and site specificity of DNA damage suggested ·OH generation via a Fenton reaction and copper-peroxide complexes in the presence of Fe(III)EDTA and Cu(II), respectively. SAL significantly increased 8-oxodG formation in normal mammary epithelial MCF-10A cells. In addition, SAL induced cell proliferation in estrogen receptor (ER)-negative MCF-10A cells, and the proliferation was inhibited by an antioxidant N-acetylcysteine and an epidermal growth factor receptor (EGFR) inhibitor AG1478, suggesting that reactive oxygen species may participate in the proliferation of MCF-10A cells via EGFR activation. Furthermore, SAL induced proliferation in estrogen-sensitive breast cancer MCF-7 cells, and a surface plasmon resonance sensor revealed that SAL significantly increased the binding activity of ERα to the estrogen response element but not ERβ. In conclusion, SAL-induced DNA damage and cell proliferation may play a role in tumor initiation and promotion of multistage mammary carcinogenesis in relation to drinking alcohol.

  1. Responses of genes involved in cell cycle control to diverse DNA damaging chemicals in human lung adenocarcinoma A549 cells

    Directory of Open Access Journals (Sweden)

    Gooderham Nigel J

    2005-08-01

    Full Text Available Abstract Background Many anticancer agents and carcinogens are DNA damaging chemicals and exposure to such chemicals results in the deregulation of cell cycle progression. The molecular mechanisms of DNA damage-induced cell cycle alteration are not well understood. We have studied the effects of etoposide (an anticancer agent, cryptolepine (CLP, a cytotoxic alkaloid, benzo [a]pyrene (BaP, a carcinogenic polycyclic aromatic hydrocarbon and 2-amino-1-methyl-6-phenylimidazo [4,5-b]pyridine (PhIP, a cooked-meat derived carcinogen on the expression of cell cycle regulatory genes to understand the molecular mechanisms of the cell cycle disturbance. Results A549 cells were treated with DMSO or chemicals for up to 72 h and periodically sampled for cell cycle analysis, mRNA and protein expression. DMSO treated cells showed a dominant G1 peak in cell cycle at all times examined. Etoposide and CLP both induced G2/M phase arrest yet the former altered the expression of genes functioning at multiple phases, whilst the latter was more effective in inhibiting the expression of genes in G2-M transition. Both etoposide and CLP induced an accumulation of p53 protein and upregulation of p53 transcriptional target genes. Neither BaP nor PhIP had substantial phase-specific cell cycle effect, however, they induced distinctive changes in gene expression. BaP upregulated the expression of CYP1B1 at 6–24 h and downregulated many cell cycle regulatory genes at 48–72 h. By contrast, PhIP increased the expression of many cell cycle regulatory genes. Changes in the expression of key mRNAs were confirmed at protein level. Conclusion Our experiments show that DNA damaging agents with different mechanisms of action induced distinctive changes in the expression pattern of a panel of cell cycle regulatory genes. We suggest that examining the genomic response to chemical exposure provides an exceptional opportunity to understand the molecular mechanism involved in cellular

  2. DNA Damage and Cell Cycle Arrest Induced by Protoporphyrin IX in Sarcoma 180 Cells

    Directory of Open Access Journals (Sweden)

    Qing Li

    2013-09-01

    Full Text Available Background: Porphyrin derivatives have been widely used in photodynamic therapy as effective sensitizers. Protoporphyrin IX (PpIX, a well-known hematoporphyrin derivative component, shows great potential to enhance light induced tumor cell damage. However, PpIX alone could also exert anti-tumor effects. The mechanisms underlying those direct effects are incompletely understood. This study thus investigated the putative mechanisms underlying the anti-tumor effects of PpIX on sarcoma 180 (S180 cells. Methods: S180 cells were treated with different concentrations of PpIX. Following the treatment, cell viability was evaluated by the 3-(4, 5- dimethylthiazol-2-yl-2, 5-diphenyltetrazoliumbromide (MTT assay; Disruption of mitochondrial membrane potential was measured by flow cytometry; The trans-location of apoptosis inducer factor (AIF from mitochondria to nucleus was visualized by confocal laser scanning microscopy; DNA damage was detected by single cell gel electrophoresis; Cell cycle distribution was analyzed by DNA content with flow cytometry; Cell cycle associated proteins were detected by western blotting. Results: PpIX (≥ 1 µg/ml significantly inhibited proliferation and reduced viability of S180 cells in a dose-dependent manner. PpIX rapidly and significantly triggered mitochondrial membrane depolarization, AIF (apoptosis inducer factor translocation from mitochondria to nucleus and DNA damage, effects partially relieved by the specific inhibitor of MPTP (mitochondrial permeability transition pore. Furthermore, S phase arrest and upregulation of the related proteins of P53 and P21 were observed following 12 and 24 h PpIX exposure. Conclusion: PpIX could inhibit tumor cell proliferation by induction of DNA damage and cell cycle arrest in the S phase.

  3. Lovastatin prevents cisplatin-induced activation of pro-apoptotic DNA damage response (DDR) of renal tubular epithelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Krüger, Katharina; Ziegler, Verena; Hartmann, Christina; Henninger, Christian [Institute of Toxicology, Medical Faculty, Heinrich Heine University Düsseldorf, 40225 Düsseldorf (Germany); Thomale, Jürgen [Institute of Cell Biology, University Duisburg-Essen, 45122 Essen (Germany); Schupp, Nicole [Institute of Toxicology, Medical Faculty, Heinrich Heine University Düsseldorf, 40225 Düsseldorf (Germany); Fritz, Gerhard, E-mail: fritz@uni-duesseldorf.de [Institute of Toxicology, Medical Faculty, Heinrich Heine University Düsseldorf, 40225 Düsseldorf (Germany)

    2016-02-01

    The platinating agent cisplatin (CisPt) is commonly used in the therapy of various types of solid tumors. The anticancer efficacy of CisPt largely depends on the formation of bivalent DNA intrastrand crosslinks, which stimulate mechanisms of the DNA damage response (DDR), thereby triggering checkpoint activation, gene expression and cell death. The clinically most relevant adverse effect associated with CisPt treatment is nephrotoxicity that results from damage to renal tubular epithelial cells. Here, we addressed the question whether the HMG-CoA-reductase inhibitor lovastatin affects the DDR of renal cells by employing rat renal proximal tubular epithelial (NRK-52E) cells as in vitro model. The data show that lovastatin has extensive inhibitory effects on CisPt-stimulated DDR of NRK-52E cells as reflected on the levels of phosphorylated ATM, Chk1, Chk2, p53 and Kap1. Mitigation of CisPt-induced DDR by lovastatin was independent of the formation of DNA damage as demonstrated by (i) the analysis of Pt-(GpG) intrastrand crosslink formation by Southwestern blot analyses and (ii) the generation of DNA strand breaks as analyzed on the level of nuclear γH2AX foci and employing the alkaline comet assay. Lovastatin protected NRK-52E cells from the cytotoxicity of high CisPt doses as shown by measuring cell viability, cellular impedance and flow cytometry-based analyses of cell death. Importantly, the statin also reduced the level of kidney DNA damage and apoptosis triggered by CisPt treatment of mice. The data show that the lipid-lowering drug lovastatin extensively counteracts pro-apoptotic signal mechanisms of the DDR of tubular epithelial cells following CisPt injury. - Highlights: • Lovastatin blocks ATM/ATR-regulated DDR of tubular cells following CisPt treatment. • Lovastatin attenuates CisPt-induced activation of protein kinase ATM in vitro. • Statin-mediated DDR inhibition is independent of initial DNA damage formation. • Statin-mediated blockage of Cis

  4. Lovastatin prevents cisplatin-induced activation of pro-apoptotic DNA damage response (DDR) of renal tubular epithelial cells

    International Nuclear Information System (INIS)

    Krüger, Katharina; Ziegler, Verena; Hartmann, Christina; Henninger, Christian; Thomale, Jürgen; Schupp, Nicole; Fritz, Gerhard

    2016-01-01

    The platinating agent cisplatin (CisPt) is commonly used in the therapy of various types of solid tumors. The anticancer efficacy of CisPt largely depends on the formation of bivalent DNA intrastrand crosslinks, which stimulate mechanisms of the DNA damage response (DDR), thereby triggering checkpoint activation, gene expression and cell death. The clinically most relevant adverse effect associated with CisPt treatment is nephrotoxicity that results from damage to renal tubular epithelial cells. Here, we addressed the question whether the HMG-CoA-reductase inhibitor lovastatin affects the DDR of renal cells by employing rat renal proximal tubular epithelial (NRK-52E) cells as in vitro model. The data show that lovastatin has extensive inhibitory effects on CisPt-stimulated DDR of NRK-52E cells as reflected on the levels of phosphorylated ATM, Chk1, Chk2, p53 and Kap1. Mitigation of CisPt-induced DDR by lovastatin was independent of the formation of DNA damage as demonstrated by (i) the analysis of Pt-(GpG) intrastrand crosslink formation by Southwestern blot analyses and (ii) the generation of DNA strand breaks as analyzed on the level of nuclear γH2AX foci and employing the alkaline comet assay. Lovastatin protected NRK-52E cells from the cytotoxicity of high CisPt doses as shown by measuring cell viability, cellular impedance and flow cytometry-based analyses of cell death. Importantly, the statin also reduced the level of kidney DNA damage and apoptosis triggered by CisPt treatment of mice. The data show that the lipid-lowering drug lovastatin extensively counteracts pro-apoptotic signal mechanisms of the DDR of tubular epithelial cells following CisPt injury. - Highlights: • Lovastatin blocks ATM/ATR-regulated DDR of tubular cells following CisPt treatment. • Lovastatin attenuates CisPt-induced activation of protein kinase ATM in vitro. • Statin-mediated DDR inhibition is independent of initial DNA damage formation. • Statin-mediated blockage of Cis

  5. Mycobacterium tuberculosis Ku can bind to nuclear DNA damage and sensitize mammalian cells to bleomycin sulfate.

    Science.gov (United States)

    Castore, Reneau; Hughes, Cameron; Debeaux, Austin; Sun, Jingxin; Zeng, Cailing; Wang, Shih-Ya; Tatchell, Kelly; Shi, Runhua; Lee, Kyung-Jong; Chen, David J; Harrison, Lynn

    2011-11-01

    Radiotherapy and chemotherapy are effective cancer treatments due to their ability to generate DNA damage. The major lethal lesion is the DNA double-strand break (DSB). Human cells predominantly repair DSBs by non-homologous end joining (NHEJ), which requires Ku70, Ku80, DNA-PKcs, DNA ligase IV and accessory proteins. Repair is initiated by the binding of the Ku heterodimer at the ends of the DSB and this recruits DNA-PKcs, which initiates damage signaling and functions in repair. NHEJ also exists in certain types of bacteria that have dormant phases in their life cycle. The Mycobacterium tuberculosis Ku (Mt-Ku) resembles the DNA-binding domain of human Ku but does not have the N- and C-terminal domains of Ku70/80 that have been implicated in binding mammalian NHEJ repair proteins. The aim of this work was to determine whether Mt-Ku could be used as a tool to bind DSBs in mammalian cells and sensitize cells to DNA damage. We generated a fusion protein (KuEnls) of Mt-Ku, EGFP and a nuclear localization signal that is able to perform bacterial NHEJ and hence bind DSBs. Using transient transfection, we demonstrated that KuEnls is able to bind laser damage in the nucleus of Ku80-deficient cells within 10 sec and remains bound for up to 2 h. The Mt-Ku fusion protein was over-expressed in U2OS cells and this increased the sensitivity of the cells to bleomycin sulfate. Hydrogen peroxide and UV radiation do not predominantly produce DSBs and there was little or no change in sensitivity to these agents. Since in vitro studies were unable to detect binding of Mt-Ku to DNA-PKcs or human Ku70/80, this work suggests that KuEnls sensitizes cells by binding DSBs, preventing human NHEJ. This study indicates that blocking or decreasing the binding of human Ku to DSBs could be a method for enhancing existing cancer treatments.

  6. DNA damage and the bystander response in tumor and normal cells exposed to X-rays.

    Science.gov (United States)

    Subhashree, M; Venkateswarlu, R; Karthik, K; Shangamithra, V; Venkatachalam, P

    2017-09-01

    Monolayer and suspension cultures of tumor (BMG-1, CCRF-CEM), normal (AG1522, HADF, lymphocytes) and ATM-mutant (GM4405) human cells were exposed to X-rays at doses used in radiotherapy (high dose and high dose-rate) or radiological imaging (low dose and low dose-rate). Radiation-induced DNA damage, its persistence, and possible bystander effects were evaluated, based on DNA damage markers (γ-H2AX, p53 ser15 ) and cell-cycle-specific cyclins (cyclin B1 and cyclin D1). Dose-dependent DNA damage and a dose-independent bystander response were seen after exposure to high dose and high dose-rate radiation. The level of induced damage (expression of p53 ser15 , γ-H2AX) depended on ATM status. However, low dose and dose-rate exposures neither increased expression of marker proteins nor induced a bystander response, except in the CCRF-CEM cells. Bystander effects after high-dose irradiation may contribute to stochastic and deterministic effects. Precautions to protect unexposed regions or to inhibit transmission of DNA damage signaling might reduce radiation risks. Copyright © 2017 Elsevier B.V. All rights reserved.

  7. Human T-Cell Leukemia Virus I Tax Protein Sensitizes p53-Mutant Cells to DNA Damage

    Science.gov (United States)

    Mihaylova, Valia T.; Green, Allison M.; Khurgel, Moshe; Semmes, Oliver J.; Kupfer, Gary M.

    2018-01-01

    Mutations in p53 are a common cause of resistance of cancers to standard chemotherapy and, thus, treatment failure. Reports have shown that Tax, a human T-cell leukemia virus type I encoded protein that has been associated with genomic instability and perturbation of transcription and cell cycle, sensitizes HeLa cells to UV treatment. The extent to which Tax can sensitize cells and the mechanism by which it exerts its effect are unknown. In this study, we show that Tax sensitizes p53-mutant cells to a broad range of DNA-damaging agents, including mitomycin C, a bifunctional alkylator, etoposide, a topoisomerase II drug, and UV light, but not ionizing radiation, a double-strand break agent, or vinblastine, a tubulin poison. Tax caused hypersensitivity in all p53-deleted cell lines and several, but not all, mutant-expressed p53–containing cell lines, while unexpectedly being protective in p53 wild-type (wt) cells. The effect observed in p53-deleted lines could be reversed for this by transfection of wt p53. We also show that Tax activates a p53-independent proapoptotic program through decreased expression of the retinoblastoma protein and subsequent increased E2F1 expression. The expression of several proapoptotic proteins was also induced by Tax, including Puma and Noxa, culminating in a substantial increase in Bax dimerization. Our results show that Tax can sensitize p53-mutant cells to DNA damage while protecting p53 wt cells, a side benefit that might result in reduced toxicity in normal cells. Such studies hold the promise of a novel adjunctive therapy that could make cancer chemotherapy more effective. PMID:18559532

  8. A model for the induction of DNA damages and their evolution into cell clonogenic inactivation

    International Nuclear Information System (INIS)

    Yamaguchi, Hiroshi; Ohara, Hiroshi; Waker, A.J.

    2006-01-01

    The dependence of the initial production of DNA damages on radiation quality was examined by using a proposed new model on the basis of target theory. For the estimation of DNA damage-production by different radiation qualities, five possible modes of radiation action, including both direct and indirect effects, were assumed inside a target the molecular structure of which was defined to consist of 10 base-pairs of DNA surrounded by water molecules. The induction of DNA damage was modeled on the basis of comparisons between the primary ionization mean free path and the distance between pairs of ionized atoms, such distance being characteristic on the mode of radiation action. The OH radicals per average energy to produce an ion pair on the nanosecond time scale was estimated and used for indirect action. Assuming a relation between estimated yields of DNA damages and experimental inactivation cross sections for AT-cells, the present model enabled the quantitative reproduction of experimental results for AT-cell killing under aerobic or hypoxic conditions. The results suggest a higher order organization of DNA in a way that there will be at least two types of water environment, one filling half the space surrounding DNA with a depth of 3.7-4.3 nm and the other filling all space with a depth 4.6-4.9 nm. (author)

  9. E2F1 and E2F2 induction in response to DNA damage preserves genomic stability in neuronal cells.

    Science.gov (United States)

    Castillo, Daniela S; Campalans, Anna; Belluscio, Laura M; Carcagno, Abel L; Radicella, J Pablo; Cánepa, Eduardo T; Pregi, Nicolás

    2015-01-01

    E2F transcription factors regulate a wide range of biological processes, including the cellular response to DNA damage. In the present study, we examined whether E2F family members are transcriptionally induced following treatment with several genotoxic agents, and have a role on the cell DNA damage response. We show a novel mechanism, conserved among diverse species, in which E2F1 and E2F2, the latter specifically in neuronal cells, are transcriptionally induced after DNA damage. This upregulation leads to increased E2F1 and E2F2 protein levels as a consequence of de novo protein synthesis. Ectopic expression of these E2Fs in neuronal cells reduces the level of DNA damage following genotoxic treatment, while ablation of E2F1 and E2F2 leads to the accumulation of DNA lesions and increased apoptotic response. Cell viability and DNA repair capability in response to DNA damage induction are also reduced by the E2F1 and E2F2 deficiencies. Finally, E2F1 and E2F2 accumulate at sites of oxidative and UV-induced DNA damage, and interact with γH2AX DNA repair factor. As previously reported for E2F1, E2F2 promotes Rad51 foci formation, interacts with GCN5 acetyltransferase and induces histone acetylation following genotoxic insult. The results presented here unveil a new mechanism involving E2F1 and E2F2 in the maintenance of genomic stability in response to DNA damage in neuronal cells.

  10. DNA damage in synchronized hela cells irradiated with ultraviolet

    International Nuclear Information System (INIS)

    Downes, C.S.; Collins, A.R.S.; Johnson, R.T.

    1979-01-01

    The lethal effect of uv radiation on HeLa cells is least in mitosis and greatest in late G 1 -early S. Photochemical damage to HeLa DNA, as measured by thymine-containing dimer formation and by alkaline sucrose sedimentation, also increases from mitosis towards early S phase. Computer simulations of uv absorption by an idealized HeLa cell at different stages of the cell cycle indicate that changes in damage could be due solely to changes in chromatin geometry. But survival is not exclusively a function of damage

  11. Ku70 inhibits gemcitabine-induced DNA damage and pancreatic cancer cell apoptosis

    International Nuclear Information System (INIS)

    Ma, Jiali; Hui, Pingping; Meng, Wenying; Wang, Na; Xiang, Shihao

    2017-01-01

    The current study focused on the role of Ku70, a DNA-dependent protein kinase (DNA-PK) complex protein, in pancreatic cancer cell resistance to gemcitabine. In both established cell lines (Mia-PaCa-2 and PANC-1) and primary human pancreatic cancer cells, shRNA/siRNA-mediated knockdown of Ku70 significantly sensitized gemcitabine-induced cell death and proliferation inhibition. Meanwhile, gemcitabine-induced DNA damage and subsequent pancreatic cancer cell apoptosis were also potentiated with Ku70 knockdown. On the other hand, exogenous overexpression of Ku70 in Mia-PaCa-2 cells suppressed gemcitabine-induced DNA damage and subsequent cell apoptosis. In a severe combined immune deficient (SCID) mice Mia-PaCa-2 xenograft model, gemcitabine-induced anti-tumor activity was remarkably pontificated when combined with Ku70 shRNA knockdown in the xenografts. The results of this preclinical study imply that Ku70 might be a primary resistance factor of gemcitabine, and Ku70 silence could significantly chemo-sensitize gemcitabine in pancreatic cancer cells. - Highlights: • Ku70 knockdown sensitizes gemcitabine-induced killing of pancreatic cancer cells. • Ku70 knockdown facilitates gemcitabine-induced DNA damage and cell apoptosis. • Ku70 overexpression deceases gemcitabine's sensitivity in pancreatic cancer cells. • Ku70 knockdown sensitizes gemcitabine-induced anti-tumor activity in vivo.

  12. rad-Dependent response of the chk1-encoded protein kinase at the DNA damage checkpoint

    NARCIS (Netherlands)

    Walworth, N.C.; Bernards, R.A.

    1996-01-01

    Exposure of eukaryotic cells to agents that generate DNA damage results in transient arrest of progression through the cell cycle. In fission yeast, the DNA damage checkpoint associated with cell cycle arrest before mitosis requires the protein kinase p56chk1. DNA damage induced by ultraviolet

  13. Radioadaptive response. Efficient repair of radiation-induced DNA damage in adapted cells

    International Nuclear Information System (INIS)

    Ikushima, Takaji; Aritomi, Hisako; Morisita, Jun

    1996-01-01

    To verify the hypothesis that the induction of a novel, efficient repair mechanism for chromosomal DNA breaks may be involved in the radioadaptive response, the repair kinetics of DNA damage has been studied in cultured Chinese hamster V79 cells with single-cell gel electrophoresis. The cells were adapted by priming exposure with 5 cGy of γ-rays and 4-h incubation at 37C. There were no indication of any difference in the initial yields of DNA double-strand breaks induced by challenging doses from non-adapted cells and from adapted cells. The rejoining of DNA double-strand breaks was monitored over 120 min after the adapted cells were challenged with 5 or 1.5 Gy, doses at the same level to those used in the cytogenetical adaptive response. The rate of DNA damage repair in adapted cells was higher than that in non-adapted cells, and the residual damage was less in adapted cells than in non-adapted cells. These results indicate that the radioadaptive response may result from the induction of a novel, efficient DNA repair mechanism which leads to less residual damage, but not from the induction of protective functions that reduce the initial DNA damage

  14. Nuclear accumulation and activation of p53 in embryonic stem cells after DNA damage

    Directory of Open Access Journals (Sweden)

    Rolletschek Alexandra

    2009-06-01

    Full Text Available Abstract Background P53 is a key tumor suppressor protein. In response to DNA damage, p53 accumulates to high levels in differentiated cells and activates target genes that initiate cell cycle arrest and apoptosis. Since stem cells provide the proliferative cell pool within organisms, an efficient DNA damage response is crucial. Results In proliferating embryonic stem cells, p53 is localized predominantly in the cytoplasm. DNA damage-induced nuclear accumulation of p53 in embryonic stem cells activates transcription of the target genes mdm2, p21, puma and noxa. We observed bi-phasic kinetics for nuclear accumulation of p53 after ionizing radiation. During the first wave of nuclear accumulation, p53 levels were increased and the p53 target genes mdm2, p21 and puma were transcribed. Transcription of noxa correlated with the second wave of nuclear accumulation. Transcriptional activation of p53 target genes resulted in an increased amount of proteins with the exception of p21. While p21 transcripts were efficiently translated in 3T3 cells, we failed to see an increase in p21 protein levels after IR in embryonal stem cells. Conclusion In embryonic stem cells where (anti-proliferative p53 activity is not necessary, or even unfavorable, p53 is retained in the cytoplasm and prevented from activating its target genes. However, if its activity is beneficial or required, p53 is allowed to accumulate in the nucleus and activates its target genes, even in embryonic stem cells.

  15. Nuclear accumulation and activation of p53 in embryonic stem cells after DNA damage.

    Science.gov (United States)

    Solozobova, Valeriya; Rolletschek, Alexandra; Blattner, Christine

    2009-06-17

    P53 is a key tumor suppressor protein. In response to DNA damage, p53 accumulates to high levels in differentiated cells and activates target genes that initiate cell cycle arrest and apoptosis. Since stem cells provide the proliferative cell pool within organisms, an efficient DNA damage response is crucial. In proliferating embryonic stem cells, p53 is localized predominantly in the cytoplasm. DNA damage-induced nuclear accumulation of p53 in embryonic stem cells activates transcription of the target genes mdm2, p21, puma and noxa. We observed bi-phasic kinetics for nuclear accumulation of p53 after ionizing radiation. During the first wave of nuclear accumulation, p53 levels were increased and the p53 target genes mdm2, p21 and puma were transcribed. Transcription of noxa correlated with the second wave of nuclear accumulation. Transcriptional activation of p53 target genes resulted in an increased amount of proteins with the exception of p21. While p21 transcripts were efficiently translated in 3T3 cells, we failed to see an increase in p21 protein levels after IR in embryonal stem cells. In embryonic stem cells where (anti-proliferative) p53 activity is not necessary, or even unfavorable, p53 is retained in the cytoplasm and prevented from activating its target genes. However, if its activity is beneficial or required, p53 is allowed to accumulate in the nucleus and activates its target genes, even in embryonic stem cells.

  16. Phospho-Ser/Thr-binding domains: navigating the cell cycle and DNA damage response.

    Science.gov (United States)

    Reinhardt, H Christian; Yaffe, Michael B

    2013-09-01

    Coordinated progression through the cell cycle is a complex challenge for eukaryotic cells. Following genotoxic stress, diverse molecular signals must be integrated to establish checkpoints specific for each cell cycle stage, allowing time for various types of DNA repair. Phospho-Ser/Thr-binding domains have emerged as crucial regulators of cell cycle progression and DNA damage signalling. Such domains include 14-3-3 proteins, WW domains, Polo-box domains (in PLK1), WD40 repeats (including those in the E3 ligase SCF(βTrCP)), BRCT domains (including those in BRCA1) and FHA domains (such as in CHK2 and MDC1). Progress has been made in our understanding of the motif (or motifs) that these phospho-Ser/Thr-binding domains connect with on their targets and how these interactions influence the cell cycle and DNA damage response.

  17. Continuous cytokine exposure of colonic epithelial cells induces DNA damage

    DEFF Research Database (Denmark)

    Seidelin, Jakob B; Nielsen, Ole Haagen

    2005-01-01

    tetrazolium bromide (MTT) test. Production of ROS was determined by the oxidation of 2',7'-dichlorodihydrofluorescein to a fluorescent 2',7'-dichlorofluorescein and measured by fluorescence reading and visualized by fluorescence microscopy. DNA stability was determined by single cell gel electrophoresis...

  18. Pedigree analyses of yeast cells recovering from DNA damage allow assignment of lethal events to individual post-treatment generations

    International Nuclear Information System (INIS)

    Klein, F.; Karwan, A.; Wintersberger, U.

    1990-01-01

    Haploid cells of Saccharomyces cerevisiae were treated with different DNA damaging agents at various doses. A study of the progeny of individual such cells allowed the assignment of lethal events to distinct post treatment generations. By microscopically inspecting those cells which were not able to form visible colonies the authors could discriminate between cells dying from immediately effective lethal hits and those generating microcolonies probably as a consequence of lethal mutation(s). The experimentally obtained numbers of lethal events were mathematically transformed into mean probabilities of lethal fixations at taking place in cells of certain post treatment generations. Such analyses give detailed insight into the kinetics of lethality as a consequence of different kinds of DNA damage. For example, X-irradiated cells lost viability mainly by lethal hits, only at a higher dose also lethal mutations fixed in the cells that were in direct contact with the mutagen, but not in later generations, occurred. Ethyl methanesulfonate (EMS)-treated cells were hit by 00-fixations in a dose dependent manner. The distribution of all sorts of lethal fixations taken together, which occurred in the EMS-damaged cell families, was not random. For comparison analyses of cells treated with methyl methanesulfonate, N-methyl-N'-nitro-N-nitrosoguanidine and nitrous acid are also reported

  19. The small molecule calactin induces DNA damage and apoptosis in human leukemia cells.

    Science.gov (United States)

    Lee, Chien-Chih; Lin, Yi-Hsiung; Chang, Wen-Hsin; Wu, Yang-Chang; Chang, Jan-Gowth

    2012-09-01

    We purified calactin from the roots of the Chinese herb Asclepias curassavica L. and analyzed its biologic effects in human leukemia cells. Our results showed that calactin treatment caused DNA damage and resulted in apoptosis. Increased phosphorylation levels of Chk2 and H2AX were observed and were reversed by the DNA damage inhibitor caffeine in calactin-treated cells. In addition, calactin treatment showed that a decrease in the expression of cell cycle regulatory proteins Cyclin B1, Cdk1, and Cdc25C was consistent with a G2/M phase arrest. Furthermore, calactin induced extracellular signal-regulated kinase (ERK) phosphorylation, activation of caspase-3, caspase-8, and caspase-9, and PARP cleavage. Pretreatment with the ERK inhibitor PD98059 significantly blocked the loss of viability in calactin-treated cells. It is indicated that calactin-induced apoptosis may occur through an ERK signaling pathway. Our data suggest that calactin is a potential anticancer compound.

  20. Cadmium Chloride Induces DNA Damage and Apoptosis of Human Liver Carcinoma Cells via Oxidative Stress

    Directory of Open Access Journals (Sweden)

    Anthony Skipper

    2016-01-01

    Full Text Available Cadmium is a heavy metal that has been shown to cause its toxicity in humans and animals. Many documented studies have shown that cadmium produces various genotoxic effects such as DNA damage and chromosomal aberrations. Ailments such as bone disease, renal damage, and several forms of cancer are attributed to overexposure to cadmium.  Although there have been numerous studies examining the effects of cadmium in animal models and a few case studies involving communities where cadmium contamination has occurred, its molecular mechanisms of action are not fully elucidated. In this research, we hypothesized that oxidative stress plays a key role in cadmium chloride-induced toxicity, DNA damage, and apoptosis of human liver carcinoma (HepG2 cells. To test our hypothesis, cell viability was determined by MTT assay. Lipid hydroperoxide content stress was estimated by lipid peroxidation assay. Genotoxic damage was tested by the means of alkaline single cell gel electrophoresis (Comet assay. Cell apoptosis was measured by flow cytometry assessment (Annexin-V/PI assay. The result of MTT assay indicated that cadmium chloride induces toxicity to HepG2 cells in a concentration-dependent manner, showing a 48 hr-LD50 of 3.6 µg/mL. Data generated from lipid peroxidation assay resulted in a significant (p < 0.05 increase of hydroperoxide production, specifically at the highest concentration tested. Data obtained from the Comet assay indicated that cadmium chloride causes DNA damage in HepG2 cells in a concentration-dependent manner. A strong concentration-response relationship (p < 0.05 was recorded between annexin V positive cells and cadmium chloride exposure. In summary, these in vitro studies provide clear evidence that cadmium chloride induces oxidative stress, DNA damage, and programmed cell death in human liver carcinoma (HepG2 cells.

  1. Regenerative capacity of old muscle stem cells declines without significant accumulation of DNA damage.

    Directory of Open Access Journals (Sweden)

    Wendy Cousin

    Full Text Available The performance of adult stem cells is crucial for tissue homeostasis but their regenerative capacity declines with age, leading to failure of multiple organs. In skeletal muscle this failure is manifested by the loss of functional tissue, the accumulation of fibrosis, and reduced satellite cell-mediated myogenesis in response to injury. While recent studies have shown that changes in the composition of the satellite cell niche are at least in part responsible for the impaired function observed with aging, little is known about the effects of aging on the intrinsic properties of satellite cells. For instance, their ability to repair DNA damage and the effects of a potential accumulation of DNA double strand breaks (DSBs on their regenerative performance remain unclear. This work demonstrates that old muscle stem cells display no significant accumulation of DNA DSBs when compared to those of young, as assayed after cell isolation and in tissue sections, either in uninjured muscle or at multiple time points after injury. Additionally, there is no significant difference in the expression of DNA DSB repair proteins or globally assayed DNA damage response genes, suggesting that not only DNA DSBs, but also other types of DNA damage, do not significantly mark aged muscle stem cells. Satellite cells from DNA DSB-repair-deficient SCID mice do have an unsurprisingly higher level of innate DNA DSBs and a weakened recovery from gamma-radiation-induced DNA damage. Interestingly, they are as myogenic in vitro and in vivo as satellite cells from young wild type mice, suggesting that the inefficiency in DNA DSB repair does not directly correlate with the ability to regenerate muscle after injury. Overall, our findings suggest that a DNA DSB-repair deficiency is unlikely to be a key factor in the decline in muscle regeneration observed upon aging.

  2. Pretreatment with UV light renders the chromatin in human fibroblasts more susceptible to the DNA-damaging agents bleomycin, gamma radiation and 8-methoxypsoralen

    International Nuclear Information System (INIS)

    Ljungman, Mats

    1989-01-01

    Confluent human fibroblast cultures were pretreated with either 254 nm UV light (UV) or methyl methanesulphonate (MMS), incubated at 37 0 C and subsequently challenged on ice with bleomycin (BLM), gamma-radiation or 8-methoxy-psoralen (MOP). The resulting number of challenge-induced DNA damages (measured as DNA strand breaks or cross-links) were compared with the numbers induced in similarly challenged but non-pretreated control cells. It was found that the timing of the subsequent challenge of cells pretreated with UV did significantly affect the amount of induced DNA damage. When the challenging agents were administered after a 10-20 min incubation period following UV pretreatment, the amount of induced DNA damage was increased 50% over control cells. In contrast, the timing of the subsequent challenge of cells pretreated with MMS has no influence on the level of challenge-induced damage. It is hypothesized that UV-irradiated chromatin undergoes a time-dependent decondensation that renders it more susceptible to the induction of strand breaks and cross-links by BLM, gamma-radiation and MOP. A possible role for chromatin decondensation in UV-induced excision repair is discussed. (author)

  3. Radiation-induced DNA damage in halogenated pyrimidine incorporated cells and its correlation with radiosensitivity

    International Nuclear Information System (INIS)

    Watanabe, R.; Nikjoo, H.

    2003-01-01

    Cells with DNA containing 5-halogenated pyrimidines in place of thymidine show significant reductions of slope (Do) and shoulder (Dq) of their radiation survival curves. Similar radiosensitization has also been observed in the yield of DNA strand breaks. The purpose of this study is to obtain an insight into the mechanism of cell lethality by examining the relationship between the spectrum of DNA damage and the cell survival. In this study we estimated the enhancement of strand breaks due to incorporation of halogenated pyrimidine, the complexity of DNA damage and the probability of the initial DNA damage leading to cell inactivation. Monte Carlo track structure methods were used to model and simulate the induction of strand breakage by X-rays. The increase of DNA strand break was estimated by assuming the excess strand break was caused by the highly reactive uracil radicals at the halouracil substituted sites. The assumption of the enhancement mechanism of strand breaks was examined and verified by comparison with experimental data for induction of SSB and DSB. The calculated DNA damage spectrum shows the increase in complexity of strand breaks is due to incorporation of halogenated pyrimidines. The increase in the yield of DSB and cell lethality show similar trend at various degrees of halogenated pyrimidine substitution. We asked the question whether this agreement supports the hypothesis that DSB is responsible for cell lethality? The estimated number of lethal damage from the cell survival using a linear-quadratic model is much less than the initial yield of DSB. This work examines the correlation of cell lethality as a function of frequencies of complex form of double strand breaks

  4. FOXO3 Transcription Factor Is Essential for Protecting Hematopoietic Stem and Progenitor Cells from Oxidative DNA Damage.

    Science.gov (United States)

    Bigarella, Carolina L; Li, Jianfeng; Rimmelé, Pauline; Liang, Raymond; Sobol, Robert W; Ghaffari, Saghi

    2017-02-17

    Accumulation of damaged DNA in hematopoietic stem cells (HSC) is associated with chromosomal abnormalities, genomic instability, and HSC aging and might promote hematological malignancies with age. Despite this, the regulatory pathways implicated in the HSC DNA damage response have not been fully elucidated. One of the sources of DNA damage is reactive oxygen species (ROS) generated by both exogenous and endogenous insults. Balancing ROS levels in HSC requires FOXO3, which is an essential transcription factor for HSC maintenance implicated in HSC aging. Elevated ROS levels result in defective Foxo3 -/- HSC cycling, among many other deficiencies. Here, we show that loss of FOXO3 leads to the accumulation of DNA damage in primitive hematopoietic stem and progenitor cells (HSPC), associated specifically with reduced expression of genes implicated in the repair of oxidative DNA damage. We provide further evidence that Foxo3 -/- HSPC are defective in DNA damage repair. Specifically, we show that the base excision repair pathway, the main pathway utilized for the repair of oxidative DNA damage, is compromised in Foxo3 -/- primitive hematopoietic cells. Treating mice in vivo with N -acetylcysteine reduces ROS levels, rescues HSC cycling defects, and partially mitigates HSPC DNA damage. These results indicate that DNA damage accrued as a result of elevated ROS in Foxo3 -/- mutant HSPC is at least partially reversible. Collectively, our findings suggest that FOXO3 serves as a protector of HSC genomic stability and health. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  5. DNA damage response and spindle assembly checkpoint function throughout the cell cycle to ensure genomic integrity.

    Directory of Open Access Journals (Sweden)

    Katherine S Lawrence

    2015-04-01

    Full Text Available Errors in replication or segregation lead to DNA damage, mutations, and aneuploidies. Consequently, cells monitor these events and delay progression through the cell cycle so repair precedes division. The DNA damage response (DDR, which monitors DNA integrity, and the spindle assembly checkpoint (SAC, which responds to defects in spindle attachment/tension during metaphase of mitosis and meiosis, are critical for preventing genome instability. Here we show that the DDR and SAC function together throughout the cell cycle to ensure genome integrity in C. elegans germ cells. Metaphase defects result in enrichment of SAC and DDR components to chromatin, and both SAC and DDR are required for metaphase delays. During persistent metaphase arrest following establishment of bi-oriented chromosomes, stability of the metaphase plate is compromised in the absence of DDR kinases ATR or CHK1 or SAC components, MAD1/MAD2, suggesting SAC functions in metaphase beyond its interactions with APC activator CDC20. In response to DNA damage, MAD2 and the histone variant CENPA become enriched at the nuclear periphery in a DDR-dependent manner. Further, depletion of either MAD1 or CENPA results in loss of peripherally associated damaged DNA. In contrast to a SAC-insensitive CDC20 mutant, germ cells deficient for SAC or CENPA cannot efficiently repair DNA damage, suggesting that SAC mediates DNA repair through CENPA interactions with the nuclear periphery. We also show that replication perturbations result in relocalization of MAD1/MAD2 in human cells, suggesting that the role of SAC in DNA repair is conserved.

  6. Protective roles of bacterioruberin and intracellular KCl in the resistance of Halobacterium salinarium against DNA-damaging agents

    International Nuclear Information System (INIS)

    Shahmohammadi, H.R.; Asgarani, E.; Terato, Hiroaki; Saito, Takeshi; Ohyama, Yoshihiko; Gekko, Kunihiko; Yamamoto, Osamu; Ide, Hiroshi

    1998-01-01

    Halobacterium salinarium, a member of the extremely halophilic archaebacteria, contains a C 50 -carotenoid namely bacterioruberin. We have previously reported the high resistance of this organism against the lethal actions of DNA-damaging agents including ionizing radiation and ultraviolet light (UV). In this study, we have examined whether bacterioruberin and the highly concentrated salts in this bacterium play protective roles against the lethal actions of ionizing radiation, UV, hydrogen peroxide, and mitomycin-C (MMC). The colourless mutant of H. salinarium deficient in bacterioruberin was more sensitive than the red-pigmented wild-type to all tested DNA-damaging agents except MMC. Circular dichroism (CD) spectra of H. salinarium chromosomal DNA at various concentrations of KCl (0-3.5 M) were similar to that of B-DNA, indicating that no conformational changes occurred as a result of high salt concentrations. However, DNA strand-breaks induced by ionizing radiation were significantly reduced by the presence of either bacterioruberin or concentrated KCl, presumably due to scavenging of free radicals. These results suggest that bacterioruberin and intracellular KCl of H. salinarium protect this organism against the lethal effects of oxidative DNA-damaging agents. (author)

  7. DNA damage and cell cycle events implicate cerebellar dentate nucleus neurons as targets of Alzheimer's disease

    Directory of Open Access Journals (Sweden)

    Yang Yan

    2010-12-01

    Full Text Available Abstract Background Although the cerebellum is considered to be predominantly involved in fine motor control, emerging evidence documents its participation in language, impulsive behavior and higher cognitive functions. While the specific connections of the cerebellar deep nuclei (CDN that are responsible for these functions are still being worked out, their deficiency has been termed "cerebellar cognitive affective syndrome" - a syndrome that bears a striking similarity to many of the symptoms of Alzheimer's disease (AD. Using ectopic cell cycle events and DNA damage markers as indexes of cellular distress, we have explored the neuropathological involvement of the CDN in human AD. Results We examined the human cerebellar dentate nucleus in 22 AD cases and 19 controls for the presence of neuronal cell cycle events and DNA damage using immunohistochemistry and fluorescence in situ hybridization. Both techniques revealed several instances of highly significant correlations. By contrast, neither amyloid plaque nor neurofibrillary tangle pathology was detected in this region, consistent with previous reports of human cerebellar pathology. Five cases of early stage AD were examined and while cell cycle and DNA damage markers were well advanced in the hippocampus of all five, few indicators of either cell cycle events (1 case or a DNA damage response (1 case were found in CDN. This implies that CDN neurons are most likely affected later in the course of AD. Clinical-pathological correlations revealed that cases with moderate to high levels of cell cycle activity in their CDN are highly likely to show deficits in unorthodox cerebellar functions including speech, language and motor planning. Conclusion Our results reveal that the CDN neurons are under cellular stress in AD and suggest that some of the non-motor symptoms found in patients with AD may be partly cerebellar in origin.

  8. Ellipticine induces apoptosis in T-cell lymphoma via oxidative DNA damage

    DEFF Research Database (Denmark)

    Savorani, Cecilia; Manfé, Valentina; Biskup, Edyta

    2015-01-01

    (CTCL), a disease that is progressive, chemoresistant and refractory to treatment. We tested the effect of ellipticine in three cell lines with different p53 status: MyLa2000 (p53(wt/wt)), SeAx ((G245S)p53) and Hut-78 ((R196Stop)p53). Ellipticine caused apoptosis in MyLa2000 and SeAx and restored...... the transcriptional activity of (G245S)p53 in SeAx. However, p53 siRNA knockdown experiments revealed that p53 was not required for ellipticine-induced apoptosis in CTCL. The lipophilic antioxidant α-tocopherol inhibited ellipticine-dependent apoptosis and we linked the apoptotic response to the oxidative DNA damage....... Our results provide evidence that ellipticine-induced apoptosis is exerted through DNA damage and does not require p53 activation in T-cell lymphoma....

  9. Variation of DNA damage levels in peripheral blood mononuclear cells isolated in different laboratories

    DEFF Research Database (Denmark)

    Godschalk, Roger W L; Ersson, Clara; Stępnik, Maciej

    2014-01-01

    This study investigated the levels of DNA strand breaks and formamidopyrimidine DNA glycosylase (FPG) sensitive sites, as assessed by the comet assay, in peripheral blood mononuclear cells (PBMC) from healthy women from five different countries in Europe. The laboratory in each country (referred...... to as 'centre') collected and cryopreserved PBMC samples from three donors, using a standardised cell isolation protocol. The samples were analysed in 13 different laboratories for DNA damage, which is measured by the comet assay. The study aim was to assess variation in DNA damage in PBMC samples that were......%) by standardisation of the primary comet assay endpoint with calibration curve samples. The level of DNA strand breaks in the samples from two of the centres (0.56-0.61 lesions/10(6) bp) was significantly higher compared with the other three centres (0.41-0.45 lesions/10(6) bp). In contrast, there was no difference...

  10. Helicobacter pylori Infection Causes Characteristic DNA Damage Patterns in Human Cells

    Directory of Open Access Journals (Sweden)

    Max Koeppel

    2015-06-01

    Full Text Available Infection with the human pathogen Helicobacter pylori (H. pylori is a major risk factor for gastric cancer. Since the bacterium exerts multiple genotoxic effects, we examined the circumstances of DNA damage accumulation and identified regions within the host genome with high susceptibility to H. pylori-induced damage. Infection impaired several DNA repair factors, the extent of which depends on a functional cagPAI. This leads to accumulation of a unique DNA damage pattern, preferentially in transcribed regions and proximal to telomeres, in both gastric cell lines and primary gastric epithelial cells. The observed pattern correlates with focal amplifications in adenocarcinomas of the stomach and partly overlaps with known cancer genes. We thus demonstrate an impact of a bacterial infection directed toward specific host genomic regions and describe underlying characteristics that make such regions more likely to acquire heritable changes during infection, which could contribute to cellular transformation.

  11. G9a coordinates with the RPA complex to promote DNA damage repair and cell survival.

    Science.gov (United States)

    Yang, Qiaoyan; Zhu, Qian; Lu, Xiaopeng; Du, Yipeng; Cao, Linlin; Shen, Changchun; Hou, Tianyun; Li, Meiting; Li, Zhiming; Liu, Chaohua; Wu, Di; Xu, Xingzhi; Wang, Lina; Wang, Haiying; Zhao, Ying; Yang, Yang; Zhu, Wei-Guo

    2017-07-25

    Histone methyltransferase G9a has critical roles in promoting cancer-cell growth and gene suppression, but whether it is also associated with the DNA damage response is rarely studied. Here, we report that loss of G9a impairs DNA damage repair and enhances the sensitivity of cancer cells to radiation and chemotherapeutics. In response to DNA double-strand breaks (DSBs), G9a is phosphorylated at serine 211 by casein kinase 2 (CK2) and recruited to chromatin. The chromatin-enriched G9a can then directly interact with replication protein A (RPA) and promote loading of the RPA and Rad51 recombinase to DSBs. This mechanism facilitates homologous recombination (HR) and cell survival. We confirmed the interaction between RPA and G9a to be critical for RPA foci formation and HR upon DNA damage. Collectively, our findings demonstrate a regulatory pathway based on CK2-G9a-RPA that permits HR in cancer cells and provide further rationale for the use of G9a inhibitors as a cancer therapeutic.

  12. DNA damage and DNA damage response in human bronchial epithelial BEAS-2B cells following exposure to 2-nitrobenzanthrone and 3-nitrobenzanthrone: role in apoptosis.

    Science.gov (United States)

    Oya, Elisabeth; Ovrevik, Johan; Arlt, Volker M; Nagy, Eszter; Phillips, David H; Holme, Jørn A

    2011-11-01

    Nitro-polycyclic aromatic hydrocarbons (nitro-PAHs) are mutagenic and carcinogenic environmental pollutants found in diesel exhaust and on urban air pollution particles. In the present study, human bronchial epithelial BEAS-2B cells were exposed to 2-nitrobenzanthrone (2-NBA) and 3-nitrobenzanthrone (3-NBA). DNA damage responses were compared to those observed after exposure to 1-nitropyrene (1-NP) and benzo[a]pyrene (B[a]P). Examination by microscopy revealed that 3-NBA was the most potent toxic compound while weaker responses were observed with 1-NP and B[a]P. Most interestingly, 2-NBA did not induce cell death or any other stress-related responses. 3-NBA induced a typical apoptotic cell death judged by nuclear condensation and little plasma membrane damage as well as cleavage of caspase 3 and poly-(ADP-ribose) polymerase (PARP). Exposure to 3-NBA resulted in an accumulation of cells in S-phase, and further analysis by Western blotting, immunocytochemistry and flow cytometry revealed that 3-NBA induced a DNA damage response characterized by phosphorylation of ATM (ataxia-telangiectasia mutated), checkpoint kinase (Chk) 2/Chk1, H2AX and p53. The p53 inhibitor pifithrin-α inhibited 3-NBA-induced apoptosis while small effects were seen using pifithrin-μ, suggesting that 3-NBA-induced cell death is a result of transcriptional activation of p53. In conclusion, 3-NBA is a potent inducer of apoptosis, which seemed to be triggered by the DNA damage response. Furthermore, a change of the nitro-group to the second position (i.e. 2-NBA) dramatically changed the cellular reactivity of the compound.

  13. Molecular mechanism of radioadaptive response: A cross-adaptive response for enhanced repair of DNA damage in adapted cells

    International Nuclear Information System (INIS)

    Takaji Ikushima

    1997-01-01

    The radioadaptive response (RAR) has been attributed to the induction of a repair mechanism by low doses of ionizing radiation, but the molecular nature of the mechanism is not yet elucidated. We have characterized RAR in a series of experiments in cultured Chinese hamster V79 cells. A 4-h interval is required for the full expression of RAR, which decays with the progression of cell proliferation. Treatments with inhibitors of poly(ADP-ribose) polymerase, protein- or RNA synthesis, and protein kinase C suppress the RAR expression. The RAR cross-reacts on clastogenic lesions induced by other physical and chemical DNA-damaging agents. The presence of newly synthesised proteins has been detected during the expression period. Experiments performed using single-cell gel electrophoresis provided more direct evidence for a faster and enhaced DNA repair rate in adapted cells. Here, using single-cell gel electrophoresis, a cross-adaptive response has been demonstrated for enhanced repair of DNA damage induced by neocarzinostatin in radio-adapted cells. (author)

  14. Systematic Analysis of the Crosstalk between Mitosis and DNA Damage by a Live Cell siRNA Screen

    DEFF Research Database (Denmark)

    Pedersen, Ronni Sølvhøi

    Recent research has shown, that the biological processes of DNA replication, DNA damage, cell cycle and mitosis cannot be considered as isolated cellular functions but are mechanistically linked in many ways. For instance, when cells are exposed to replication stress and enter mitosis...... propose that this strong p53 response, which often occurs without detectable increase in DNA damage, is caused by the acute increase in chromosomal aneuploidy. Finally, our systematic approach to the DNA damage-mitosis crosstalk reveals widespread cell death in response to mitotic pertubations, showing...

  15. Low doses of ionizing radiation to mammalian cells may rather control than cause DNA damage

    International Nuclear Information System (INIS)

    Feinendegen, L.E.; Sondhaus, C.A.; Altman, K.I.

    1998-01-01

    This report examines the origin of tissue effects that may follow from different cellular responses to low-dose irradiation, using published data. Two principal categories of cellular responses are considered. One response category relates to the probability of radiation-induced DNA damage. The other category consists of low-dose induced metabolic changes that induce mechanisms of DNA damage mitigation, which do not operate at high levels of exposure. Modeled in this way, tissue is treated as a complex adaptive system. The interaction of the various cellular responses results in a net tissue dose-effect relation that is likely to deviate from linearity in the low-dose region. This suggests that the LNT hypothesis should be reexamined. This paper aims at demonstrating tissue effects as an expression of cellular responses, both damaging and defensive, in relation to the energy deposited in cell mass, by use of microdosimetric concepts

  16. Low doses of ionizing radiation to mammalian cells may rather control than cause DNA damage

    Energy Technology Data Exchange (ETDEWEB)

    Feinendegen, L.E. [Brookhaven National Lab., Upton, NY (United States). Medical Dept.; Bond, V.P. [Washington State Univ., Richland, WA (United States); Sondhaus, C.A. [Univ. of Arizona, Tucson, AZ (United States). Dept. of Radiology and Radiation Control Office; Altman, K.I. [Univ. of Rochester Medical Center, NY (United States). Dept. of Biochemistry and Biophysics

    1998-12-31

    This report examines the origin of tissue effects that may follow from different cellular responses to low-dose irradiation, using published data. Two principal categories of cellular responses are considered. One response category relates to the probability of radiation-induced DNA damage. The other category consists of low-dose induced metabolic changes that induce mechanisms of DNA damage mitigation, which do not operate at high levels of exposure. Modeled in this way, tissue is treated as a complex adaptive system. The interaction of the various cellular responses results in a net tissue dose-effect relation that is likely to deviate from linearity in the low-dose region. This suggests that the LNT hypothesis should be reexamined. This paper aims at demonstrating tissue effects as an expression of cellular responses, both damaging and defensive, in relation to the energy deposited in cell mass, by use of microdosimetric concepts.

  17. Higher-Density Culture in Human Embryonic Stem Cells Results in DNA Damage and Genome Instability

    Directory of Open Access Journals (Sweden)

    Kurt Jacobs

    2016-03-01

    Full Text Available Human embryonic stem cells (hESC show great promise for clinical and research applications, but their well-known proneness to genomic instability hampers the development to their full potential. Here, we demonstrate that medium acidification linked to culture density is the main cause of DNA damage and genomic alterations in hESC grown on feeder layers, and this even in the short time span of a single passage. In line with this, we show that increasing the frequency of the medium refreshments minimizes the levels of DNA damage and genetic instability. Also, we show that cells cultured on laminin-521 do not present this increase in DNA damage when grown at high density, although the (long-term impact on their genomic stability remains to be elucidated. Our results explain the high levels of genome instability observed over the years by many laboratories worldwide, and show that the development of optimal culture conditions is key to solving this problem.

  18. Alpha-phellandrene-induced DNA damage and affect DNA repair protein expression in WEHI-3 murine leukemia cells in vitro.

    Science.gov (United States)

    Lin, Jen-Jyh; Wu, Chih-Chung; Hsu, Shu-Chun; Weng, Shu-Wen; Ma, Yi-Shih; Huang, Yi-Ping; Lin, Jaung-Geng; Chung, Jing-Gung

    2015-11-01

    Although there are few reports regarding α-phellandrene (α-PA), a natural compound from Schinus molle L. essential oil, there is no report to show that α-PA induced DNA damage and affected DNA repair associated protein expression. Herein, we investigated the effects of α-PA on DNA damage and repair associated protein expression in murine leukemia cells. Flow cytometric assay was used to measure the effects of α-PA on total cell viability and the results indicated that α-PA induced cell death. Comet assay and 4,6-diamidino-2-phenylindole dihydrochloride staining were used for measuring DNA damage and condensation, respectively, and the results indicated that α-PA induced DNA damage and condensation in a concentration-dependent manner. DNA gel electrophoresis was used to examine the DNA damage and the results showed that α-PA induced DNA damage in WEHI-3 cells. Western blotting assay was used to measure the changes of DNA damage and repair associated protein expression and the results indicated that α-PA increased p-p53, p-H2A.X, 14-3-3-σ, and MDC1 protein expression but inhibited the protein of p53, MGMT, DNA-PK, and BRCA-1. © 2014 Wiley Periodicals, Inc.

  19. Apoptosis-like yeast cell death in response to DNA damage and replication defects

    Energy Technology Data Exchange (ETDEWEB)

    Burhans, William C.; Weinberger, Martin; Marchetti, Maria A.; Ramachandran, Lakshmi; D' Urso, Gennaro; Huberman, Joel A

    2003-11-27

    In budding (Saccharomyces cerevisiae) and fission (Schizosaccharomyces pombe) yeast and other unicellular organisms, DNA damage and other stimuli can induce cell death resembling apoptosis in metazoans, including the activation of a recently discovered caspase-like molecule in budding yeast. Induction of apoptotic-like cell death in yeasts requires homologues of cell cycle checkpoint proteins that are often required for apoptosis in metazoan cells. Here, we summarize these findings and our unpublished results which show that an important component of metazoan apoptosis recently detected in budding yeast - reactive oxygen species (ROS) - can also be detected in fission yeast undergoing an apoptotic-like cell death. ROS were detected in fission and budding yeast cells bearing conditional mutations in genes encoding DNA replication initiation proteins and in fission yeast cells with mutations that deregulate cyclin-dependent kinases (CDKs). These mutations may cause DNA damage by permitting entry of cells into S phase with a reduced number of replication forks and/or passage through mitosis with incompletely replicated chromosomes. This may be relevant to the frequent requirement for elevated CDK activity in mammalian apoptosis, and to the recent discovery that the initiation protein Cdc6 is destroyed during apoptosis in mammals and in budding yeast cells exposed to lethal levels of DNA damage. Our data indicate that connections between apoptosis-like cell death and DNA replication or CDK activity are complex. Some apoptosis-like pathways require checkpoint proteins, others are inhibited by them, and others are independent of them. This complexity resembles that of apoptotic pathways in mammalian cells, which are frequently deregulated in cancer. The greater genetic tractability of yeasts should help to delineate these complex pathways and their relationships to cancer and to the effects of apoptosis-inducing drugs that inhibit DNA replication.

  20. Apoptosis-like yeast cell death in response to DNA damage and replication defects

    International Nuclear Information System (INIS)

    Burhans, William C.; Weinberger, Martin; Marchetti, Maria A.; Ramachandran, Lakshmi; D'Urso, Gennaro; Huberman, Joel A.

    2003-01-01

    In budding (Saccharomyces cerevisiae) and fission (Schizosaccharomyces pombe) yeast and other unicellular organisms, DNA damage and other stimuli can induce cell death resembling apoptosis in metazoans, including the activation of a recently discovered caspase-like molecule in budding yeast. Induction of apoptotic-like cell death in yeasts requires homologues of cell cycle checkpoint proteins that are often required for apoptosis in metazoan cells. Here, we summarize these findings and our unpublished results which show that an important component of metazoan apoptosis recently detected in budding yeast - reactive oxygen species (ROS) - can also be detected in fission yeast undergoing an apoptotic-like cell death. ROS were detected in fission and budding yeast cells bearing conditional mutations in genes encoding DNA replication initiation proteins and in fission yeast cells with mutations that deregulate cyclin-dependent kinases (CDKs). These mutations may cause DNA damage by permitting entry of cells into S phase with a reduced number of replication forks and/or passage through mitosis with incompletely replicated chromosomes. This may be relevant to the frequent requirement for elevated CDK activity in mammalian apoptosis, and to the recent discovery that the initiation protein Cdc6 is destroyed during apoptosis in mammals and in budding yeast cells exposed to lethal levels of DNA damage. Our data indicate that connections between apoptosis-like cell death and DNA replication or CDK activity are complex. Some apoptosis-like pathways require checkpoint proteins, others are inhibited by them, and others are independent of them. This complexity resembles that of apoptotic pathways in mammalian cells, which are frequently deregulated in cancer. The greater genetic tractability of yeasts should help to delineate these complex pathways and their relationships to cancer and to the effects of apoptosis-inducing drugs that inhibit DNA replication

  1. Establishing a model for assessing DNA damage in murine brain cells as a molecular marker of chemotherapy-associated cognitive impairment.

    Science.gov (United States)

    Krynetskiy, Evgeny; Krynetskaia, Natalia; Rihawi, Diana; Wieczerzak, Katarzyna; Ciummo, Victoria; Walker, Ellen

    2013-10-17

    Chemotherapy-associated cognitive impairment often follows cancer chemotherapy. We explored chemotherapy-induced DNA damage in the brain cells of mice treated with 5-fluorouracil (5FU), an antineoplastic agent, to correlate the extent of DNA damage to behavioral functioning in an autoshaping-operant mouse model of chemotherapy-induced learning and memory deficits (Foley et al., 2008). Male, Swiss-Webster mice were injected once with saline or 75 mg/kg 5FU at 0, 12, and 24h and weighed every 24h. Twenty-four h after the last injection, the mice were tested in a two-day acquisition and the retention of a novel response task for food reinforcement. Murine brain cells were analyzed for the presence of single- and double-strand DNA breaks by the single cell gel electrophoresis assay (the Comet assay). We detected significant differences (p<0.0001) for all DNA damage characteristics (DNA "comet" tail shape, migration pattern, tail moment and olive moments) between control mice cohort and 5FU-treated mice cohort: tail length - 119 vs. 153; tail moment - 101 vs. 136; olive moment - 60 vs. 82, correspondingly. We found a positive correlation between increased response rates (r=0.52, p<0.05) and increased rate of errors (r=0.51, p<0.05), and DNA damage on day 1. For all 15 mice (saline-treated and 5FU-treated mice), we found negative correlations between DNA damage and weight (r=-0.75, p<0.02). Our results indicate that chemotherapy-induced DNA damage changes the physiological status of the brain cells and may provide insights to the mechanisms for cognitive impairment after cancer chemotherapy. Copyright © 2013 Elsevier Inc. All rights reserved.

  2. Umbelliferone suppresses radiation induced DNA damage and apoptosis in hematopoietic cells of mice

    International Nuclear Information System (INIS)

    Jayakumar, S.; Bhilwade, H.N.; Chaubey, R.C.

    2012-01-01

    Radiotherapy is one of the major modes of treatment for different types of cancers. But the success of radiotherapy is limited by injury to the normal cells. Protection of the normal cells from radiation damage by radioprotectors can increase therapeutic efficiency. These radioprotectors can also be used during nuclear emergency situations. Umbelliferone (UMB) is a wide spread natural product of the coumarin family. It occurs in many plants from the Apiaceae family. In the present study radioprotective effect of UMB was investigated in vitro and in vivo. Anti genotoxic effect of Umbelliferone was tested by treating the splenic lymphocytes with various doses of UMB (6.5 μM - 50 μM) prior to radiation (6Gy) exposure. After the radiation exposure, extent of DNA damage was assessed by comet assay at 5 mm and two hours after radiation exposure. At both the time points, it was observed that the pretreatment of UMB reduced the radiation induced DNA damage to a significant extent in comparison to radiation control. UMB pretreatment also significantly reduced the radiation induced apoptosis enumerated by propidium iodide staining assay. Results of clonogenic survival assay using intestinal cell line showed that pretreatment with UMB significantly protected against radiation induced loss of colony forming units. To assess the anti genotoxic role of umbelliferone in vivo two different doses of UMB (20 mg/Kg and 40 mg/Kg of body weight) were injected into Swiss mice or with vehicle and exposed to radiation. Thirty minutes after the radiation comet assay was performed in peripheral leukocytes. Frequency of micro nucleated erythrocytes was scored in bone marrow cells. It was observed that UMB alone did not cause any significant increase in DNA damage in comparison to control. Animals which are exposed to radiation alone showed significant increase in DNA damage and micronuclei frequency. But animals treated with UMB prior to the radiation exposure showed significant decrease

  3. A germline FANCA alteration that is associated with increased sensitivity to DNA damaging agents.

    OpenAIRE

    Wilkes, David C; Sailer, Verena; Xue, Hui; Cheng, Hongwei; Collins, Colin C; Gleave, Martin; Wang, Yuzhuo; Demichelis, Francesca; Beltran, Himisha; Rubin, Mark Andrew; Rickman, David S

    2017-01-01

    Defects in genes involved in DNA damage repair (DDR) pathway are emerging as novel biomarkers and targets for new prostate cancer drug therapies. A previous report revealed an association between an exceptional response to cisplatin treatment and a somatic loss of heterozygosity (LOH) of FANCA in a patient with metastatic prostate cancer who also harbored a germline FANCA variant (S1088F). Although germline FANCA mutations are the most frequent alterations in patients with Fanconi anemia, ger...

  4. DNA Damage: A Sensible Mediator of the Differentiation Decision in Hematopoietic Stem Cells and in Leukemia

    Directory of Open Access Journals (Sweden)

    Cary N. Weiss

    2015-03-01

    Full Text Available In the adult, the source of functionally diverse, mature blood cells are hematopoietic stem cells, a rare population of quiescent cells that reside in the bone marrow niche. Like stem cells in other tissues, hematopoietic stem cells are defined by their ability to self-renew, in order to maintain the stem cell population for the lifetime of the organism, and to differentiate, in order to give rise to the multiple lineages of the hematopoietic system. In recent years, increasing evidence has suggested a role for the accumulation of reactive oxygen species and DNA damage in the decision for hematopoietic stem cells to exit quiescence and to differentiate. In this review, we will examine recent work supporting the idea that detection of cell stressors, such as oxidative and genetic damage, is an important mediator of cell fate decisions in hematopoietic stem cells. We will explore the benefits of such a system in avoiding the development and progression of malignancies, and in avoiding tissue exhaustion and failure. Additionally, we will discuss new work that examines the accumulation of DNA damage and replication stress in aging hematopoietic stem cells and causes us to rethink ideas of genoprotection in the bone marrow niche.

  5. DNA damage in oral cancer cells induced by nitrogen atmospheric pressure plasma jets

    Energy Technology Data Exchange (ETDEWEB)

    Han, Xu; Ptasinska, Sylwia [Radiation Laboratory, University of Notre Dame, Notre Dame, Indiana 46556 (United States); Department of Physics, University of Notre Dame, Notre Dame, Indiana 46556 (United States); Klas, Matej [Radiation Laboratory, University of Notre Dame, Notre Dame, Indiana 46556 (United States); Liu, Yueying [Harper Cancer Research Institute, University of Notre Dame, Notre Dame, Indiana 46556 (United States); Sharon Stack, M. [Harper Cancer Research Institute, University of Notre Dame, Notre Dame, Indiana 46556 (United States); Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana 46556 (United States)

    2013-06-10

    The nitrogen atmospheric pressure plasma jet (APPJ) was applied to induce DNA damage of SCC-25 oral cancer cells. Optical emission spectra were taken to characterize the reactive species produced in APPJ. In order to explore the spatial distribution of plasma effects, cells were placed onto photo-etched grid slides and the antibody H2A.X was used to locate double strand breaks of DNA inside nuclei using an immunofluorescence assay. The number of cells with double strand breaks in DNA was observed to be varied due to the distance from the irradiation center and duration of plasma treatment.

  6. DNA damage in oral cancer cells induced by nitrogen atmospheric pressure plasma jets

    International Nuclear Information System (INIS)

    Han, Xu; Ptasinska, Sylwia; Klas, Matej; Liu, Yueying; Sharon Stack, M.

    2013-01-01

    The nitrogen atmospheric pressure plasma jet (APPJ) was applied to induce DNA damage of SCC-25 oral cancer cells. Optical emission spectra were taken to characterize the reactive species produced in APPJ. In order to explore the spatial distribution of plasma effects, cells were placed onto photo-etched grid slides and the antibody H2A.X was used to locate double strand breaks of DNA inside nuclei using an immunofluorescence assay. The number of cells with double strand breaks in DNA was observed to be varied due to the distance from the irradiation center and duration of plasma treatment.

  7. [DNA damage in human pleural mesothelial cells induced by exposure to carbon nanotubes].

    Science.gov (United States)

    Ogasawara, Yuki; Umezu, Noriaki; Ishii, Kazuyuki

    2012-01-01

    Nanomaterials are currently used in electronics, industrial materials, cosmetics, and medicine because they have useful physicochemical properties, such as strength, conductivity, durability, and chemical stability. As these materials have become widespread, many questions have arisen regarding their effects on health and the environment. In particular, recent studies have demonstrated that carbon nanotubes (CNTs) cause significant inflammation and mesothelioma in vivo. In this study, we investigated the potential risk posed by singlewalled carbon nanotube (SWCNT) and multiwalled carbon nanotube (MWCNT) exposure in human pleural mesothelial cells. CNT cytotoxicity was determined by a trypan blue exclusion assay, and DNA damage was detected by an alkaline comet assay. The concentration of 8-oxodeoxyguanosine (8-OHdG) in DNA was measured by high perhormance liquid chromatography with electrochemical detection. The expression of base excision repair enzymes in the cell was estimated by immunoblot analysis. We observed inhibitory effects on cell proliferation and the induction of DNA damage following exposure of cells to purified CNTs that were suspended in dispersion medium. However, accumulation of 8-OHdG in DNA was not found. In addition, the expression levels of base excision enzymes that are involved in hOGG1, hMTH1, and MYH in MeT-5A cells remained unchanged for 24 h after carbon nanotube exposure. CNTs significantly inhibit cell proliferation and decrease DNA damage in human pleural mesothelial cells. Our results indicate that the mechanism of CNT-induced genotoxicity is different from that following exposure to reactive oxygen species, which causes oxidative DNA modifications and 8-OHdG production. Further investigation is required to characterize the specific DNA mutations that occur following CNT exposure.

  8. NEIL2 protects against oxidative DNA damage induced by sidestream smoke in human cells.

    Directory of Open Access Journals (Sweden)

    Altaf H Sarker

    Full Text Available Secondhand smoke (SHS is a confirmed lung carcinogen that introduces thousands of toxic chemicals into the lungs. SHS contains chemicals that have been implicated in causing oxidative DNA damage in the airway epithelium. Although DNA repair is considered a key defensive mechanism against various environmental attacks, such as cigarette smoking, the associations of individual repair enzymes with susceptibility to lung cancer are largely unknown. This study investigated the role of NEIL2, a DNA glycosylase excising oxidative base lesions, in human lung cells treated with sidestream smoke (SSS, the main component of SHS. To do so, we generated NEIL2 knockdown cells using siRNA-technology and exposed them to SSS-laden medium. Representative SSS chemical compounds in the medium were analyzed by mass spectrometry. An increased production of reactive oxygen species (ROS in SSS-exposed cells was detected through the fluorescent detection and the induction of HIF-1α. The long amplicon-quantitative PCR (LA-QPCR assay detected significant dose-dependent increases of oxidative DNA damage in the HPRT gene of cultured human pulmonary fibroblasts (hPF and BEAS-2B epithelial cells exposed to SSS for 24 h. These data suggest that SSS exposure increased oxidative stress, which could contribute to SSS-mediated toxicity. siRNA knockdown of NEIL2 in hPF and HEK 293 cells exposed to SSS for 24 h resulted in significantly more oxidative DNA damage in HPRT and POLB than in cells with control siRNA. Taken together, our data strongly suggest that decreased repair of oxidative DNA base lesions due to an impaired NEIL2 expression in non-smokers exposed to SSS would lead to accumulation of mutations in genomic DNA of lung cells over time, thus contributing to the onset of SSS-induced lung cancer.

  9. TGF-β1 accelerates the DNA damage response in epithelial cells via Smad signaling

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Jeeyong; Kim, Mi-Ra; Kim, Hyun-Ji; An, You Sun; Yi, Jae Youn, E-mail: yjy_71@kcch.re.kr

    2016-08-05

    The evidence suggests that transforming growth factor-beta (TGF-β) regulates the DNA-damage response (DDR) upon irradiation, and we previously reported that TGF-β1 induced DNA ligase IV (Lig4) expression and enhanced the nonhomologous end-joining repair pathway in irradiated cells. In the present study, we investigated the effects of TGF-β1 on the irradiation-induced DDRs of A431 and HaCaT cells. Cells were pretreated with or without TGF-β1 and irradiated. At 30 min post-irradiation, DDRs were detected by immunoblotting of phospho-ATM, phospho-Chk2, and the presence of histone foci (γH2AX). The levels of all three factors were similar right after irradiation regardless of TGF-β1 pretreatment. However, they soon thereafter exhibited downregulation in TGF-β1-pretreated cells, indicating the acceleration of the DDR. Treatment with a TGF-β type I receptor inhibitor (SB431542) or transfections with siRNAs against Smad2/3 or DNA ligase IV (Lig4) reversed this acceleration of the DDR. Furthermore, the frequency of irradiation-induced apoptosis was decreased by TGF-β1 pretreatment in vivo, but this effect was abrogated by SB431542. These results collectively suggest that TGF-β1 could enhance cell survival by accelerating the DDR via Smad signaling and Lig4 expression. -- Highlights: •TGF-β1 pretreatment accelerates γ-radiation-induced DNA damage response. •TGF-β1-accelerated DNA damage response is dependent on Smad signaling and DNA Ligase IV. •TGF-β1 pretreatment protects epithelial cells from γ-radiation in vivo.

  10. DNA Damage during G2 Phase Does Not Affect Cell Cycle Progression of the Green Alga Scenedesmus quadricauda

    Science.gov (United States)

    Vítová, Milada; Bišová, Kateřina; Zachleder, Vilém

    2011-01-01

    DNA damage is a threat to genomic integrity in all living organisms. Plants and green algae are particularly susceptible to DNA damage especially that caused by UV light, due to their light dependency for photosynthesis. For survival of a plant, and other eukaryotic cells, it is essential for an organism to continuously check the integrity of its genetic material and, when damaged, to repair it immediately. Cells therefore utilize a DNA damage response pathway that is responsible for sensing, reacting to and repairing damaged DNA. We have studied the effect of 5-fluorodeoxyuridine, zeocin, caffeine and combinations of these on the cell cycle of the green alga Scenedesmus quadricauda. The cells delayed S phase and underwent a permanent G2 phase block if DNA metabolism was affected prior to S phase; the G2 phase block imposed by zeocin was partially abolished by caffeine. No cell cycle block was observed if the treatment with zeocin occurred in G2 phase and the cells divided normally. CDKA and CDKB kinases regulate mitosis in S. quadricauda; their kinase activities were inhibited by Wee1. CDKA, CDKB protein levels were stabilized in the presence of zeocin. In contrast, the protein level of Wee1 was unaffected by DNA perturbing treatments. Wee1 therefore does not appear to be involved in the DNA damage response in S. quadricauda. Our results imply a specific reaction to DNA damage in S. quadricauda, with no cell cycle arrest, after experiencing DNA damage during G2 phase. PMID:21603605

  11. DNA damage during G2 phase does not affect cell cycle progression of the green alga Scenedesmus quadricauda.

    Directory of Open Access Journals (Sweden)

    Monika Hlavová

    Full Text Available DNA damage is a threat to genomic integrity in all living organisms. Plants and green algae are particularly susceptible to DNA damage especially that caused by UV light, due to their light dependency for photosynthesis. For survival of a plant, and other eukaryotic cells, it is essential for an organism to continuously check the integrity of its genetic material and, when damaged, to repair it immediately. Cells therefore utilize a DNA damage response pathway that is responsible for sensing, reacting to and repairing damaged DNA. We have studied the effect of 5-fluorodeoxyuridine, zeocin, caffeine and combinations of these on the cell cycle of the green alga Scenedesmus quadricauda. The cells delayed S phase and underwent a permanent G2 phase block if DNA metabolism was affected prior to S phase; the G2 phase block imposed by zeocin was partially abolished by caffeine. No cell cycle block was observed if the treatment with zeocin occurred in G2 phase and the cells divided normally. CDKA and CDKB kinases regulate mitosis in S. quadricauda; their kinase activities were inhibited by Wee1. CDKA, CDKB protein levels were stabilized in the presence of zeocin. In contrast, the protein level of Wee1 was unaffected by DNA perturbing treatments. Wee1 therefore does not appear to be involved in the DNA damage response in S. quadricauda. Our results imply a specific reaction to DNA damage in S. quadricauda, with no cell cycle arrest, after experiencing DNA damage during G2 phase.

  12. Deficiency in DNA damage response of enterocytes accelerates intestinal stem cell aging in Drosophila.

    Science.gov (United States)

    Park, Joung-Sun; Jeon, Ho-Jun; Pyo, Jung-Hoon; Kim, Young-Shin; Yoo, Mi-Ae

    2018-03-07

    Stem cell dysfunction is closely linked to tissue and organismal aging and age-related diseases, and heavily influenced by the niche cells' environment. The DNA damage response (DDR) is a key pathway for tissue degeneration and organismal aging; however, the precise protective role of DDR in stem cell/niche aging is unclear. The Drosophila midgut is an excellent model to study the biology of stem cell/niche aging because of its easy genetic manipulation and its short lifespan. Here, we showed that deficiency of DDR in Drosophila enterocytes (ECs) accelerates intestinal stem cell (ISC) aging. We generated flies with knockdown of Mre11 , Rad50 , Nbs1 , ATM , ATR , Chk1 , and Chk2 , which decrease the DDR system in ECs. EC-specific DDR depletion induced EC death, accelerated the aging of ISCs, as evidenced by ISC hyperproliferation, DNA damage accumulation, and increased centrosome amplification, and affected the adult fly's survival. Our data indicated a distinct effect of DDR depletion in stem or niche cells on tissue-resident stem cell proliferation. Our findings provide evidence of the essential role of DDR in protecting EC against ISC aging, thus providing a better understanding of the molecular mechanisms of stem cell/niche aging.

  13. The DNA damage response during mitosis

    International Nuclear Information System (INIS)

    Heijink, Anne Margriet; Krajewska, Małgorzata; Vugt, Marcel A.T.M. van

    2013-01-01

    Cells are equipped with a cell-intrinsic signaling network called the DNA damage response (DDR). This signaling network recognizes DNA lesions and initiates various downstream pathways to coordinate a cell cycle arrest with the repair of the damaged DNA. Alternatively, the DDR can mediate clearance of affected cells that are beyond repair through apoptosis or senescence. The DDR can be activated in response to DNA damage throughout the cell cycle, although the extent of DDR signaling is different in each cell cycle phase. Especially in response to DNA double strand breaks, only a very marginal response was observed during mitosis. Early on it was recognized that cells which are irradiated during mitosis continued division without repairing broken chromosomes. Although these initial observations indicated diminished DNA repair and lack of an acute DNA damage-induced cell cycle arrest, insight into the mechanistic re-wiring of DDR signaling during mitosis was only recently provided. Different mechanisms appear to be at play to inactivate specific signaling axes of the DDR network in mitosis. Importantly, mitotic cells not simply inactivate the entire DDR, but appear to mark their DNA damage for repair after mitotic exit. Since the treatment of cancer frequently involves agents that induce DNA damage as well as agents that block mitotic progression, it is clinically relevant to obtain a better understanding of how cancer cells deal with DNA damage during interphase versus mitosis. In this review, the molecular details concerning DDR signaling during mitosis as well as the consequences of encountering DNA damage during mitosis for cellular fate are discussed

  14. The DNA damage response during mitosis

    Energy Technology Data Exchange (ETDEWEB)

    Heijink, Anne Margriet; Krajewska, Małgorzata; Vugt, Marcel A.T.M. van, E-mail: m.vugt@umcg.nl

    2013-10-15

    Cells are equipped with a cell-intrinsic signaling network called the DNA damage response (DDR). This signaling network recognizes DNA lesions and initiates various downstream pathways to coordinate a cell cycle arrest with the repair of the damaged DNA. Alternatively, the DDR can mediate clearance of affected cells that are beyond repair through apoptosis or senescence. The DDR can be activated in response to DNA damage throughout the cell cycle, although the extent of DDR signaling is different in each cell cycle phase. Especially in response to DNA double strand breaks, only a very marginal response was observed during mitosis. Early on it was recognized that cells which are irradiated during mitosis continued division without repairing broken chromosomes. Although these initial observations indicated diminished DNA repair and lack of an acute DNA damage-induced cell cycle arrest, insight into the mechanistic re-wiring of DDR signaling during mitosis was only recently provided. Different mechanisms appear to be at play to inactivate specific signaling axes of the DDR network in mitosis. Importantly, mitotic cells not simply inactivate the entire DDR, but appear to mark their DNA damage for repair after mitotic exit. Since the treatment of cancer frequently involves agents that induce DNA damage as well as agents that block mitotic progression, it is clinically relevant to obtain a better understanding of how cancer cells deal with DNA damage during interphase versus mitosis. In this review, the molecular details concerning DDR signaling during mitosis as well as the consequences of encountering DNA damage during mitosis for cellular fate are discussed.

  15. The DNA damage response during mitosis.

    Science.gov (United States)

    Heijink, Anne Margriet; Krajewska, Małgorzata; van Vugt, Marcel A T M

    2013-10-01

    Cells are equipped with a cell-intrinsic signaling network called the DNA damage response (DDR). This signaling network recognizes DNA lesions and initiates various downstream pathways to coordinate a cell cycle arrest with the repair of the damaged DNA. Alternatively, the DDR can mediate clearance of affected cells that are beyond repair through apoptosis or senescence. The DDR can be activated in response to DNA damage throughout the cell cycle, although the extent of DDR signaling is different in each cell cycle phase. Especially in response to DNA double strand breaks, only a very marginal response was observed during mitosis. Early on it was recognized that cells which are irradiated during mitosis continued division without repairing broken chromosomes. Although these initial observations indicated diminished DNA repair and lack of an acute DNA damage-induced cell cycle arrest, insight into the mechanistic re-wiring of DDR signaling during mitosis was only recently provided. Different mechanisms appear to be at play to inactivate specific signaling axes of the DDR network in mitosis. Importantly, mitotic cells not simply inactivate the entire DDR, but appear to mark their DNA damage for repair after mitotic exit. Since the treatment of cancer frequently involves agents that induce DNA damage as well as agents that block mitotic progression, it is clinically relevant to obtain a better understanding of how cancer cells deal with DNA damage during interphase versus mitosis. In this review, the molecular details concerning DDR signaling during mitosis as well as the consequences of encountering DNA damage during mitosis for cellular fate are discussed. Copyright © 2013 Elsevier B.V. All rights reserved.

  16. Autophagy in DNA Damage Response

    Directory of Open Access Journals (Sweden)

    Piotr Czarny

    2015-01-01

    Full Text Available DNA damage response (DDR involves DNA repair, cell cycle regulation and apoptosis, but autophagy is also suggested to play a role in DDR. Autophagy can be activated in response to DNA-damaging agents, but the exact mechanism underlying this activation is not fully understood, although it is suggested that it involves the inhibition of mammalian target of rapamycin complex 1 (mTORC1. mTORC1 represses autophagy via phosphorylation of the ULK1/2–Atg13–FIP200 complex thus preventing maturation of pre-autophagosomal structures. When DNA damage occurs, it is recognized by some proteins or their complexes, such as poly(ADPribose polymerase 1 (PARP-1, Mre11–Rad50–Nbs1 (MRN complex or FOXO3, which activate repressors of mTORC1. SQSTM1/p62 is one of the proteins whose levels are regulated via autophagic degradation. Inhibition of autophagy by knockout of FIP200 results in upregulation of SQSTM1/p62, enhanced DNA damage and less efficient damage repair. Mitophagy, one form of autophagy involved in the selective degradation of mitochondria, may also play role in DDR. It degrades abnormal mitochondria and can either repress or activate apoptosis, but the exact mechanism remains unknown. There is a need to clarify the role of autophagy in DDR, as this process may possess several important biomedical applications, involving also cancer therapy.

  17. Sinularin Selectively Kills Breast Cancer Cells Showing G2/M Arrest, Apoptosis, and Oxidative DNA Damage

    Directory of Open Access Journals (Sweden)

    Hurng-Wern Huang

    2018-04-01

    Full Text Available The natural compound sinularin, isolated from marine soft corals, is antiproliferative against several cancers, but its possible selective killing effect has rarely been investigated. This study investigates the selective killing potential and mechanisms of sinularin-treated breast cancer cells. In 3-(4,5-dimethylthiazol-2-yl-5-(3-carboxymethoxyphenyl-2-(4-sulfophenyl-2H- tetrazolium, inner salt (MTS assay, sinularin dose-responsively decreased the cell viability of two breast cancer (SKBR3 and MDA-MB-231 cells, but showed less effect on breast normal (M10 cells after a 24 h treatment. According to 7-aminoactinomycin D (7AAD flow cytometry, sinularin dose-responsively induced the G2/M cycle arrest of SKBR3 cells. Sinularin dose-responsively induced apoptosis on SKBR3 cells in terms of a flow cytometry-based annexin V/7AAD assay and pancaspase activity, as well as Western blotting for cleaved forms of poly(ADP-ribose polymerase (PARP, caspases 3, 8, and 9. These caspases and PARP activations were suppressed by N-acetylcysteine (NAC pretreatment. Moreover, sinularin dose-responsively induced oxidative stress and DNA damage according to flow cytometry analyses of reactive oxygen species (ROS, mitochondrial membrane potential (MitoMP, mitochondrial superoxide, and 8-oxo-2′-deoxyguanosine (8-oxodG. In conclusion, sinularin induces selective killing, G2/M arrest, apoptosis, and oxidative DNA damage of breast cancer cells.

  18. DNA-damage-induced differentiation of leukaemic cells as an anti-cancer barrier.

    Science.gov (United States)

    Santos, Margarida A; Faryabi, Robert B; Ergen, Aysegul V; Day, Amanda M; Malhowski, Amy; Canela, Andres; Onozawa, Masahiro; Lee, Ji-Eun; Callen, Elsa; Gutierrez-Martinez, Paula; Chen, Hua-Tang; Wong, Nancy; Finkel, Nadia; Deshpande, Aniruddha; Sharrow, Susan; Rossi, Derrick J; Ito, Keisuke; Ge, Kai; Aplan, Peter D; Armstrong, Scott A; Nussenzweig, André

    2014-10-02

    Self-renewal is the hallmark feature both of normal stem cells and cancer stem cells. Since the regenerative capacity of normal haematopoietic stem cells is limited by the accumulation of reactive oxygen species and DNA double-strand breaks, we speculated that DNA damage might also constrain leukaemic self-renewal and malignant haematopoiesis. Here we show that the histone methyl-transferase MLL4, a suppressor of B-cell lymphoma, is required for stem-cell activity and an aggressive form of acute myeloid leukaemia harbouring the MLL-AF9 oncogene. Deletion of MLL4 enhances myelopoiesis and myeloid differentiation of leukaemic blasts, which protects mice from death related to acute myeloid leukaemia. MLL4 exerts its function by regulating transcriptional programs associated with the antioxidant response. Addition of reactive oxygen species scavengers or ectopic expression of FOXO3 protects MLL4(-/-) MLL-AF9 cells from DNA damage and inhibits myeloid maturation. Similar to MLL4 deficiency, loss of ATM or BRCA1 sensitizes transformed cells to differentiation, suggesting that myeloid differentiation is promoted by loss of genome integrity. Indeed, we show that restriction-enzyme-induced double-strand breaks are sufficient to induce differentiation of MLL-AF9 blasts, which requires cyclin-dependent kinase inhibitor p21(Cip1) (Cdkn1a) activity. In summary, we have uncovered an unexpected tumour-promoting role of genome guardians in enforcing the oncogene-induced differentiation blockade in acute myeloid leukaemia.

  19. Phytometabolite Dehydroleucodine Induces Cell Cycle Arrest, Apoptosis, and DNA Damage in Human Astrocytoma Cells through p73/p53 Regulation.

    Directory of Open Access Journals (Sweden)

    Natalia Bailon-Moscoso

    Full Text Available Accumulating evidence supports the idea that secondary metabolites obtained from medicinal plants (phytometabolites may be important contributors in the development of new chemotherapeutic agents to reduce the occurrence or recurrence of cancer. Our study focused on Dehydroleucodine (DhL, a sesquiterpene found in the provinces of Loja and Zamora-Chinchipe. In this study, we showed that DhL displayed cytostatic and cytotoxic activities on the human cerebral astrocytoma D384 cell line. With lactone isolated from Gynoxys verrucosa Wedd, a medicinal plant from Ecuador, we found that DhL induced cell death in D384 cells by triggering cell cycle arrest and inducing apoptosis and DNA damage. We further found that the cell death resulted in the increased expression of CDKN1A and BAX proteins. A marked induction of the levels of total TP73 and phosphorylated TP53, TP73, and γ-H2AX proteins was observed in D384 cells exposed to DhL, but no increase in total TP53 levels was detected. Overall these studies demonstrated the marked effect of DhL on the diminished survival of human astrocytoma cells through the induced expression of TP73 and phosphorylation of TP73 and TP53, suggesting their key roles in the tumor cell response to DhL treatment.

  20. Phytometabolite Dehydroleucodine Induces Cell Cycle Arrest, Apoptosis, and DNA Damage in Human Astrocytoma Cells through p73/p53 Regulation.

    Science.gov (United States)

    Bailon-Moscoso, Natalia; González-Arévalo, Gabriela; Velásquez-Rojas, Gabriela; Malagon, Omar; Vidari, Giovanni; Zentella-Dehesa, Alejandro; Ratovitski, Edward A; Ostrosky-Wegman, Patricia

    2015-01-01

    Accumulating evidence supports the idea that secondary metabolites obtained from medicinal plants (phytometabolites) may be important contributors in the development of new chemotherapeutic agents to reduce the occurrence or recurrence of cancer. Our study focused on Dehydroleucodine (DhL), a sesquiterpene found in the provinces of Loja and Zamora-Chinchipe. In this study, we showed that DhL displayed cytostatic and cytotoxic activities on the human cerebral astrocytoma D384 cell line. With lactone isolated from Gynoxys verrucosa Wedd, a medicinal plant from Ecuador, we found that DhL induced cell death in D384 cells by triggering cell cycle arrest and inducing apoptosis and DNA damage. We further found that the cell death resulted in the increased expression of CDKN1A and BAX proteins. A marked induction of the levels of total TP73 and phosphorylated TP53, TP73, and γ-H2AX proteins was observed in D384 cells exposed to DhL, but no increase in total TP53 levels was detected. Overall these studies demonstrated the marked effect of DhL on the diminished survival of human astrocytoma cells through the induced expression of TP73 and phosphorylation of TP73 and TP53, suggesting their key roles in the tumor cell response to DhL treatment.

  1. Adaptive response to DNA-damaging agents in natural Saccharomyces cerevisiae populations from "Evolution Canyon", Mt. Carmel, Israel.

    Directory of Open Access Journals (Sweden)

    Gabriel A Lidzbarsky

    2009-06-01

    Full Text Available Natural populations of most organisms, especially unicellular microorganisms, are constantly exposed to harsh environmental factors which affect their growth. UV radiation is one of the most important physical parameters which influences yeast growth in nature. Here we used 46 natural strains of Saccharomyces cerevisiae isolated from several natural populations at the "Evolution Canyon" microsite (Nahal Oren, Mt. Carmel, Israel. The opposing slopes of this canyon share the same geology, soil, and macroclimate, but they differ in microclimatic conditions. The interslope differences in solar radiation (200%-800% more on the "African" slope caused the development of two distinct biomes. The south-facing slope is sunnier and has xeric, savannoid "African" environment while the north-facing slope is represented by temperate, "European" forested environment. Here we studied the phenotypic response of the S. cerevisiae strains to UVA and UVC radiations and to methyl methanesulfonate (MMS in order to evaluate the interslope effect on the strains' ability to withstand DNA-damaging agents.We exposed our strains to the different DNA-damaging agents and measured survival by counting colony forming units. The strains from the "African" slope were more resilient to both UVA and MMS than the strains from the "European" slope. In contrast, we found that there was almost no difference between strains (with similar ploidy from the opposite slopes, in their sensitivity to UVC radiation. These results suggest that the "African" strains are more adapted to higher solar radiation than the "European" strains. We also found that the tetraploids strains were more tolerant to all DNA-damaging agents than their neighboring diploid strains, which suggest that high ploidy level might be a mechanism of adaptation to high solar radiation.Our results and the results of parallel studies with several other organisms, suggest that natural selection appears to select, at a

  2. 10-Acetylirciformonin B, A Sponge Furanoterpenoid, Induces DNA Damage and Apoptosis in Leukemia Cells

    Directory of Open Access Journals (Sweden)

    Fu-Wen Kuo

    2012-10-01

    Full Text Available 10-Acetylirciformonin B, a furanoterpenoid derived from irciformonin B found in a marine sponge, has been reported to possess potent cytotoxic activity against several cancer cell lines. However, the mechanism of its apoptotic activity against human leukemia cells has never been reported. The purpose of this study was to investigate the cytotoxic effects of 10-acetylirciformonin B and its possible mechanism of action against leukemia HL 60 cells. We found that 10-acetylirciformonin B decreased cell viability through the inhibition of cell growth as well as the induction of DNA damage and apoptosis in a dose-dependent manner. The induction of DNA damage was mediated by the increase of p-CHK2 and γ-H2A.X, which was suggested from the increase of tail movement in the neutral Comet assay. Induction of apoptosis was mediated with the increase in caspases 8, 9 and 3 activation as well as PARP cleavage. In summary, our resultsindicate that 10-acetylirciformonin B treatment causes apoptosis in leukaemia cells; probably through a caspase-dependent regulatory pathway.

  3. Chloroethylating nitrosoureas in cancer therapy: DNA damage, repair and cell death signaling.

    Science.gov (United States)

    Nikolova, Teodora; Roos, Wynand P; Krämer, Oliver H; Strik, Herwig M; Kaina, Bernd

    2017-08-01

    Chloroethylating nitrosoureas (CNU), such as lomustine, nimustine, semustine, carmustine and fotemustine are used for the treatment of malignant gliomas, brain metastases of different origin, melanomas and Hodgkin disease. They alkylate the DNA bases and give rise to the formation of monoadducts and subsequently interstrand crosslinks (ICL). ICL are critical cytotoxic DNA lesions that link the DNA strands covalently and block DNA replication and transcription. As a result, S phase progression is inhibited and cells are triggered to undergo apoptosis and necrosis, which both contribute to the effectiveness of CNU-based cancer therapy. However, tumor cells resist chemotherapy through the repair of CNU-induced DNA damage. The suicide enzyme O 6 -methylguanine-DNA methyltransferase (MGMT) removes the precursor DNA lesion O 6 -chloroethylguanine prior to its conversion into ICL. In cells lacking MGMT, the formed ICL evoke complex enzymatic networks to accomplish their removal. Here we discuss the mechanism of ICL repair as a survival strategy of healthy and cancer cells and DNA damage signaling as a mechanism contributing to CNU-induced cell death. We also discuss therapeutic implications and strategies based on sequential and simultaneous treatment with CNU and the methylating drug temozolomide. Copyright © 2017 Elsevier B.V. All rights reserved.

  4. Delayed chromosomal instability induced by DNA damage

    International Nuclear Information System (INIS)

    Morgan, W.F.; Marder, B.A.; Day, J.P.

    1994-01-01

    Cellular exposure to DNA damaging agents rapidly results in a dose dependent increase in chromosomal breakage and gross structural chromosomal rearrangements. Over recent years, evidence has been accumulating indicating genomic instability can manifest multiple generations after cellular exposure to physical and chemical DNA damaging agents. Genomic instability manifests in the progeny of surviving cells, and has been implicated in mutation, gene application, cellular transformation, and cell killing. To investigate chromosome instability following DNA damage, we have used fluorescence in situ hybridization to detect chromosomal rearrangements in a human/hamster somatic hybrid cell line following exposure to ionizing radiation. Delayed chromosomal instability was detected when multiple populations of uniquely arranged metaphases were observed in clonal isolates raised from single cells surviving X-irradiation many generations after exposure. At higher radiation doses, chromosomal instability was observed in a relatively high frequency of surviving clones and, in general, those clones showed delayed chromosome instability also showed reduced survival as measured by colony forming ability

  5. MicroRNAs, the DNA damage response and cancer

    International Nuclear Information System (INIS)

    Wouters, Maikel D.; Gent, Dik C. van; Hoeijmakers, Jan H.J.; Pothof, Joris

    2011-01-01

    Many carcinogenic agents such as ultra-violet light from the sun and various natural and man-made chemicals act by damaging the DNA. To deal with these potentially detrimental effects of DNA damage, cells induce a complex DNA damage response (DDR) that includes DNA repair, cell cycle checkpoints, damage tolerance systems and apoptosis. This DDR is a potent barrier against carcinogenesis and defects within this response are observed in many, if not all, human tumors. DDR defects fuel the evolution of precancerous cells to malignant tumors, but can also induce sensitivity to DNA damaging agents in cancer cells, which can be therapeutically exploited by the use of DNA damaging treatment modalities. Regulation of and coordination between sub-pathways within the DDR is important for maintaining genome stability. Although regulation of the DDR has been extensively studied at the transcriptional and post-translational level, less is known about post-transcriptional gene regulation by microRNAs, the topic of this review. More specifically, we highlight current knowledge about DNA damage responsive microRNAs and microRNAs that regulate DNA damage response genes. We end by discussing the role of DNA damage response microRNAs in cancer etiology and sensitivity to ionizing radiation and other DNA damaging therapeutic agents.

  6. The phytochemical 3,3'-diindolylmethane decreases expression of AR-controlled DNA damage repair genes through repressive chromatin modifications and is associated with DNA damage in prostate cancer cells.

    Science.gov (United States)

    Palomera-Sanchez, Zoraya; Watson, Gregory W; Wong, Carmen P; Beaver, Laura M; Williams, David E; Dashwood, Roderick H; Ho, Emily

    2017-09-01

    Androgen receptor (AR) is a transcription factor involved in normal prostate physiology and prostate cancer (PCa) development. 3,3'-Diindolylmethane (DIM) is a promising phytochemical agent against PCa that affects AR activity and epigenetic regulators in PCa cells. However, whether DIM suppresses PCa via epigenetic regulation of AR target genes is unknown. We assessed epigenetic regulation of AR target genes in LNCaP PCa cells and showed that DIM treatment led to epigenetic suppression of AR target genes involved in DNA repair (PARP1, MRE11, DNA-PK). Decreased expression of these genes was accompanied by an increase in repressive chromatin marks, loss of AR occupancy and EZH2 recruitment to their regulatory regions. Decreased DNA repair gene expression was associated with an increase in DNA damage (γH2Ax) and up-regulation of genomic repeat elements LINE1 and α-satellite. Our results suggest that DIM suppresses AR-dependent gene transcription through epigenetic modulation, leading to DNA damage and genome instability in PCa cells. Published by Elsevier Inc.

  7. Ionizing Radiation-Induced DNA Damage and Its Repair in Human Cells

    Energy Technology Data Exchange (ETDEWEB)

    Dizdaroglu, Miral

    1999-05-12

    DNA damage in mammalian chromatin in vitro and in cultured mammalian cells including human cells was studied. In the first phase of these studies, a cell culture laboratory was established. Necessary equipment including an incubator, a sterile laminar flow hood and several centrifuges was purchased. We have successfully grown several cell lines such as murine hybridoma cells, V79 cells and human K562 leukemia cells. This was followed by the establishment of a methodology for the isolation of chromatin from cells. This was a very important step, because a routine and successful isolation of chromatin was a prerequisite for the success of the further studies in this project, the aim of which was the measurement of DNA darnage in mammalian chromatin in vitro and in cultured cells. Chromatin isolation was accomplished using a slightly modified procedure of the one described by Mee & Adelstein (1981). For identification and quantitation of DNA damage in cells, analysis of chromatin was preferred over the analysis of "naked DNA" for the following reasons: i. DNA may not be extracted efficiently from nucleoprotein in exposed cells, due to formation of DNA-protein cross-links, ii. the extractability of DNA is well known to decrease with increasing doses of radiation, iii. portions of DNA may not be extracted due to fragmentation, iv. unextracted DNA may contain a significant portion of damaged DNA bases and DNA-protein cross-links. The technique of gas chromatography/mass spectrometry (GC/MS), which was used in the present project, permits the identification and quantitation of modified DNA bases in chromatin in the presence of proteins without the necessity of first isolating DNA from chromatin. This has been demonstrated previously by the results from our laboratory and by the results obtained during the course of the present project. The quality of isolated chromatin was tested by measurement of its content of DNA, proteins, and RNA, by analysis of its protein

  8. Ionizing Radiation-Induced DNA Damage and Its Repair in Human Cells

    International Nuclear Information System (INIS)

    Dizdaroglu, Miral

    1999-01-01

    DNA damage in mammalian chromatin in vitro and in cultured mammalian cells including human cells was studied. In the first phase of these studies, a cell culture laboratory was established. Necessary equipment including an incubator, a sterile laminar flow hood and several centrifuges was purchased. We have successfully grown several cell lines such as murine hybridoma cells, V79 cells and human K562 leukemia cells. This was followed by the establishment of a methodology for the isolation of chromatin from cells. This was a very important step, because a routine and successful isolation of chromatin was a prerequisite for the success of the further studies in this project, the aim of which was the measurement of DNA darnage in mammalian chromatin in vitro and in cultured cells. Chromatin isolation was accomplished using a slightly modified procedure of the one described by Mee ampersand Adelstein (1981). For identification and quantitation of DNA damage in cells, analysis of chromatin was preferred over the analysis of ''naked DNA'' for the following reasons: i. DNA may not be extracted efficiently from nucleoprotein in exposed cells, due to formation of DNA-protein cross-links, ii. the extractability of DNA is well known to decrease with increasing doses of radiation, iii. portions of DNA may not be extracted due to fragmentation, iv. unextracted DNA may contain a significant portion of damaged DNA bases and DNA-protein cross-links. The technique of gas chromatography/mass spectrometry (GC/MS), which was used in the present project, permits the identification and quantitation of modified DNA bases in chromatin in the presence of proteins without the necessity of first isolating DNA from chromatin. This has been demonstrated previously by the results from our laboratory and by the results obtained during the course of the present project. The quality of isolated chromatin was tested by measurement of its content of DNA, proteins, and RNA, by analysis of its protein

  9. Sulforaphane enhances irradiation effects in terms of perturbed cell cycle progression and increased DNA damage in pancreatic cancer cells.

    Directory of Open Access Journals (Sweden)

    Patrick Naumann

    Full Text Available Sulforaphane (SFN, an herbal isothiocyanate enriched in cruciferous vegetables like broccoli and cauliflower, has gained popularity for its antitumor effects in cell lines such as pancreatic cancer. Antiproliferative as well as radiosensitizing properties were reported for head and neck cancer but little is known about its effects in pancreatic cancer cells in combination with irradiation (RT.In four established pancreatic cancer cell lines we investigated clonogenic survival, analyzed cell cycle distribution and compared DNA damage via flow cytometry and western blot after treatment with SFN and RT.Both SFN and RT show a strong and dose dependent survival reduction in clonogenic assays, an induction of a G2/M cell cycle arrest and an increase in γH2AX protein level indicating DNA damage. Effects were more pronounced in combined treatment and both cell cycle perturbation and DNA damage persisted for a longer period than after SFN or RT alone. Moreover, SFN induced a loss of DNA repair proteins Ku 70, Ku 80 and XRCC4.Our results suggest that combination of SFN and RT exerts a more distinct DNA damage and growth inhibition than each treatment alone. SFN seems to be a viable option to improve treatment efficacy of chemoradiation with hopefully higher rates of secondary resectability after neoadjuvant treatment for pancreatic cancer.

  10. Fasting protects mice from lethal DNA damage by promoting small intestinal epithelial stem cell survival.

    Science.gov (United States)

    Tinkum, Kelsey L; Stemler, Kristina M; White, Lynn S; Loza, Andrew J; Jeter-Jones, Sabrina; Michalski, Basia M; Kuzmicki, Catherine; Pless, Robert; Stappenbeck, Thaddeus S; Piwnica-Worms, David; Piwnica-Worms, Helen

    2015-12-22

    Short-term fasting protects mice from lethal doses of chemotherapy through undetermined mechanisms. Herein, we demonstrate that fasting preserves small intestinal (SI) architecture by maintaining SI stem cell viability and SI barrier function following exposure to high-dose etoposide. Nearly all SI stem cells were lost in fed mice, whereas fasting promoted sufficient SI stem cell survival to preserve SI integrity after etoposide treatment. Lineage tracing demonstrated that multiple SI stem cell populations, marked by Lgr5, Bmi1, or HopX expression, contributed to fasting-induced survival. DNA repair and DNA damage response genes were elevated in SI stem/progenitor cells of fasted etoposide-treated mice, which importantly correlated with faster resolution of DNA double-strand breaks and less apoptosis. Thus, fasting preserved SI stem cell viability as well as SI architecture and barrier function suggesting that fasting may reduce host toxicity in patients undergoing dose intensive chemotherapy.

  11. Dysregulation of the DNA Damage Response and KMT2A Rearrangement in Fetal Liver Hematopoietic Cells.

    Directory of Open Access Journals (Sweden)

    Mai Nanya

    Full Text Available Etoposide, a topoisomerase 2 (TOP2 inhibitor, is associated with the development of KMT2A (MLL-rearranged infant leukemia. An epidemiological study suggested that in utero exposure to TOP2 inhibitors may be involved in generation of KMT2A (MLL rearrangement. The present study examined the mechanism underlying the development of KMT2A (MLL-rearranged infant leukemia in response to in utero exposure to etoposide in a mouse model. Fetal liver hematopoietic stem cells were more susceptible to etoposide than maternal bone marrow mononuclear cells. Etoposide-induced Kmt2a breakage was detected in fetal liver hematopoietic stem cells using a newly developed chromatin immunoprecipitation (ChIP assay. Assessment of etoposide-induced chromosomal translocation by next-generation RNA sequencing (RNA-seq identified several chimeric fusion messenger RNAs that were generated by etoposide treatment. However, Kmt2a (Mll-rearranged fusion mRNA was detected in Atm-knockout mice, which are defective in the DNA damage response, but not in wild-type mice. The present findings suggest that in utero exposure to TOP2 inhibitors induces Kmt2a rearrangement when the DNA damage response is defective.

  12. Colon Cancer Chemoprevention by Sage Tea Drinking: Decreased DNA Damage and Cell Proliferation.

    Science.gov (United States)

    Pedro, Dalila F N; Ramos, Alice A; Lima, Cristovao F; Baltazar, Fatima; Pereira-Wilson, Cristina

    2016-02-01

    Salvia officinalis and some of its isolated compounds have been found to be preventive of DNA damage and increased proliferation in vitro in colon cells. In the present study, we used the azoxymethane model to test effects of S. officinalis on colon cancer prevention in vivo. The results showed that sage treatment reduced the number of ACF formed only if administered before azoxymethane injection, demonstrating that sage tea drinking has a chemopreventive effect on colorectal cancer. A decrease in the proliferation marker Ki67 and in H2 O2 -induced and azoxymethane-induced DNA damage to colonocytes and lymphocytes were found with sage treatment. This confirms in vivo the chemopreventive effects of S. officinalis. Taken together, our results show that sage treatment prevented initiation phases of colon carcinogenesis, an effect due, at least in part, to DNA protection, and reduced proliferation rates of colon epithelial cell that prevent mutations and their fixation through cell replication. These chemopreventive effects of S. officinalis on colon cancer add to the many health benefits attributed to sage and encourage its consumption. Copyright © 2015 John Wiley & Sons, Ltd.

  13. DNA damage in blood cells in relation to chemotherapy and nutritional status in colorectal cancer patients-A pilot study.

    Science.gov (United States)

    Kværner, Ane Sørlie; Minaguchi, Jun; Yamani, Naouale El; Henriksen, Christine; Ræder, Hanna; Paur, Ingvild; Henriksen, Hege Berg; Wiedswang, Gro; Smeland, Sigbjørn; Blomhoff, Rune; Collins, Andrew Richard; Bøhn, Siv Kjølsrud

    2018-03-01

    DNA damage can be considered as a biomarker for toxicity and response to chemotherapy. It is not known whether the chemotherapy-induced genotoxicity is associated with malnutrition. In this pilot study, we assess genotoxicity by means of DNA damage in patients with lymph-node positive colorectal cancer (CRC) and explore associations with chemotherapy treatment and nutritional status. DNA damage was compared between patients receiving chemotherapy (n = 24) and those not receiving chemotherapy (n = 20). DNA damage was measured in frozen whole blood by the comet assay. Associations between DNA damage and various indicators of malnutrition were also explored, including Patient-Generated Subjective Global Assessment (PG-SGA), bioelectrical impedance analysis (BIA) and anthropometric measurements, using multiple linear regression models. Patients on chemotherapy have higher levels of DNA damage in blood cells than patients not receiving chemotherapy (median of 16.9 and 7.9% tail DNA respectively, p = 0.001). The moderately malnourished patients (PG-SGA category B), representing 41% of the patients, have higher levels of cellular DNA damage than patients with good nutritional status (mean difference of 7.5% tail DNA, p = 0.033). In conclusion, adjuvant chemotherapy and malnutrition are both associated with increased levels of DNA damage in blood cells of CRC patients. Carefully controlled longitudinal studies or randomized controlled trials should be performed to determine whether good nutritional status may protect against chemotherapy-induced genotoxicity and enhance compliance to therapy in CRC patients. Copyright © 2018 Elsevier B.V. All rights reserved.

  14. Cell Survival and DNA Damage in Normal Prostate Cells Irradiated Out-of-Field.

    LENUS (Irish Health Repository)

    Shields, L

    2014-10-31

    Interest in out-of-field radiation dose has been increasing with the introduction of new techniques, such as volumetric modulated arc therapy (VMAT). These new techniques offer superior conformity of high-dose regions to the target compared to conventional techniques, however more normal tissue is exposed to low-dose radiation with VMAT. There is a potential increase in radiobiological effectiveness associated with lower energy photons delivered during VMAT as normal cells are exposed to a temporal change in incident photon energy spectrum. During VMAT deliveries, normal cells can be exposed to the primary radiation beam, as well as to transmission and scatter radiation. The impact of low-dose radiation, radiation-induced bystander effect and change in energy spectrum on normal cells are not well understood. The current study examined cell survival and DNA damage in normal prostate cells after exposure to out-of-field radiation both with and without the transfer of bystander factors. The effect of a change in energy spectrum out-of-field compared to in-field was also investigated. Prostate cancer (LNCaP) and normal prostate (PNT1A) cells were placed in-field and out-of-field, respectively, with the PNT1A cells being located 1 cm from the field edge when in-field cells were being irradiated with 2 Gy. Clonogenic and γ-H2AX assays were performed postirradiation to examine cell survival and DNA damage. The assays were repeated when bystander factors from the LNCaP cells were transferred to the PNT1A cells and also when the PNT1A cells were irradiated in-field to a different energy spectrum. An average out-of-field dose of 10.8 ± 4.2 cGy produced a significant reduction in colony volume and increase in the number of γ-H2AX foci\\/cell in the PNT1A cells compared to the sham-irradiated control cells. An adaptive response was observed in the PNT1A cells having first received a low out-of-field dose and then the bystander factors. The PNT1A cells showed a significant

  15. Increased sensitivity of DNA damage response-deficient cells to stimulated microgravity-induced DNA lesions.

    Directory of Open Access Journals (Sweden)

    Nan Li

    Full Text Available Microgravity is a major stress factor that astronauts have to face in space. In the past, the effects of microgravity on genomic DNA damage were studied, and it seems that the effect on genomic DNA depends on cell types and the length of exposure time to microgravity or simulated microgravity (SMG. In this study we used mouse embryonic stem (MES and mouse embryonic fibroblast (MEF cells to assess the effects of SMG on DNA lesions. To acquire the insight into potential mechanisms by which cells resist and/or adapt to SMG, we also included Rad9-deleted MES and Mdc1-deleted MEF cells in addition to wild type cells in this study. We observed significant SMG-induced DNA double strand breaks (DSBs in Rad9-/- MES and Mdc1-/- MEF cells but not in their corresponding wild type cells. A similar pattern of DNA single strand break or modifications was also observed in Rad9-/- MES. As the exposure to SMG was prolonged, Rad9-/- MES cells adapted to the SMG disturbance by reducing the induced DNA lesions. The induced DNA lesions in Rad9-/- MES were due to SMG-induced reactive oxygen species (ROS. Interestingly, Mdc1-/- MEF cells were only partially adapted to the SMG disturbance. That is, the induced DNA lesions were reduced over time, but did not return to the control level while ROS returned to a control level. In addition, ROS was only partially responsible for the induced DNA lesions in Mdc1-/- MEF cells. Taken together, these data suggest that SMG is a weak genomic DNA stress and can aggravate genomic instability in cells with DNA damage response (DDR defects.

  16. EGFR-targeted plasmonic magnetic nanoparticles suppress lung tumor growth by abrogating G2/M cell-cycle arrest and inducing DNA damage

    Directory of Open Access Journals (Sweden)

    Kuroda S

    2014-08-01

    G2/M checkpoint by inhibiting BRCA1, Chk1, and phospho-Cdc2/CDK1 protein expression. In vivo therapy studies showed 225-NP treatment reduced EGFR phosphorylation, increased γH2AX foci, and induced tumor cell apoptosis, resulting in suppression of tumor growth. Conclusion: The 225-NP treatment induces DNA damage and abrogates G2/M phase of the cell cycle, leading to cellular apoptosis and suppression of lung tumor growth both in vitro and in vivo. Our findings provide a rationale for combining 225-NP with other DNA-damaging agents for achieving enhanced anticancer activity. Keywords: lung cancer, epidermal growth factor receptor, autophagy

  17. [Effect of DNA-damaging agents on the aerobic methylobacteria capable and incapable of utilizing dichloromethane].

    Science.gov (United States)

    Firsova, Iu E; Torgonskaia, M L; Doronina, N V; Trotsenko, Iu A

    2005-01-01

    Methylobacterium dichloromethanicum DM4, a degrader of dichloromethane (DCM), was more tolerant to the effect of H2O2 and UV irradiation than Methylobacterium extorquens AM1, which does not consume DCM. Addition of CH2Cl2 to methylobacteria with active serine, ribulose monophosphate, and ribulose bisphosphate pathways of C1 metabolism, grown on methanol, resulted in a 1.1- to 2.5-fold increase in the incorporation of [alpha-32P]dATP into DNA Klenow fragment (exo-). As DCM dehalogenase was not induced in this process, the increase in total lengths of DNA gaps resulted from the action of DCM rather than S-chloromethylglutathione (intermediate of primary dehalogenation). The degree of DNA damage in the presence of CH2Cl2 was lower in DCM degraders than methylobacteria incapable of degrading this pollutant. This suggests that DCM degraders possess a more efficient mechanism of DNA repair.

  18. Methotrexate induces DNA damage and inhibits homologous recombination repair in choriocarcinoma cells

    Directory of Open Access Journals (Sweden)

    Xie L

    2016-11-01

    Full Text Available Lisha Xie,1,* Tiancen Zhao,1,2,* Jing Cai,1 You Su,1 Zehua Wang,1 Weihong Dong1 1Department of Obstetrics and Gynecology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, 2Department of Obstetrics and Gynecology, Central Hospital of Wuhan, Wuhan, China *These authors contributed equally to this work Objective: The objective of this study was to investigate the mechanism of sensitivity to methotrexate (MTX in human choriocarcinoma cells regarding DNA damage response. Methods: Two choriocarcinoma cancer cell lines, JAR and JEG-3, were utilized in this study. An MTX-sensitive osteosarcoma cell line MG63, an MTX-resistant epithelial ovarian cancer cell line A2780 and an MTX-resistant cervical adenocarcinoma cell line Hela served as controls. Cell viability assay was carried out to assess MTX sensitivity of cell lines. MTX-induced DNA damage was evaluated by comet assay. Quantitative reverse transcription polymerase chain reaction was used to detect the mRNA levels of BRCA1, BRCA2, RAD51 and RAD52. The protein levels of γH2AX, RAD 51 and p53 were analyzed by Western blot. Results: Remarkable DNA strand breaks were observed in MTX-sensitive cell lines (JAR, JEG-3 and MG63 but not in MTX-resistant cancer cells (A2780 and Hela after 48 h of MTX treatment. Only in the choriocarcinoma cells, the expression of homologous recombination (HR repair gene RAD51 was dramatically suppressed by MTX in a dose- and time-dependent manner, accompanied with the increase in p53. Conclusion: The MTX-induced DNA strand breaks accompanied by deficiencies in HR repair may contribute to the hypersensitivity to chemotherapy in choriocarcinoma. Keywords: choriocarcinoma, chemotherapy hypersensitivity, DNA double-strand break, RAD51, p53

  19. Pharmacological targeting of valosin containing protein (VCP) induces DNA damage and selectively kills canine lymphoma cells

    International Nuclear Information System (INIS)

    Nadeau, Marie-Ève; Rico, Charlène; Tsoi, Mayra; Vivancos, Mélanie; Filimon, Sabin; Paquet, Marilène; Boerboom, Derek

    2015-01-01

    Valosin containing protein (VCP) is a critical mediator of protein homeostasis and may represent a valuable therapeutic target for several forms of cancer. Overexpression of VCP occurs in many cancers, and often in a manner correlating with malignancy and poor outcome. Here, we analyzed VCP expression in canine lymphoma and assessed its potential as a therapeutic target for this disease. VCP expression in canine lymphomas was evaluated by immunoblotting and immunohistochemistry. The canine lymphoma cell lines CLBL-1, 17–71 and CL-1 were treated with the VCP inhibitor Eeyarestatin 1 (EER-1) at varying concentrations and times and were assessed for viability by trypan blue exclusion, apoptosis by TUNEL and caspase activity assays, and proliferation by propidium iodide incorporation and FACS. The mechanism of EER-1 action was determined by immunoblotting and immunofluorescence analyses of Lys48 ubiquitin and markers of ER stress (DDIT3), autophagy (SQSTM1, MAP1LC3A) and DNA damage (γH2AFX). TRP53/ATM-dependent signaling pathway activity was assessed by immunoblotting for TRP53 and phospho-TRP53 and real-time RT-PCR measurement of Cdkn1a mRNA. VCP expression levels in canine B cell lymphomas were found to increase with grade. EER-1 treatment killed canine lymphoma cells preferentially over control peripheral blood mononuclear cells. EER-1 treatment of CLBL-1 cells was found to both induce apoptosis and cell cycle arrest in G1. Unexpectedly, EER-1 did not appear to act either by inducing ER stress or inhibiting the aggresome-autophagy pathway. Rather, a rapid and dramatic increase in γH2AFX expression was noted, indicating that EER-1 may act by promoting DNA damage accumulation. Increased TRP53 phosphorylation and Cdkn1a mRNA levels indicated an activation of the TRP53/ATM DNA damage response pathway in response to EER-1, likely contributing to the induction of apoptosis and cell cycle arrest. These results correlate VCP expression with malignancy in canine B cell

  20. Fibroblasts Cultured on Nanowires Exhibit Low Motility, Impaired Cell Division, and DNA Damage

    DEFF Research Database (Denmark)

    Persson, H.; Købler, Carsten; Mølhave, Kristian

    2013-01-01

    beam milling and scanning electron microscopy, highly curved but intact nuclear membranes are observed, showing no direct contact between the nanowires and the DNA. The nanowires possibly induce cellular stress and high respiration rates, which trigger the formation of ROS, which in turn results in DNA......Nanowires are commonly used as tools for interfacing living cells, acting as biomolecule-delivery vectors or electrodes. It is generally assumed that the small size of the nanowires ensures a minimal cellular perturbation, yet the effects of nanowires on cell migration and proliferation remain...... largely unknown. Fibroblast behaviour on vertical nanowire arrays is investigated, and it is shown that cell motility and proliferation rate are reduced on nanowires. Fibroblasts cultured on long nanowires exhibit failed cell division, DNA damage, increased ROS content and respiration. Using focused ion...

  1. Cytotoxic and DNA-damaging effects of methyl tert-butyl ether and its metabolites on HL-60 cells in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Tang, G.H. [Xian Medical Univ. (China); Shen, Y.; Shen, H.M. [National Univ. of Singapore (Singapore)] [and others

    1996-12-31

    Methyl tert-butyl ether (MTBE) is a widely used oxygenate in unleaded gasoline; however, few studies have been conducted on the toxicity of this compound. This study evaluates the cytotoxic and DNA-damaging effects of MTBE and its metabolites in a human haemopoietic cell line, HL-60. The metabolites of MTBE studied include tertiary butyl alcohol (TBA), {alpha}-hydroxyisobutyric acid (HIBA), and formaldehyde. Comet assay is used to assess DNA damage, and the cytotoxicity is investigated by lactate dehydrogenease (LDH) release. The results show no significant cytotoxic effects of MTBE, TBA, and HIBA over a concentration ranging from 1 to 30 mM. Formaldehyde, in contrast, causes a substantial LDH release at a concentration of 5 {mu}M. Hydrogen peroxide, a known oxidative agent, at concentrations ranging from 10 to 100 {mu}M, produces a significant dose-related increase in DNA damage, whereas a much higher concentration of MTBE (1 to 30 mM) is required to produce a similar observation. The genotoxic effects of TBA and HIBA appear to be identical to that of MTBE. Conversely, DNA damage is observed for formaldehyde at a relatively low concentration range (5 to 100 {mu}M). These findings suggest that MTBE and its metabolites, except formaldehyde, have relatively low cytotoxic and genotoxic effects. 16 refs., 4 figs.

  2. γ irradiation with different dose rates induces different DNA damage responses in Petunia x hybrida cells.

    Science.gov (United States)

    Donà, Mattia; Ventura, Lorenzo; Macovei, Anca; Confalonieri, Massimo; Savio, Monica; Giovannini, Annalisa; Carbonera, Daniela; Balestrazzi, Alma

    2013-05-15

    In plants, there is evidence that different dose rate exposures to gamma (γ) rays can cause different biological effects. The dynamics of DNA damage accumulation and molecular mechanisms that regulate recovery from radiation injury as a function of dose rate are poorly explored. To highlight dose-rate dependent differences in DNA damage, single cell gel electrophoresis was carried out on regenerating Petunia x hybrida leaf discs exposed to LDR (total dose 50 Gy, delivered at 0.33 Gy min(-1)) and HDR (total doses 50 and 100 Gy, delivered at 5.15 Gy min(-1)) γ-ray in the 0-24h time period after treatments. Significant fluctuations of double strand breaks and different repair capacities were observed between treatments in the 0-4h time period following irradiation. Dose-rate-dependent changes in the expression of the PhMT2 and PhAPX genes encoding a type 2 metallothionein and the cytosolic isoform of ascorbate peroxidase, respectively, were detected by Quantitative RealTime-Polymerase Chain Reaction. The PhMT2 and PhAPX genes were significantly up-regulated (3.0- and 0.7-fold) in response to HDR. The results are discussed in light of the potential practical applications of LDR-based treatments in mutation breeding. Copyright © 2013 Elsevier GmbH. All rights reserved.

  3. Periovulatory follicular fluid levels of estradiol trigger inflammatory and DNA damage responses in oviduct epithelial cells.

    Directory of Open Access Journals (Sweden)

    Sergio E Palma-Vera

    Full Text Available Ovarian steroid hormones (mainly E2 and P4 regulate oviduct physiology. Serum-E2 acts on the oviduct epithelium from the basolateral cell compartment. Upon ovulation, the apical compartment of the oviduct epithelium is temporarily exposed to follicular fluid, which contains much higher levels of E2 than serum. The aim of this study was to evaluate the effects of human periovulatory follicular fluid levels of E2 on oviduct epithelial cells using two porcine in vitro models.A cell line derived from the porcine oviductal epithelium (CCLV-RIE270 was characterized (lineage markers, proliferation characteristics and transformation status. Primary porcine oviduct epithelial cells (POEC were cultured in air-liquid interface and differentiation was assessed histologically. Both cultures were exposed to E2 (10 ng/ml and 200 ng/ml. Proliferation of CCLV-RIE270 and POEC was determined by real-time impedance monitoring and immunohistochemical detection of Ki67. Furthermore, marker gene expression for DNA damage response (DDR and inflammation was quantified.CCLV-RIE270 was not transformed and exhibited properties of secretory oviduct epithelial cells. Periovulatory follicular fluid levels of E2 (200 ng/ml upregulated the expression of inflammatory genes in CCLV-RIE270 but not in POEC (except for IL8. Expression of DDR genes was elevated in both models. A significant increase in cell proliferation could not be detected in response to E2.CCLV-RIE270 and POEC are complementary models to evaluate the consequences of oviduct exposure to follicular fluid components. Single administration of periovulatory follicular fluid E2 levels trigger inflammatory and DNA damage responses, but not proliferation in oviduct epithelial cells.

  4. Assessment of okadaic acid effects on cytotoxicity, DNA damage and DNA repair in human cells.

    Science.gov (United States)

    Valdiglesias, Vanessa; Méndez, Josefina; Pásaro, Eduardo; Cemeli, Eduardo; Anderson, Diana; Laffon, Blanca

    2010-07-07

    Okadaic acid (OA) is a phycotoxin produced by several types of dinoflagellates causing diarrheic shellfish poisoning (DSP) in humans. Symptoms induced by DSP toxins are mainly gastrointestinal, but the intoxication does not appear to be fatal. Despite this, this toxin presents a potential threat to human health even at concentrations too low to induce acute toxicity, since previous animal studies have shown that OA has very potent tumour promoting activity. However, its concrete action mechanism has not been described yet and the results reported with regard to OA cytotoxicity and genotoxicity are often contradictory. In the present study, the genotoxic and cytotoxic effects of OA on three different types of human cells (peripheral blood leukocytes, HepG2 hepatoma cells, and SHSY5Y neuroblastoma cells) were evaluated. Cells were treated with a range of OA concentrations in the presence and absence of S9 fraction, and MTT test and Comet assay were performed in order to evaluate cytotoxicity and genotoxicity, respectively. The possible effects of OA on DNA repair were also studied by means of the DNA repair competence assay, using bleomycin as DNA damage inductor. Treatment with OA in absence of S9 fraction induced not statistically significant decrease in cell viability and significant increase in DNA damage in all cell types at the highest concentrations investigated. However, only SHSY5Y cells showed OA induced genotoxic and cytotoxic effects in presence of S9 fraction. Furthermore, we found that OA can induce modulations in DNA repair processes when exposure was performed prior to BLM treatment, in co-exposure, or during the subsequent DNA repair process. Copyright 2010 Elsevier B.V. All rights reserved.

  5. Clustered DNA damages induced in human hematopoietic cells by low doses of ionizing radiation

    Science.gov (United States)

    Sutherland, Betsy M.; Bennett, Paula V.; Cintron-Torres, Nela; Hada, Megumi; Trunk, John; Monteleone, Denise; Sutherland, John C.; Laval, Jacques; Stanislaus, Marisha; Gewirtz, Alan

    2002-01-01

    Ionizing radiation induces clusters of DNA damages--oxidized bases, abasic sites and strand breaks--on opposing strands within a few helical turns. Such damages have been postulated to be difficult to repair, as are double strand breaks (one type of cluster). We have shown that low doses of low and high linear energy transfer (LET) radiation induce such damage clusters in human cells. In human cells, DSB are about 30% of the total of complex damages, and the levels of DSBs and oxidized pyrimidine clusters are similar. The dose responses for cluster induction in cells can be described by a linear relationship, implying that even low doses of ionizing radiation can produce clustered damages. Studies are in progress to determine whether clusters can be produced by mechanisms other than ionizing radiation, as well as the levels of various cluster types formed by low and high LET radiation.

  6. DNA Damages and White Blood Cell Death Processes in Victims with Severe Injury

    Directory of Open Access Journals (Sweden)

    V. V. Moroz

    2014-01-01

    Full Text Available Objective. To study the mechanisms of posttraumatic changes in the blood cells, by investigating DNA damages associat ed with hypoxia caused by massive blood loss (BL in severe injury.Subjects and methods. Ninetyfive patients aged 40.6±16.5 years (from 20 to 79 years who had sustained severe mechanical injury with different BL volumes (BLV (from 100 to 4000 ml and hemodynamic disorders were examined to study DNA damages and white blood cell necrotic and apop totic processes. In terms of the victims' weight, the mean BL was 21.5±16.5 ml/kg (from 1.4 to 61.5 ml/kg. The victimswere divided into 4 groups according to BLV: 1 26 victims whose BLV was less than 750 ml (5.93±2.41 ml/kg (grade I BL; 2 23 victims whose BLV was 750—1500 ml (11.5±1.5 ml/kg (grade 2 BL; 3 23 victims whose BLV was 1500—2000 ml (23.8±4.0 ml/kg (grade 3 BL; 4 23 victims whose BLV was over 2000 ml (45.6±10.1 ml/kg (grade 4 BL, according to the type of injury: 1 severe skeletal injury (SSI (n=17; 2 brain injury (BI (n=43; 3 a concurrence of SSI and BI (SSI+BI (n=35; according to the development of infectious complications: 1 69 victims who developed infectious com plications on days 5—7 postinjury; 2 26 victims who did not. To evaluate the impact of hypoxia on DNA damages, white blood cell apoptotic and necrotic processes, the victims were divided into 2 groups: 1 hypoxia (18 of the 95 victims who had 4 altered indicators, such as capillary blood pO2, plasma lactate levels, pH, and BE; 2 no hypoxia (10 of the 95 victims whose indicators were within the normal range. DNA damages and necrotic and apoptotic changes in the white blood cells were assessed by the DNA comet assay. The plasma concentration of extracellular DNA was fluorometrically determined using a QuantiTTM HS DNA Assay Kit (Invitrogen, USA. That of 8hydroxy2deoxyguanosine was estimated by enzyme immunoassay employing an 8hydroxy2deoxyGuanosine EIA Kit (Cayman Chemical, USA. The levels of cas

  7. Activation of protein kinase A and clustering of cell surface receptors by N-methyl-N'-nitro-N-nitrosoguanidine are independent of genomic DNA damage

    Energy Technology Data Exchange (ETDEWEB)

    Wang Zheng; Wang Guliang; Yang Jun; Guo Lei; Yu Yingnian

    2003-07-25

    Alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) induces cellular stress leading to chromosomal aberrations, mutations and cell death. Previous reports from our laboratory have shown that low concentration of MNNG induces untargeted mutation (UTM), which occurs on intact DNA in mammalian cells through changes in gene expression profile. It also causes the activation of cAMP-protein kinase A (PKA) and up-regulation of POL-{beta}, which is demonstrated to play a role in DNA repair system. In order to find out the possible initial signal involved in UTM, we try to investigate whether the activation of PKA-CREB signal pathway is closely related to DNA damage. Our data shows that the treatment of low concentration MNNG (0.2 {mu}M) activates PKA-CREB pathway in a comparable level both in a nuclear and enucleated cell system. And similar to the cell response caused by UV, the clustering of cell surface receptors of epidermal growth factor (EGF) and tumor necrosis factor {alpha} (TNF{alpha}) was also observed in cells exposed to MNNG. It was further demonstrated that the clustering of the surface receptors is independent of the genomic DNA damage, as this phenomenon was also observed in enucleated cells. These observations indicate that the initiation of signal cascades induced by low concentration of MNNG might be associated with its interaction with cell surface receptors and/or direct activation of related signal proteins but not its DNA damaging property.

  8. Polymyxin B causes DNA damage in HK-2 cells and mice.

    Science.gov (United States)

    Yun, B; Zhang, T; Azad, M A K; Wang, J; Nowell, C J; Kalitsis, P; Velkov, T; Hudson, D F; Li, J

    2018-03-20

    Increasing incidence of multidrug-resistant bacteria presents an imminent risk to global health. Polymyxins are 'last-resort' antibiotics against Gram-negative 'superbugs'; however, nephrotoxicity remains a key impediment in their clinical use. Molecular mechanisms underlying this nephrotoxicity remain poorly defined. Here, we examined the pathways which led to polymyxin B induced cell death in vitro and in vivo. Human proximal tubular cells were treated with polymyxin B (12.5-100 μM) for up to 24 h and showed a significant increase in micronuclei frequency, as well as abnormal mitotic events (over 40% in treated cells, p < 0.05). Time-course studies were performed using a mouse nephrotoxicity model (cumulative 72 mg/kg). Kidneys were collected over 48 h and investigated for histopathology and DNA damage. Notable increases in γH2AX foci (indicative of double-stranded breaks) were observed in both cell culture (up to ~ 44% cells with 5+ foci at 24 h, p < 0.05) and mice treated with polymyxin B (up to ~ 25%, p < 0.05). Consistent with these results, in vitro assays showed high binding affinity of polymyxin B to DNA. Together, our results indicate that polymyxin B nephrotoxicity is associated with DNA damage, leading to chromosome missegregation and genome instability. This novel mechanistic information may lead to new strategies to overcome the nephrotoxicity of this important last-line class of antibiotics.

  9. Radiation-induced DNA damage in tumors and normal tissues. II. Influence of dose, residual DNA damage and physiological factors in oxygenated cells

    International Nuclear Information System (INIS)

    Zhang, H.; Wheeler, K.T.

    1994-01-01

    Detection and quantification of hypoxic cells in solid tumors is important for many experimental and clinical situations. Several laboratories, including ours, have suggested that assays which measure radiation-induced DNA strand breaks and DNA-protein crosslinks (DPCs) might be used to detect or quantify hypoxic cells in tumors and normal tissues. Recently, we demonstrated the feasibility of using an alkaline elution assay that measures strand breaks and DPCs to detect and/or quantify hypoxic cells in tissues. For this approach to be valid, DPCs must not be formed to any great extent in irradiated oxygenated cells, and the formation and repair of strand breaks and DPCs in oxygenated cells must not be modified appreciably by physiological factors (e.g., temperature, pH and nutrient depletion) that are often found in solid tumors. To address these issues, two sets of experiments were performed. In one set of experiments, oxygenated 9L cells in tissue culture, subcutaneous 9L tumors and rat cerebella were irradiated with doses of 15 or 50 Gy and allowed to repair until the residual strand break damage was low enough to detect DPCs. In another set of experiments, oxygenated exponentially growing or plateau-phase 9L cells in tissue culture were irradiated with a dose of 15 Gy at 37 or 20 degrees C, while the cells were maintained at a pH of either 6.6 or 7.3. DNA-protein crosslinks were formed in oxygenated cells about 100 times less efficiently than in hypoxic cells. In addition, temperature, pH, nutrient depletion and growth phase did not appreciably alter the formation and repair of strand breaks or the formation of DPCs in oxygenated 9L cells. These results support the use of this DNA damage assay for the detection and quantification of hypoxic cells in solid tumors. 27 refs., 5 tabs

  10. Modulation of DNA damage response and induction of apoptosis mediates synergism between doxorubicin and a new imidazopyridine derivative in breast and lung cancer cells.

    Science.gov (United States)

    El-Awady, Raafat A; Semreen, Mohammad H; Saber-Ayad, Maha M; Cyprian, Farhan; Menon, Varsha; Al-Tel, Taleb H

    2016-01-01

    DNA damage response machinery (DDR) is an attractive target of cancer therapy. Modulation of DDR network may alter the response of cancer cells to DNA damaging anticancer drugs such as doxorubicin. The aim of the present study is to investigate the effects of a newly developed imidazopyridine (IAZP) derivative on the DDR after induction of DNA damage in cancer cells by doxorubicin. Cytotoxicity sulphrhodamine-B assay showed a weak anti-proliferative effect of IAZP alone on six cancer cell lines (MCF7, A549, A549DOX11, HepG2, HeLa and M8) and a normal fibroblast strain. Combination of IAZP with doxorubicin resulted in synergism in lung (A549) and breast (MCF7) cancer cells but neither in the other cancer cell lines nor in normal fibroblasts. Molecular studies revealed that synergism is mediated by modulation of DNA damage response and induction of apoptosis. Using constant-field gel electrophoresis and immunofluorescence detection of γ-H2AX foci, IAZP was shown to inhibit the repair of doxorubicin-induced DNA damage in A549 and MCF7 cells. Immunoblot analysis showed that IAZP suppresses the phosphorylation of the ataxia lelangiectasia and Rad3 related (ATR) protein, which is an important player in the response of cancer cells to chemotherapy-induced DNA damage. Moreover, IAZP augmented the doxorubicin-induced degradation of p21, activation of p53, CDK2, caspase 3/7 and phosphorylation of Rb protein. These effects enhanced doxorubicin-induced apoptosis in both cell lines. Our results indicate that IAZP is a promising agent that may enhance the cytotoxic effects of doxorubicin on some cancer cells through targeting the DDR. It is a preliminary step toward the clinical application of IAZP in combination with anticancer drugs and opens the avenue for the development of compounds targeting the DDR pathway that might improve the therapeutic index of anticancer drugs and enhance their cure rate. Copyright © 2015 Elsevier B.V. All rights reserved.

  11. Ochratoxin A: induction of (oxidative) DNA damage, cytotoxicity and apoptosis in mammalian cell lines and primary cells

    International Nuclear Information System (INIS)

    Kamp, Hennicke G.; Eisenbrand, Gerhard; Schlatter, Josef; Wuerth, Kirsten; Janzowski, Christine

    2005-01-01

    Ochratoxin A (OTA) is a nephrotoxic/-carcinogenic mycotoxin, produced by several Aspergillus- and Penicillium-strains. Humans are exposed to OTA via food contamination, a causal relationship of OTA to human endemic Balkan nephropathy is still under debate. Since DNA-adducts of OTA or its metabolites could not be identified unambiguously, its carcinogenic effectiveness might be related to secondary effects, such as oxidative cell damage or cell proliferation. In this study, OTA mediated induction of (oxidative) DNA damage, cytotoxicity (necrosis, growth inhibition, apoptosis) and modulation of glutathione were investigated in cell lines (V79, CV-1) and primary rat kidney cells. After 24 h incubation, viability of V79 cells was strongly decreased by OTA concentrations >2.5 μmol/L, whereas CV-1 cells were clearly less sensitive. Strong growth inhibition occurred in both cell lines (IC 50 ∼2 μmol/L). Apoptosis, detected with an immunochemical test and with flow cytometry, was induced by >1 μmol/L OTA. Oxidative DNA damage, detected by comet assay after additional treatment with repair enzymes, was induced in all cell systems already at five-fold lower concentrations. Glutathione in CV-1 cells was depleted after 1 h incubation (>100 μmol/L). In contrast, an increase was measured after 24 h incubation (>0.5 μmol/L). In conclusion, OTA induces oxidative DNA damage at low, not yet cytotoxic concentrations. Oxidative DNA damage might initiate cell transformation eventually in connection with proliferative response following cytotoxic cell death. Both events might represent pivotal factors in the chain of cellular events leading into nephro-carcinogenicity of OTA

  12. Telomeric Allelic Imbalance Indicates Defective DNA Repair and Sensitivity to DNA-Damaging Agents

    DEFF Research Database (Denmark)

    Birkbak, Nicolai J.; Wang, Zhigang C.; Kim, Ji-Young

    2012-01-01

    with triple-negative breast cancer (TNBC). In serous ovarian cancer treated with platinum-based chemotherapy, higher levels of NtAI forecast a better initial response. We found an inverse relationship between BRCA1 expression and NtAI in sporadic TNBC and serous ovarian cancers without BRCA1 or BRCA2 mutation...... of defective DNA repair in cell lines and tumors and correlated these signatures to platinum sensitivity. The number of subchromosomal regions with allelic imbalance extending to the telomere (NtAI) predicted cisplatin sensitivity in vitro and pathologic response to preoperative cisplatin treatment in patients...... also benefit from these agents. NtAI, a genomic measure of unfaithfully repaired DNA, may identify cancer patients likely to benefit from treatments targeting defective DNA repair. Cancer Discov; 2(4); 366–75. ©2012 AACR. This article is highlighted in the In This Issue feature, p. 288...

  13. 2- and 4-Aminobiphenyls induce oxidative DNA damage in human hepatoma (Hep G2) cells via different mechanisms

    International Nuclear Information System (INIS)

    Wang Shuchi; Chung, Jing-Gung; Chen, C.-H.; Chen, S.-C.

    2006-01-01

    4-Aminobiphenyl (4-ABP) and its analogue, 2-aminobiphenyl (2-ABP), were examined for their ability to induce oxidative DNA damage in Hep G2 cells. Using the alkaline comet assay, we showed that 2-ABP and 4-ABP (25-200 μM) were able to induce the DNA damage in Hep G2 cells. With both compounds, formation of intracellular reactive oxygen species (ROS) was detected using flow cytometry analysis. Post-treatment of 2-ABP and 4-ABP-treated cells by endonuclease III (Endo III) or formamidopyrimidine-DNA glycosylase (Fpg) to determine the formation of oxidized pyrimidines or oxidized purines showed a significant increase of the extent of DNA migration. This indicated that oxidative DNA damage occurs in Hep G2 cells after exposure to 2-ABP and 4-ABP. This assumption was further substantiated by the fact that the spin traps, 5,5-dimethyl-pyrroline-N-oxide (DMPO) and N-tert-butyl-α-phenylnitrone (PBN), decreased DNA damage significantly. Furthermore, addition of the catalase (100 U/ml) caused a decrease in the DNA damage induced by 2-ABP or 4-ABP, indicating that H 2 O 2 is involved in ABP-induced DNA damage. Pre-incubation of the cells with the iron chelator desferrioxamine (DFO) (1 mM) and with the copper chelator neocupronine (NC) (100 μM) also decreased DNA damage in cells treated with 200 μM 2-ABP or 200 μM 4-ABP, while the calcium chelator {1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester}(BAPTA/AM) (10 μM) decreased only DNA strand breaks in cells exposed to 4-ABP. This suggested that ions are involved in the formation of DNA strand breaks. Using RT-PCR and Western blotting, lower inhibition of the expression of the OGG1 gene and of the OGG1 protein was observed in cells treated with 4-ABP, and 2-ABP-treated cells showed a marked reduction in the expression of OGG1 gene and OGG1 protein. Taken together, our finding indicated the mechanisms of induced oxidative DNA damage in Hep G2 cell by 2-ABP and 4-ABP are different, although both

  14. C/EBPβ represses p53 to promote cell survival downstream of DNA damage independent of oncogenic Ras and p19Arf

    Science.gov (United States)

    Ewing, SJ; Zhu, S; Zhu, F; House, JS; Smart, RC

    2013-01-01

    CCAAT/enhancer-binding protein-β (C/EBPβ) is a mediator of cell survival and tumorigenesis. When C/EBPβ−/− mice are treated with carcinogens that produce oncogenic Ras mutations in keratinocytes, they respond with abnormally elevated keratinocyte apoptosis and a block in skin tumorigenesis. Although this aberrant carcinogen-induced apoptosis results from abnormal upregulation of p53, it is not known whether upregulated p53 results from oncogenic Ras and its ability to induce p19Arf and/or activate DNA-damage response pathways or from direct carcinogen-induced DNA damage. We report that p19Arf is dramatically elevated in C/EBPβ−/− epidermis and that C/EBPβ represses a p19Arf promoter reporter. To determine whether p19Arf is responsible for the proapoptotic phenotype in C/EBPβ−/− mice, C/EBPβ−/−;p19Arf−/− mice were generated. C/EBPβ−/−;p19Arf−/− mice responded to carcinogen treatment with increased p53 and apoptosis, indicating p19Arf is not essential. To ascertain whether oncogenic Ras activation induces aberrant p53 and apoptosis in C/EBPβ−/− epidermis, we generated K14-ER:Ras; C/EBPβ−/− mice. Oncogenic Ras activation induced by 4-hydroxytamoxifen did not produce increased p53 or apoptosis. Finally, when C/EBPβ−/− mice were treated with differing types of DNA-damaging agents, including alkylating chemotherapeutic agents, they displayed aberrant levels of p53 and apoptosis. These results indicate that C/EBPβ represses p53 to promote cell survival downstream of DNA damage and suggest that inhibition of C/EBPβ may be a target for cancer cotherapy to increase the efficacy of alkylating chemotherapeutic agents. PMID:18636078

  15. Dissociation of DNA damage and mitochondrial injury caused by hydrogen peroxide in SV-40 transformed lung epithelial cells

    Directory of Open Access Journals (Sweden)

    Adcock Ian M

    2002-11-01

    Full Text Available Abstract Background Since lung epithelial cells are constantly being exposed to reactive oxygen intermediates (ROIs, the alveolar surface is a major site of oxidative stress, and each cell type may respond differently to oxidative stress. We compared the extent of oxidative DNA damage with that of mitochondrial injury in lung epithelial cells at the single cell level. Result DNA damage and mitochondrial injury were measured after oxidative stress in the SV-40 transformed lung epithelial cell line challenged with hydrogen peroxide (H2O2. Single cell analysis of DNA damage was determined by assessing the number of 8-oxo-2-deoxyguanosine (8-oxo-dG positive cells, a marker of DNA modification, and the length of a comet tail. Mitochondrial membrane potential, ΔΨm, was determined using JC-1. A 1 h pulse of H2O2 induced small amounts of apoptosis (3%. 8-oxo-dG-positive cells and the length of the comet tail increased within 1 h of exposure to H2O2. The number of cells with reduced ΔΨm increased after the addition of H2O2 in a concentration-dependent manner. In spite of a continual loss of ΔΨm, DNA fragmentation was reduced 2 h after exposure to H2O2. Conclusion The data suggest that SV-40 transformed lung epithelial cells are resistant to oxidative stress, showing that DNA damage can be dissociated from mitochondrial injury.

  16. Patterns of DNA damage response in intracranial germ cell tumors versus glioblastomas reflect cell of origin rather than brain environment

    DEFF Research Database (Denmark)

    Bartkova, Jirina; Hoei-Hansen, Christina E; Krizova, Katerina

    2014-01-01

    The DNA damage response (DDR) machinery becomes commonly activated in response to oncogenes and during early stages of development of solid malignancies, with an exception of testicular germ cell tumors (TGCTs). The active DDR signaling evokes cell death or senescence but this anti-tumor barrier ...... checkpoints in intracranial tumorigenesis, with implications for the differential biological responses of diverse tumor types to endogenous stress as well as to genotoxic treatments such as ionizing radiation or chemotherapy....

  17. 4β-Hydroxywithanolide E selectively induces oxidative DNA damage for selective killing of oral cancer cells.

    Science.gov (United States)

    Tang, Jen-Yang; Huang, Hurng-Wern; Wang, Hui-Ru; Chan, Ya-Ching; Haung, Jo-Wen; Shu, Chih-Wen; Wu, Yang-Chang; Chang, Hsueh-Wei

    2018-03-01

    Reactive oxygen species (ROS) induction had been previously reported in 4β-hydroxywithanolide (4βHWE)-induced selective killing of oral cancer cells, but the mechanism involving ROS and the DNA damage effect remain unclear. This study explores the role of ROS and oxidative DNA damage of 4βHWE in the selective killing of oral cancer cells. Changes in cell viability, morphology, ROS, DNA double strand break (DSB) signaling (γH2AX foci in immunofluorescence and DSB signaling in western blotting), and oxidative DNA damage (8-oxo-2'deoxyguanosine [8-oxodG]) were detected in 4βHWE-treated oral cancer (Ca9-22) and/or normal (HGF-1) cells. 4βHWE decreased cell viability, changed cell morphology and induced ROS generation in oral cancer cells rather than oral normal cells, which were recovered by a free radical scavenger N-acetylcysteine (NAC). For immunofluorescence, 4βHWE also accumulated more of the DSB marker, γH2AX foci, in oral cancer cells than in oral normal cells. For western blotting, DSB signaling proteins such as γH2AX and MRN complex (MRE11, RAD50, and NBS1) were overexpressed in 4βHWE-treated oral cancer cells in different concentrations and treatment time. In the formamidopyrimidine-DNA glycolyase (Fpg)-based comet assay and 8-oxodG-based flow cytometry, the 8-oxodG expressions were higher in 4βHWE-treated oral cancer cells than in oral normal cells. All the 4βHWE-induced DSB and oxidative DNA damage to oral cancer cells were recovered by NAC pretreatment. Taken together, the 4βHWE selectively induced DSB and oxidative DNA damage for the ROS-mediated selective killing of oral cancer cells. © 2017 Wiley Periodicals, Inc.

  18. Co-visualization of DNA damage and ion traversals in live mammalian cells using a fluorescent nuclear track detector

    International Nuclear Information System (INIS)

    Kodaira, Satoshi; Konishi, Teruaki; Kobayashi, Alisa

    2015-01-01

    The geometric locations of ion traversals in mammalian cells constitute important information in the study of heavy ion-induced biological effect. Single ion traversal through a cellular nucleus produces complex and massive DNA damage at a nanometer level, leading to cell inactivation, mutations and transformation. We present a novel approach that uses a fluorescent nuclear track detector (FNTD) for the simultaneous detection of the geometrical images of ion traversals and DNA damage in single cells using confocal microscopy. HT1080 or HT1080–53BP1-GFP cells were cultured on the surface of a FNTD and exposed to 5.1-MeV/n neon ions. The positions of the ion traversals were obtained as fluorescent images of a FNTD. Localized DNA damage in cells was identified as fluorescent spots of γ-H2AX or 53BP1-GFP. These track images and images of damaged DNA were obtained in a short time using a confocal laser scanning microscope. The geometrical distribution of DNA damage indicated by fluorescent γ-H2AX spots in fixed cells or fluorescent 53BP1-GFP spots in living cells was found to correlate well with the distribution of the ion traversals. This method will be useful for evaluating the number of ion hits on individual cells, not only for micro-beam but also for random-beam experiments. (author)

  19. Echinacoside Induces Apoptosis in Human SW480 Colorectal Cancer Cells by Induction of Oxidative DNA Damages

    Directory of Open Access Journals (Sweden)

    Liwei Dong

    2015-06-01

    Full Text Available Echinacoside is a natural compound with potent reactive oxygen species (ROS-scavenging and anti-oxidative bioactivities, which protect cells from oxidative damages. As cancer cells are often under intense oxidative stress, we therefore tested if Echinacoside treatment would promote cancer development. Surprisingly, we found that Echinacoside significantly inhibited the growth and proliferation of a panel of cancer cell lines. Treatment of the human SW480 cancer cells with Echinacoside resulted in marked apoptosis and cell cycle arrest, together with a significant increase in active caspase 3 and cleaved PARP, and upregulation of the G1/S-CDK blocker CDKN1B (p21. Interestingly, immunocytochemistry examination of drug-treated cancer cells revealed that Echinacoside caused a significant increase of intracellular oxidized guanine, 8-oxoG, and dramatic upregulation of the double-strand DNA break (DSB-binding protein 53BP1, suggesting that Echinacoside induced cell cycle arrest and apoptosis in SW480 cancer cells via induction of oxidative DNA damages. These results establish Echinacoside as a novel chemical scaffold for development of anticancer drugs.

  20. Oncogenic RAS enables DNA damage- and p53-dependent differentiation of acute myeloid leukemia cells in response to chemotherapy.

    Directory of Open Access Journals (Sweden)

    Mona Meyer

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

  1. Mitochondrial catalase overexpressed transgenic mice are protected against lung fibrosis in part via preventing alveolar epithelial cell mitochondrial DNA damage.

    Science.gov (United States)

    Kim, Seok-Jo; Cheresh, Paul; Jablonski, Renea P; Morales-Nebreda, Luisa; Cheng, Yuan; Hogan, Erin; Yeldandi, Anjana; Chi, Monica; Piseaux, Raul; Ridge, Karen; Michael Hart, C; Chandel, Navdeep; Scott Budinger, G R; Kamp, David W

    2016-12-01

    Alveolar epithelial cell (AEC) injury and mitochondrial dysfunction are important in the development of lung fibrosis. Our group has shown that in the asbestos exposed lung, the generation of mitochondrial reactive oxygen species (ROS) in AEC mediate mitochondrial DNA (mtDNA) damage and apoptosis which are necessary for lung fibrosis. These data suggest that mitochondrial-targeted antioxidants should ameliorate asbestos-induced lung. To determine whether transgenic mice that express mitochondrial-targeted catalase (MCAT) have reduced lung fibrosis following exposure to asbestos or bleomycin and, if so, whether this occurs in association with reduced AEC mtDNA damage and apoptosis. Crocidolite asbestos (100µg/50µL), TiO 2 (negative control), bleomycin (0.025 units/50µL), or PBS was instilled intratracheally in 8-10 week-old wild-type (WT - C57Bl/6J) or MCAT mice. The lungs were harvested at 21d. Lung fibrosis was quantified by collagen levels (Sircol) and lung fibrosis scores. AEC apoptosis was assessed by cleaved caspase-3 (CC-3)/Surfactant protein C (SFTPC) immunohistochemistry (IHC) and semi-quantitative analysis. AEC (primary AT2 cells from WT and MCAT mice and MLE-12 cells) mtDNA damage was assessed by a quantitative PCR-based assay, apoptosis was assessed by DNA fragmentation, and ROS production was assessed by a Mito-Sox assay. Compared to WT, crocidolite-exposed MCAT mice exhibit reduced pulmonary fibrosis as measured by lung collagen levels and lung fibrosis score. The protective effects in MCAT mice were accompanied by reduced AEC mtDNA damage and apoptosis. Similar findings were noted following bleomycin exposure. Euk-134, a mitochondrial SOD/catalase mimetic, attenuated MLE-12 cell DNA damage and apoptosis. Finally, compared to WT, asbestos-induced MCAT AT2 cell ROS production was reduced. Our finding that MCAT mice have reduced pulmonary fibrosis, AEC mtDNA damage and apoptosis following exposure to asbestos or bleomycin suggests an important role

  2. Hydroxychavicol, a key ingredient of Piper betle induces bacterial cell death by DNA damage and inhibition of cell division.

    Science.gov (United States)

    Singh, Deepti; Narayanamoorthy, Shwetha; Gamre, Sunita; Majumdar, Ananda Guha; Goswami, Manish; Gami, Umesh; Cherian, Susan; Subramanian, Mahesh

    2018-05-20

    Antibiotic resistance is a global problem and there is an urgent need to augment the arsenal against pathogenic bacteria. The emergence of different drug resistant bacteria is threatening human lives to be pushed towards the pre-antibiotic era. Botanical sources remain a vital source of diverse organic molecules that possess antibacterial property as well as augment existing antibacterial molecules. Piper betle, a climber, is widely used in south and south-east Asia whose leaves and nuts are consumed regularly. Hydroxychavicol (HC) isolated from Piper betle has been reported to possess antibacterial activity. It is currently not clear how the antibacterial activity of HC is manifested. In this investigation we show HC generates superoxide in E. coli cells. Antioxidants protected E. coli against HC induced cell death while gshA mutant was more sensitive to HC than wild type. DNA damage repair deficient mutants are hypersensitive to HC and HC induces the expression of DNA damage repair genes that repair oxidative DNA damage. HC treated E. coli cells are inhibited from growth and undergo DNA condensation. In vitro HC binds to DNA and cleaves it in presence of copper. Our data strongly indicates HC mediates bacterial cell death by ROS generation and DNA damage. Damage to iron sulfur proteins in the cells contribute to amplification of oxidative stress initiated by HC. Further HC is active against a number of Gram negative bacteria isolated from patients with a wide range of clinical symptoms and varied antibiotic resistance profiles. Copyright © 2018 Elsevier Inc. All rights reserved.

  3. A comparative study of radiation induced DNA damage and repair in buccal cells and lymphocytes assessed by single cell gel electrophoresis (comet) assay

    International Nuclear Information System (INIS)

    Dhillon, V.S.; Fenech, M.

    2003-01-01

    Full text: During the past few years, there has been increasing interest in epithelial cells from buccal mucosa for genotoxicity evaluation of different chemical and/or physical agents. In the present study we used the buccal and sublingual epithelial cells to detect both inter- and intra-individual variation in radiation induced DNA damage and repair. For this purpose we used the single cell gel electrophoresis assay which over the years has gained wide spread acceptance as a simple, sensitive and reliable assay to measure genotoxicity related effects as well as kinetics of DNA repair. Buccal and sublingual epithelial cells from six individuals (3 male and 3 females; 35-45 years old) were collected. Cells were then irradiated for 0, 2 and 4 Gy doses using 137 Cs-source (5.58 Gy min-1). After irradiation the cells were either placed immediately on ice or incubated at 37 deg C for 2 1/2 hour to allow cellular repair. We also studied G0 and G1 lymphocytes from the same individuals to compare the radiation-induced DNA damage and repair potential with the two types of buccal cells. Baseline DNA damage rate was significantly greater (p < 0.001) in buccal (28.18%) and sublingual epithelial cells (30.66) as compared to G0 (22.02%) and G1 (21.46%) lymphocytes. Radiation-induced DNA damage in buccal (19.34%, 2Gy; 21.41%, 4 Gy) and sublingual epithelial cells (18.11% and 20.60%) was very similar and significantly lower than that observed in lymphocytes (29.76%, 56.77% for G0 and 32.66%, 59.32% for G1). The extent of DNA repair in buccal and sublingual epithelial cells was significantly lower than that observed in lymphocytes. The results for buccal and sublingual epithelial cells were highly correlated with each other (r 0.9541) as were those of G0 and G1 lymphocytes (r 0.9868). The results suggest a much reduced capacity for cellular repair in buccal and sublingual epithelial cells

  4. DNA damage in oral mucosa cells of patients with fixed orthodontic appliances.

    Directory of Open Access Journals (Sweden)

    Farzin Heravi

    2013-12-01

    Full Text Available The release of toxic metal ions from orthodontic alloys has induced concerns regarding the biocompatibility of fixed appliances. This study investigated the genotoxic effect of metal appliances in a sample of patients undergoing fixed orthodontic treatment.The study included twenty-five healthy individuals requiring orthodontic therapy in both jaws. The patients were treated by stainless steel orthodontic brackets and nickel-titanium or stainless steel arch wires. The oral mucosa cells were gathered just before the appliance placement and 9 months later. The cells were centrifuged, fixed and dropped onto slides. After staining, the micronucleus (MN assay was used to determine genome alteration. The data were analyzed by paired sample t-test.The mean micronuclei frequency in the buccal mucosa was 10.6 ± 5.7 per 1000 cells before the appliance placement and 9.2 ± 6.37 per 1000 cells 9 months later. No significant difference was found in the MN count before and 9 months after therapy (p=0.336.Under the conditions used in this study, application of fixed orthodontic appliances did not expose healthy individuals to increased risk of DNA damage in oral mucosa cells.

  5. DNA damage in the gill cells of the marine scallop Mizuhopecten yessoensis during anoxic stress and aerobic recovery

    Science.gov (United States)

    Slobodskova, Valentina V.; Zhukovskaya, Avianna F.; Chelomin, Victor P.

    2012-06-01

    Anoxia-induced DNA damage in the gill cells of the marine scallop Mizuhopecten yessoensis was assessed with the alkaline comet assay (single-cell gel electrophoresis). The alkaline comet assay method for detecting DNA strand breaks and alkali labile sites in individual cells. DNA damage was determened in the scallops ( M. yessoensis) gill cells. The scallops were exposed to air for 8 h showing a clear increase in the levels of DNA damage. After the air exposure, M. yessoensis were re-submersed for a period of 12 h, leading values to return to a pre-aerial exposure level. Control animals were kept immersed during the whole period. The resulting data demonstrate that natural influences, such as oxygen depletion (anoxia) in seawater, can be responsible for the induction of DNA damage. If the scallops were re-immersed in oxic conditions, the anoxically induced breaks were repaired. The main mechanisms influencing the integrity of the DNA structure are discussed in this paper.

  6. The Fanconi anemia pathway: Repairing the link between DNA damage and squamous cell carcinoma

    International Nuclear Information System (INIS)

    Romick-Rosendale, Lindsey E.; Lui, Vivian W.Y.; Grandis, Jennifer R.; Wells, Susanne I.

    2013-01-01

    Fanconi anemia (FA) is a rare inherited recessive disease caused by mutations in one of fifteen genes known to encode FA pathway components. In response to DNA damage, nuclear FA proteins associate into high molecular weight complexes through a cascade of post-translational modifications and physical interactions, followed by the repair of damaged DNA. Hematopoietic cells are particularly sensitive to the loss of these interactions, and bone marrow failure occurs almost universally in FA patients. FA as a disease is further characterized by cancer susceptibility, which highlights the importance of the FA pathway in tumor suppression, and will be the focus of this review. Acute myeloid leukemia is the most common cancer type, often subsequent to bone marrow failure. However, FA patients are also at an extreme risk of squamous cell carcinoma (SCC) of the head and neck and gynecological tract, with an even greater incidence in those individuals who have received a bone marrow transplant and recovered from hematopoietic disease. FA tumor suppression in hematopoietic versus epithelial compartments could be mechanistically similar or distinct. Definition of compartment specific FA activities is now critical to assess the effects of today's bone marrow failure treatments on tomorrow's solid tumor development. It is our hope that current therapies can then be optimized to decrease the risk of malignant transformation in both hematopoietic and epithelial cells. Here we review our current understanding of the mechanisms of action of the Fanconi anemia pathway as it contributes to stress responses, DNA repair and squamous cell carcinoma susceptibility

  7. DNA damage does not appear to be a trigger for thermotolerance in mammalian cells

    Energy Technology Data Exchange (ETDEWEB)

    Anderson, R.L.; Shiu, E.; Fisher, G.A.; Hahn, G.M.

    1988-08-01

    The hypothesis that DNA damage is the trigger for thermotolerance in mammalian cells was tested in Chinese hamster ovary cells by looking for evidence of thermotolerance after ionizing radiation or ultraviolet light exposure. As previous studies have demonstrated that relatively non-toxic radiation exposures do not induce thermotolerance in mammalian cells (Li et al. 1976), higher doses, comparable to those used in yeast to induce thermotolerance (Mitchel and Morison 1984), were tested in this study. Doses of this magnitude are lethal to mammalian cells, thereby precluding the use of clonogenic survival as an endpoint. The authors used three alternative assays as indicators of the subsequent development of thermotolerance: (a) heat-induced inhibition of total protein synthesis, (b) heat-induced uptake of dansyl lysine, and (c) synthesis of heat shock proteins. Only total protein synthesis revealed evidence of a small degree of thermotolerance which occurred immediately after ..gamma..-radiation exposure. By 4 h postirradiation the tolerance, as measured by this assay, was no longer evident. No evidence of thermotolerance was seen following UV exposure. In addition, when a large radiation dose was given either immediately before or after a heat treatment used to induce thermotolerance, there was no alteration in the level of heat-induced tolerance, despite the extensive number of DNA strand breaks caused by the radiation.

  8. DNA damage does not appear to be a trigger for thermotolerance in mammalian cells

    International Nuclear Information System (INIS)

    Anderson, R.L.; Shiu, E.; Fisher, G.A.; Hahn, G.M.

    1988-01-01

    The hypothesis that DNA damage is the trigger for thermotolerance in mammalian cells was tested in Chinese hamster ovary cells by looking for evidence of thermotolerance after ionizing radiation or ultraviolet light exposure. As previous studies have demonstrated that relatively non-toxic radiation exposures do not induce thermotolerance in mammalian cells (Li et al. 1976), higher doses, comparable to those used in yeast to induce thermotolerance (Mitchel and Morison 1984), were tested in this study. Doses of this magnitude are lethal to mammalian cells, thereby precluding the use of clonogenic survival as an endpoint. The authors used three alternative assays as indicators of the subsequent development of thermotolerance: (a) heat-induced inhibition of total protein synthesis, (b) heat-induced uptake of dansyl lysine, and (c) synthesis of heat shock proteins. Only total protein synthesis revealed evidence of a small degree of thermotolerance which occurred immediately after γ-radiation exposure. By 4 h postirradiation the tolerance, as measured by this assay, was no longer evident. No evidence of thermotolerance was seen following UV exposure. In addition, when a large radiation dose was given either immediately before or after a heat treatment used to induce thermotolerance, there was no alteration in the level of heat-induced tolerance, despite the extensive number of DNA strand breaks caused by the radiation. (author)

  9. Electromagnetic noise inhibits radiofrequency radiation-induced DNA damage and reactive oxygen species increase in human lens epithelial cells

    Science.gov (United States)

    Wu, Wei; Wang, KaiJun; Ni, Shuang; Ye, PanPan; Yu, YiBo; Ye, Juan; Sun, LiXia

    2008-01-01

    Purpose The goal of this study was to investigate whether superposing of electromagnetic noise could block or attenuate DNA damage and intracellular reactive oxygen species (ROS) increase of cultured human lens epithelial cells (HLECs) induced by acute exposure to 1.8 GHz radiofrequency field (RF) of the Global System for Mobile Communications (GSM). Methods An sXc-1800 RF exposure system was used to produce a GSM signal at 1.8 GHz (217 Hz amplitude-modulated) with the specific absorption rate (SAR) of 1, 2, 3, and 4 W/kg. After 2 h of intermittent exposure, the ROS level was assessed by the fluorescent probe, 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA). DNA damage to HLECs was examined by alkaline comet assay and the phosphorylated form of histone variant H2AX (γH2AX) foci formation assay. Results After exposure to 1.8 GHz RF for 2 h, HLECs exhibited significant intracellular ROS increase in the 2, 3, and 4 W/kg groups. RF radiation at the SAR of 3 W/kg and 4 W/kg could induce significant DNA damage, examined by alkaline comet assay, which was used to detect mainly single strand breaks (SSBs), while no statistical difference in double strand breaks (DSBs), evaluated by γH2AX foci, was found between RF exposure (SAR: 3 and 4 W/kg) and sham exposure groups. When RF was superposed with 2 μT electromagnetic noise could block RF-induced ROS increase and DNA damage. Conclusions DNA damage induced by 1.8 GHz radiofrequency field for 2 h, which was mainly SSBs, may be associated with the increased ROS production. Electromagnetic noise could block RF-induced ROS formation and DNA damage. PMID:18509546

  10. Characterization of environmental chemicals with potential for DNA damage using isogenic DNA repair-deficient chicken DT40 cell lines.

    Science.gov (United States)

    Yamamoto, Kimiyo N; Hirota, Kouji; Kono, Koichi; Takeda, Shunichi; Sakamuru, Srilatha; Xia, Menghang; Huang, Ruili; Austin, Christopher P; Witt, Kristine L; Tice, Raymond R

    2011-08-01

    Included among the quantitative high throughput screens (qHTS) conducted in support of the US Tox21 program are those being evaluated for the detection of genotoxic compounds. One such screen is based on the induction of increased cytotoxicity in seven isogenic chicken DT40 cell lines deficient in DNA repair pathways compared to the parental DNA repair-proficient cell line. To characterize the utility of this approach for detecting genotoxic compounds and identifying the type(s) of DNA damage induced, we evaluated nine of 42 compounds identified as positive for differential cytotoxicity in qHTS (actinomycin D, adriamycin, alachlor, benzotrichloride, diglycidyl resorcinol ether, lovastatin, melphalan, trans-1,4-dichloro-2-butene, tris(2,3-epoxypropyl)isocyanurate) and one non-cytotoxic genotoxic compound (2-aminothiamine) for (1) clastogenicity in mutant and wild-type cells; (2) the comparative induction of γH2AX positive foci by melphalan; (3) the extent to which a 72-hr exposure duration increased assay sensitivity or specificity; (4) the use of 10 additional DT40 DNA repair-deficient cell lines to better analyze the type(s) of DNA damage induced; and (5) the involvement of reactive oxygen species in the induction of DNA damage. All compounds but lovastatin and 2-aminothiamine were more clastogenic in at least one DNA repair-deficient cell line than the wild-type cells. The differential responses across the various DNA repair-deficient cell lines provided information on the type(s) of DNA damage induced. The results demonstrate the utility of this DT40 screen for detecting genotoxic compounds, for characterizing the nature of the DNA damage, and potentially for analyzing mechanisms of mutagenesis. Copyright © 2011 Wiley-Liss, Inc.

  11. Reconstitution of the cellular response to DNA damage in vitro using damage-activated extracts from mammalian cells

    International Nuclear Information System (INIS)

    Roper, Katherine; Coverley, Dawn

    2012-01-01

    In proliferating mammalian cells, DNA damage is detected by sensors that elicit a cellular response which arrests the cell cycle and repairs the damage. As part of the DNA damage response, DNA replication is inhibited and, within seconds, histone H2AX is phosphorylated. Here we describe a cell-free system that reconstitutes the cellular response to DNA double strand breaks using damage-activated cell extracts and naïve nuclei. Using this system the effect of damage signalling on nuclei that do not contain DNA lesions can be studied, thereby uncoupling signalling and repair. Soluble extracts from G1/S phase cells that were treated with etoposide before isolation, or pre-incubated with nuclei from etoposide-treated cells during an in vitro activation reaction, restrain both initiation and elongation of DNA replication in naïve nuclei. At the same time, H2AX is phosphorylated in naïve nuclei in a manner that is dependent upon the phosphatidylinositol 3-kinase-like protein kinases. Notably, phosphorylated H2AX is not focal in naïve nuclei, but is evident throughout the nucleus suggesting that in the absence of DNA lesions the signal is not amplified such that discrete foci can be detected. This system offers a novel screening approach for inhibitors of DNA damage response kinases, which we demonstrate using the inhibitors wortmannin and LY294002. -- Highlights: ► A cell free system that reconstitutes the response to DNA damage in the absence of DNA lesions. ► Damage-activated extracts impose the cellular response to DNA damage on naïve nuclei. ► PIKK-dependent response impacts positively and negatively on two separate fluorescent outputs. ► Can be used to screen for inhibitors that impact on the response to damage but not on DNA repair. ► LY294002 and wortmannin demonstrate the system's potential as a pathway focused screening approach.

  12. Persistent DNA Damage in Spermatogonial Stem Cells After Fractionated Low-Dose Irradiation of Testicular Tissue

    Energy Technology Data Exchange (ETDEWEB)

    Grewenig, Angelika; Schuler, Nadine; Rübe, Claudia E., E-mail: claudia.ruebe@uks.eu

    2015-08-01

    Purpose: Testicular spermatogenesis is extremely sensitive to radiation-induced damage, and even low scattered doses to testis from radiation therapy may pose reproductive risks with potential treatment-related infertility. Radiation-induced DNA double-strand breaks (DSBs) represent the greatest threat to the genomic integrity of spermatogonial stem cells (SSCs), which are essential to maintain spermatogenesis and prevent reproduction failure. Methods and Materials: During daily low-dose radiation with 100 mGy or 10 mGy, radiation-induced DSBs were monitored in mouse testis by quantifying 53 binding protein 1 (53BP-1) foci in SSCs within their stem cell niche. The accumulation of DSBs was correlated with proliferation, differentiation, and apoptosis of testicular germ cell populations. Results: Even very low doses of ionizing radiation arrested spermatogenesis, primarily by inducing apoptosis in spermatogonia. Eventual recovery of spermatogenesis depended on the survival of SSCs and their functional ability to proliferate and differentiate to provide adequate numbers of differentiating spermatogonia. Importantly, apoptosis-resistant SSCs resulted in increased 53BP-1 foci levels during, and even several months after, fractionated low-dose radiation, suggesting that surviving SSCs have accumulated an increased load of DNA damage. Conclusions: SSCs revealed elevated levels of DSBs for weeks after radiation, and if these DSBs persist through differentiation to spermatozoa, this may have severe consequences for the genomic integrity of the fertilizing sperm.

  13. Persistent DNA Damage in Spermatogonial Stem Cells After Fractionated Low-Dose Irradiation of Testicular Tissue

    International Nuclear Information System (INIS)

    Grewenig, Angelika; Schuler, Nadine; Rübe, Claudia E.

    2015-01-01

    Purpose: Testicular spermatogenesis is extremely sensitive to radiation-induced damage, and even low scattered doses to testis from radiation therapy may pose reproductive risks with potential treatment-related infertility. Radiation-induced DNA double-strand breaks (DSBs) represent the greatest threat to the genomic integrity of spermatogonial stem cells (SSCs), which are essential to maintain spermatogenesis and prevent reproduction failure. Methods and Materials: During daily low-dose radiation with 100 mGy or 10 mGy, radiation-induced DSBs were monitored in mouse testis by quantifying 53 binding protein 1 (53BP-1) foci in SSCs within their stem cell niche. The accumulation of DSBs was correlated with proliferation, differentiation, and apoptosis of testicular germ cell populations. Results: Even very low doses of ionizing radiation arrested spermatogenesis, primarily by inducing apoptosis in spermatogonia. Eventual recovery of spermatogenesis depended on the survival of SSCs and their functional ability to proliferate and differentiate to provide adequate numbers of differentiating spermatogonia. Importantly, apoptosis-resistant SSCs resulted in increased 53BP-1 foci levels during, and even several months after, fractionated low-dose radiation, suggesting that surviving SSCs have accumulated an increased load of DNA damage. Conclusions: SSCs revealed elevated levels of DSBs for weeks after radiation, and if these DSBs persist through differentiation to spermatozoa, this may have severe consequences for the genomic integrity of the fertilizing sperm

  14. Cytotoxicity and DNA damage in the neutrophils of patients with sickle cell anaemia treated with hydroxyurea

    Directory of Open Access Journals (Sweden)

    Alano Martins Pedrosa

    2014-04-01

    Full Text Available Hydroxyurea (HU is the most important advance in the treatment of sickle cell anaemia (SCA for preventing complications and improving quality of life for patients. However, some aspects of treatment with HU remain unclear, including their effect on and potential toxicity to other blood cells such as neutrophils. This study used the measurement of Lactate Dehydrogenase (LDH and Methyl ThiazolTetrazolium (MTT and the comet assay to investigate the cytotoxicity and damage index (DI of the DNA in the neutrophils of patients with SCA using HU.In the LDH and MTT assays, a cytoprotective effect was observed in the group of patients treated, as well as an absence of toxicity. When compared to patients without the treatment, the SS group (n=20, 13 women and 07 men, aged 18-69 years, and the group of healthy individuals (AA used as a control group (n=52, 28 women and 24 men, aged 19-60 years, The SSHU group (n=21, 11 women and 10 men, aged 19-63 years showed a significant reduction (p20 months, demonstrating that despite the cytoprotective effects in terms of cell viability, the use of HU can induce DNA damage in neutrophils.

  15. DNA damage and apoptosis of endometrial cells cause loss of the early embryo in mice exposed to carbon disulfide

    International Nuclear Information System (INIS)

    Zhang, Bingzhen; Shen, Chunzi; Yang, Liu; Li, Chunhui; Yi, Anji; Wang, Zhiping

    2013-01-01

    Carbon disulfide (CS 2 ) may lead to spontaneous abortion and very early pregnancy loss in women exposed in the workplace, but the mechanism remains unclear. We designed an animal model in which gestating Kunming strain mice were exposed to CS 2 via i.p. on gestational day 4 (GD4). We found that the number of implanted blastocysts on GD8 was significantly reduced by each dose of 0.1 LD 50 (157.85 mg/kg), 0.2 LD 50 (315.7 mg/kg) and 0.4 LD 50 (631.4 mg/kg). In addition, both the level of DNA damage and apoptosis rates of endometrial cells on GD4.5 were increased, showed definite dose–response relationships, and inversely related to the number of implanted blastocysts. The expressions of mRNA and protein for the Bax and caspase-3 genes in the uterine tissues on GD4.5 were up-regulated, while the expressions of mRNA and protein for the Bcl-2 gene were dose-dependently down-regulated. Our results indicated that DNA damage and apoptosis of endometrial cells were important reasons for the loss of implanted blastocysts induced by CS 2 . - Highlights: • We built an animal model of CS2 exposure during blastocyst implantation. • Endometrial cells were used in the comet assay to detect DNA damage. • CS2 exposure caused DNA damage and endometrial cell apoptosis. • DNA damage and endometrial cell apoptosis were responsible for embryo loss

  16. DNA damage and apoptosis of endometrial cells cause loss of the early embryo in mice exposed to carbon disulfide

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Bingzhen [Department of Epidemiology and Health Statistics, School of Public Health, Shandong University, Jinan (China); Shen, Chunzi [Centers for Disease Control and Prevention, Zibo (China); Yang, Liu; Li, Chunhui; Yi, Anji [Department of Epidemiology and Health Statistics, School of Public Health, Shandong University, Jinan (China); Wang, Zhiping, E-mail: zhipingw@sdu.edu.cn [Department of Epidemiology and Health Statistics, School of Public Health, Shandong University, Jinan (China)

    2013-12-01

    Carbon disulfide (CS{sub 2}) may lead to spontaneous abortion and very early pregnancy loss in women exposed in the workplace, but the mechanism remains unclear. We designed an animal model in which gestating Kunming strain mice were exposed to CS{sub 2} via i.p. on gestational day 4 (GD4). We found that the number of implanted blastocysts on GD8 was significantly reduced by each dose of 0.1 LD{sub 50} (157.85 mg/kg), 0.2 LD{sub 50} (315.7 mg/kg) and 0.4 LD{sub 50} (631.4 mg/kg). In addition, both the level of DNA damage and apoptosis rates of endometrial cells on GD4.5 were increased, showed definite dose–response relationships, and inversely related to the number of implanted blastocysts. The expressions of mRNA and protein for the Bax and caspase-3 genes in the uterine tissues on GD4.5 were up-regulated, while the expressions of mRNA and protein for the Bcl-2 gene were dose-dependently down-regulated. Our results indicated that DNA damage and apoptosis of endometrial cells were important reasons for the loss of implanted blastocysts induced by CS{sub 2}. - Highlights: • We built an animal model of CS2 exposure during blastocyst implantation. • Endometrial cells were used in the comet assay to detect DNA damage. • CS2 exposure caused DNA damage and endometrial cell apoptosis. • DNA damage and endometrial cell apoptosis were responsible for embryo loss.

  17. The immune receptor Trem1 cooperates with diminished DNA damage response to induce preleukemic stem cell expansion.

    Science.gov (United States)

    Du, W; Amarachintha, S; Wilson, A; Pang, Q

    2017-02-01

    Fanconi anemia (FA) is an inherited bone marrow failure syndrome with extremely high risk of leukemic transformation. Here we investigate the relationship between DNA damage response (DDR) and leukemogenesis using the Fanca knockout mouse model. We found that chronic exposure of the Fanca -/- hematopoietic stem cells to DNA crosslinking agent mitomycin C in vivo leads to diminished DDR, and the emergence/expansion of pre-leukemia stem cells (pre-LSCs). Surprisingly, although genetic correction of Fanca deficiency in the pre-LSCs restores DDR and reduces genomic instability, but fails to prevent pre-LSC expansion or delay leukemia development in irradiated recipients. Furthermore, we identified transcription program underlying dysregulated DDR and cell migration, myeloid proliferation, and immune response in the Fanca -/- pre-LSCs. Forced expression of the downregulated DNA repair genes, Rad51c or Trp53i13, in the Fanca -/- pre-LSCs partially rescues DDR but has no effect on leukemia, whereas shRNA knockdown of the upregulated immune receptor genes Trem1 or Pilrb improves leukemia-related survival, but not DDR or genomic instability. Furthermore, Trem1 cooperates with diminished DDR in vivo to promote Fanca -/- pre-LSC expansion and leukemia development. Our study implicates diminishing DDR as a root cause of FA leukemogenesis, which subsequently collaborates with other signaling pathways for leukemogenic transformation.

  18. The prevention of radiation-induced DNA damage and apoptosis in human intestinal epithelial cells by salvianic acid A

    Directory of Open Access Journals (Sweden)

    Yanjun Zhang

    2014-07-01

    Full Text Available The topic of radiation always provokes public debate, and the uses of radiation for therapeutic and other purposes have always been associated with some anxiety. Salvia miltiorrhiza Bunge has been widely used for the treatment of various diseases including cerebrovascular diseases, coronary artery diseases, and myocardial infarction. Salvianolic acid A (SAA d (+-(3,4-dihydroxyphenyl lactic acid is the principal effective, watersoluble constituent of Salvia miltiorrhiza Bunge. In our present study, radiation-induced DNA damage and apoptosis in human intestinal epithelial cells (HIEC in the presence and absence of SAA were examined. We investigated the effects of SAA on ROS formation and the activity of enzymatic antioxidants (SOD, the lipid peroxidative index and the levels of non-enzymatic antioxidant (GSH. Finally, we investigated whether the reduction of radiation-induced cell death caused by SAA might be related to mitochondria-dependent apoptosis. Present findings indicate that SAA is a promising radioprotective agent with a strong antioxidant activity. SAA exerted its protective action on the proliferative activity of HIEC cells as evidenced by decreased cytotoxicity after exposure to γ-radiation. It is possible that SAA achieved its radioprotective action, at least in part, by enhancing DNA repair and the activity of antioxidant enzymes, by scavenging ROS and by inhibiting the mitochondria-dependent apoptotic pathway.

  19. DNA damage in human germ cell exposed to the some food additives in vitro.

    Science.gov (United States)

    Pandir, Dilek

    2016-08-01

    The use of food additives has increased enormously in modern food technology but they have adverse effects in human healthy. The aim of this study was to investigate the DNA damage of some food additives such as citric acid (CA), benzoic acid (BA), brilliant blue (BB) and sunset yellow (SY) which were investigated in human male germ cells using comet assay. The sperm cells were incubated with different concentrations of these food additives (50, 100, 200 and 500 μg/mL) for 1 h at 32 °C. The results showed for CA, BA, BB and SY a dose dependent increase in tail DNA%, tail length and tail moment in human sperm when compared to control group. When control values were compared in the studied parameters in the treatment concentrations, SY was found to exhibit the highest level of DNA damage followed by BB > BA > CA. However, none of the food additives affected the tail DNA%, tail length and tail moment at 50 and 100 μg/mL. At 200 μg/mL of SY, the tail DNA% and tail length of sperm were 95.80 ± 0.28 and 42.56 ± 4.66, for BB the values were 95.06 ± 2.30 and 39.56 ± 3.78, whereas for BA the values were 89.05 ± 2.78 and 31.50 ± 0.71, for CA the values were 88.59 ± 6.45 and 13.59 ± 2.74, respectively. However, only the highest concentration of the used food additives significantly affected the studied parameters of sperm DNA. The present results indicate that SY and BB are more harmful than BA and CA to human sperm in vitro.

  20. Mitochondrial DNA damage and oxidative damage in HL-60 cells exposed to 900 MHz radiofrequency fields

    Energy Technology Data Exchange (ETDEWEB)

    Sun, Yulong; Zong, Lin; Gao, Zhen [School of Public Health, Soochow University, Suzhou, Jiangsu Province (China); Zhu, Shunxing [Laboratory Animal Center, Nantong University, Nantong, Jiangsu Province (China); Tong, Jian [School of Public Health, Soochow University, Suzhou, Jiangsu Province (China); Cao, Yi, E-mail: yicao@suda.edu.cn [School of Public Health, Soochow University, Suzhou, Jiangsu Province (China)

    2017-03-15

    Highlights: • Increased reactive oxygen species. • Decreased mitochondrial transcription Factor A and polymerase gamma. • Decreased mitochondrial transcripts (ND1 and 16S) and mtDNA copy number. • Increased 8-hydroxy-2′deoxyguanosine. • Decreased adenosine triphosphate. - Abstract: HL-60 cells, derived from human promyelocytic leukemia, were exposed to continuous wave 900 MHz radiofrequency fields (RF) at 120 μW/cm{sup 2} power intensity for 4 h/day for 5 consecutive days to examine whether such exposure is capable damaging the mitochondrial DNA (mtDNA) mediated through the production of reactive oxygen species (ROS). In addition, the effect of RF exposure was examined on 8-hydroxy-2′-dexoyguanosine (8-OHdG) which is a biomarker for oxidative damage and on the mitochondrial synthesis of adenosine triphosphate (ATP) which is the energy required for cellular functions. The results indicated a significant increase in ROS and significant decreases in mitochondrial transcription factor A, mtDNA polymerase gamma, mtDNA transcripts and mtDNA copy number in RF-exposed cells compared with those in sham-exposed control cells. In addition, there was a significant increase in 8-OHdG and a significant decrease in ATP in RF-exposed cells. The response in positive control cells exposed to gamma radiation (GR, which is also known to induce ROS) was similar to those in RF-exposed cells. Thus, the overall data indicated that RF exposure was capable of inducing mtDNA damage mediated through ROS pathway which also induced oxidative damage. Prior-treatment of RF- and GR-exposed the cells with melatonin, a well-known free radical scavenger, reversed the effects observed in RF-exposed cells.

  1. Significant Suppression of CT Radiation-Induced DNA Damage in Normal Human Cells by the PrC-210 Radioprotector.

    Science.gov (United States)

    Jermusek, Frank; Benedict, Chelsea; Dreischmeier, Emma; Brand, Michael; Uder, Michael; Jeffery, Justin J; Ranallo, Frank N; Fahl, William E

    2018-05-21

    While computed tomography (CT) is now commonly used and considered to be clinically valuable, significant DNA double-strand breaks (γ-H2AX foci) in white blood cells from adult and pediatric CT patients have been frequently reported. In this study to determine whether γ-H2AX foci and X-ray-induced naked DNA damage are suppressed by administration of the PrC-210 radioprotector, human blood samples were irradiated in a CT scanner at 50-150 mGy with or without PrC-210, and γ-H2AX foci were scored. X-ray-induced naked DNA damage was also studied, and the DNA protective efficacy of PrC-210 was compared against 12 other common "antioxidants." PrC-210 reduced CT radiation-induced γ-H2AX foci in white blood cells to near background ( P 95% DNA damage. A systemic PrC-210 dose known to confer 100% survival in irradiated mice had no discernible effect on micro-CT image signal-to-noise ratio and CT image integrity. PrC-210 suppressed DNA damage to background or near background in each of these assay systems, thus supporting its development as a radioprotector for humans in multiple radiation exposure settings.

  2. DisA and c-di-AMP act at the intersection between DNA-damage response and stress homeostasis in exponentially growing Bacillus subtilis cells.

    Science.gov (United States)

    Gándara, Carolina; Alonso, Juan C

    2015-03-01

    Bacillus subtilis contains two vegetative diadenylate cyclases, DisA and CdaA, which produce cyclic di-AMP (c-di-AMP), and one phosphodiesterase, GdpP, that degrades it into a linear di-AMP. We report here that DisA and CdaA contribute to elicit repair of DNA damage generated by alkyl groups and H2O2, respectively, during vegetative growth. disA forms an operon with radA (also termed sms) that encodes a protein distantly related to RecA. Among different DNA damage agents tested, only methyl methane sulfonate (MMS) affected disA null strain viability, while radA showed sensitivity to all of them. A strain lacking both disA and radA was as sensitive to MMS as the most sensitive single parent (epistasis). Low c-di-AMP levels (e.g. by over-expressing GdpP) decreased the ability of cells to repair DNA damage caused by MMS and in less extent by H2O2, while high levels of c-di-AMP (absence of GdpP or expression of sporulation-specific diadenylate cyclase, CdaS) increased cell survival. Taken together, our results support the idea that c-di-AMP is a crucial signalling molecule involved in DNA repair with DisA and CdaA contributing to modulate different DNA damage responses during exponential growth. Copyright © 2015 Elsevier B.V. All rights reserved.

  3. Radiosensitization of mammalian cells by misonidazole and oxygen: DNA damage exposed by Micrococcus luteus enzymes

    International Nuclear Information System (INIS)

    Skov, K.A.; Palcic, B.; Skarsgard, L.D.

    1979-01-01

    When misonidazole is present during irradiation of hypoxic mammalian cells, an enhancement of single-strand breaks (SSB) in DNA is observed. Oxygen also enhances SSB, presumably in a manner similar to that of misonidazole. The dose-modifying factor (DMF) for 15 mM misonidazole was found to be 3.4, compared to an oxygen enhancement ratio (OER) of 3.5. Another class of DNA damage, namely, sites exposed by an extract of Micrococcus luteus, was examined. Radiation-induced M. luteus extract-sensitive sites (MLS) were also found to be enhanced by the presence of misonidazole or molecular oxygen. The DMF for this damage by 15 mM misonidazole was 1.6 while the OER was 2.5. The ratio of MLS to SSB is approximately 1.25 under hypoxia, 0.9 in the presence of oxygen, and 0.6 in the presence of 15 mM misonidazole under hypoxic conditions. Incubation with misonidazole under conditions which are toxic to mammalian cells (37 0 C, hypoxia), and which result in many SSB, produces no detectable lesions sensitive to the M. luteus extract

  4. Radiation-induced DNA damage in canine hemopoietic cells and stromal cells as measured by the comet assay

    International Nuclear Information System (INIS)

    Kreja, L.; Selig, C.; Plappert, U.; Nothdurft, W.

    1996-01-01

    Stromal cell progenitors (fibroblastoid colony-forming unit; CFU-Fs) are representative of the progenitor cell population of the hemopoietic microenvironment in bone marrow (BM). Previous studies of the radiation dose-effect relationships for colony formation have shown that canine CFU-Fs are relatively radioresistant as characterized by a D 0 value of about 2.4 Gy. In contrast, hemopoietic progenitors are particularly radiosensitive (D 0 values = 0.12-0.60 Gy). In the present study, the alkaline single-cell gel electrophoresis technique for the in situ quantitation of DNA strand breaks and alkalilabile site was employed. Canine buffy coat cells from BM aspirates and cells harvested from CFU-F colonies or from mixed populations of adherent BM stromal cell (SC) layers were exposed to increasing doses of X-rays, embedded in agarose gel on slides, lysed with detergents, and placed in an electric field. DNA migrating from single cells in the gel was made visible as open-quotes cometsclose quotes by ethidium bromide staining. Immediate DNA damage was much less in cultured stromal cells than in hemopoietic cells in BM aspirates. These results suggest that the observed differences in clonogenic survival could be partly due to differences in the type of the initial DNA damage between stromal cells and hemopoietic cells. 37 refs., 2 figs., 1 tab

  5. Mercuric dichloride induces DNA damage in human salivary gland tissue cells and lymphocytes

    Energy Technology Data Exchange (ETDEWEB)

    Schmid, Katharina; Kroemer, Susanne [University of Regensburg, Regensburg (Germany); Sassen, Andrea [University of Regensburg, Department of Pathology, Regensburg (Germany); Staudenmaier, Rainer [Technical University of Munich, Department of Otorhinolaryngology, Head and Neck Surgery, Munich (Germany); Reichl, Franz-Xaver [University of Munich, Institute of Pharmacology and Toxicology, Munich (Germany); Harreus, Ulrich [University of Munich, Department of Otorhinolaryngology, Head and Neck Surgery, Munich (Germany); Hagen, Rudolf; Kleinsasser, Norbert [University of Wuerzburg, Department of Otorhinolaryngology, Head and Neck Surgery, Wuerzburg (Germany)

    2007-11-15

    Amalgam is still one of the most frequently used dental filling materials. However, the possible adverse effects especially that of the mercuric component have led to continued controversy. Considering that mercury may be released from amalgam fillings into the oral cavity and also reach the circulating blood after absorption and resorption, it eventually may contribute to tumorigenesis in a variety of target cells. The present investigation focuses on genotoxic effects below a cytotoxic dose level of mercuric dichloride (HgCl{sub 2}) in human samples of salivary glands and lymphocytes to elucidate a possible role in tumor initiation. DNA migration due to single strand breaks, alkali labile sites and incomplete excision repair was quantified with the aid of the single cell microgel electrophoresis (Comet) assay. The concepts of Olive Tail Moment, percentage of DNA in the Tail and Tail Length were used as measures of DNA damage. To control for cytotoxic effects, the trypan blue exclusion test was applied. Human samples of the parotid salivary gland and lymphocytes of ten donors were exposed to HgCl{sub 2} concentrations from 1 to 50 {mu}M. N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and dimethyl sulfoxide (DMSO) served as controls. Increasing dose-dependent DNA migration could be demonstrated after exposure to HgCl{sub 2} in cells of the salivary glands and lymphocytes. In both cell types a significant increase in DNA migration could be shown starting from HgCl{sub 2} concentrations of 5 {mu}M in comparison to the negative control. The viability of the cell systems was not affected except at the highest concentration (50 {mu}M) tested. These data indicate genotoxic effects of mercuric dichloride in human salivary glands and lymphocytes at concentrations not leading to cytotoxic effects or cell death. Consequently, a contributory role in oral salivary gland tumor initiation warrants further investigation. (orig.)

  6. The amount of DNA damage needed to activate the radiation-induced G2 checkpoint varies between single cells

    International Nuclear Information System (INIS)

    Tkacz-Stachowska, Kinga; Lund-Andersen, Christin; Velissarou, Angeliki; Myklebust, June H.; Stokke, Trond; Syljuåsen, Randi G.

    2011-01-01

    Background and purpose: The radiation-induced G2 checkpoint helps facilitate DNA repair before cell division. However, recent work has revealed that human cells often escape the G2 checkpoint with unrepaired DNA breaks. The purpose was to explore whether G2 checkpoint activation occurs according to a threshold level of DNA damage. Materials and methods: G2 checkpoint activation was assayed at 75–90 min and 24–48 h after X-ray irradiation of BJ diploid fibroblasts and U2OS osteosarcoma cells. Multiparameter flow cytometry with pacific blue barcoding, and flow cytometry-based sorting of phospho-H3 positive cells to microscope slides, were used to examine the DNA damage marker γ-H2AX in individual mitotic cells that had escaped the G2 checkpoint. Results: For all radiation doses and times tested, the number of γ-H2AX foci varied between individual mitotic cells. At 75 min the median levels of γ-H2AX in mitotic cells increased with higher radiation doses. At 24–48 h, following a prolonged G2 checkpoint, cells were more resistant to checkpoint re-activation by a second dose of radiation. Conclusion: Our results suggest that different amounts of DNA damage are needed to activate the G2 checkpoint in individual cells. Such single cell variation in checkpoint activation may potentially contribute to radiation-induced genomic instability.

  7. 4beta-Hydroxywithanolide E from Physalis peruviana (golden berry) inhibits growth of human lung cancer cells through DNA damage, apoptosis and G2/M arrest.

    Science.gov (United States)

    Yen, Ching-Yu; Chiu, Chien-Chih; Chang, Fang-Rong; Chen, Jeff Yi-Fu; Hwang, Chi-Ching; Hseu, You-Cheng; Yang, Hsin-Ling; Lee, Alan Yueh-Luen; Tsai, Ming-Tz; Guo, Zong-Lun; Cheng, Yu-Shan; Liu, Yin-Chang; Lan, Yu-Hsuan; Chang, Yu-Ching; Ko, Ying-Chin; Chang, Hsueh-Wei; Wu, Yang-Chang

    2010-02-18

    The crude extract of the fruit bearing plant, Physalis peruviana (golden berry), demonstrated anti-hepatoma and anti-inflammatory activities. However, the cellular mechanism involved in this process is still unknown. Herein, we isolated the main pure compound, 4beta-Hydroxywithanolide (4betaHWE) derived from golden berries, and investigated its antiproliferative effect on a human lung cancer cell line (H1299) using survival, cell cycle, and apoptosis analyses. An alkaline comet-nuclear extract (NE) assay was used to evaluate the DNA damage due to the drug. It was shown that DNA damage was significantly induced by 1, 5, and 10 microg/mL 4betaHWE for 2 h in a dose-dependent manner (p < 0.005). A trypan blue exclusion assay showed that the proliferation of cells was inhibited by 4betaHWE in both dose- and time-dependent manners (p < 0.05 and 0.001 for 24 and 48 h, respectively). The half maximal inhibitory concentrations (IC50) of 4betaHWE in H1299 cells for 24 and 48 h were 0.6 and 0.71 microg/mL, respectively, suggesting it could be a potential therapeutic agent against lung cancer. In a flow cytometric analysis, 4betaHWE produced cell cycle perturbation in the form of sub-G1 accumulation and slight arrest at the G2/M phase with 1 microg/mL for 12 and 24 h, respectively. Using flow cytometric and annexin V/propidium iodide immunofluorescence double-staining techniques, these phenomena were proven to be apoptosis and complete G2/M arrest for H1299 cells treated with 5 microg/mL for 24 h. In this study, we demonstrated that golden berry-derived 4betaHWE is a potential DNA-damaging and chemotherapeutic agent against lung cancer.

  8. 4β-Hydroxywithanolide E from Physalis peruviana (golden berry) inhibits growth of human lung cancer cells through DNA damage, apoptosis and G2/M arrest

    International Nuclear Information System (INIS)

    Yen, Ching-Yu; Guo, Zong-Lun; Cheng, Yu-Shan; Liu, Yin-Chang; Lan, Yu-Hsuan; Chang, Yu-Ching; Ko, Ying-Chin; Chang, Hsueh-Wei; Wu, Yang-Chang; Chiu, Chien-Chih; Chang, Fang-Rong; Chen, Jeff Yi-Fu; Hwang, Chi-Ching; Hseu, You-Cheng; Yang, Hsin-Ling; Lee, Alan Yueh-Luen; Tsai, Ming-Tz

    2010-01-01

    The crude extract of the fruit bearing plant, Physalis peruviana (golden berry), demonstrated anti-hepatoma and anti-inflammatory activities. However, the cellular mechanism involved in this process is still unknown. Herein, we isolated the main pure compound, 4β-Hydroxywithanolide (4βHWE) derived from golden berries, and investigated its antiproliferative effect on a human lung cancer cell line (H1299) using survival, cell cycle, and apoptosis analyses. An alkaline comet-nuclear extract (NE) assay was used to evaluate the DNA damage due to the drug. It was shown that DNA damage was significantly induced by 1, 5, and 10 μg/mL 4βHWE for 2 h in a dose-dependent manner (p < 0.005). A trypan blue exclusion assay showed that the proliferation of cells was inhibited by 4βHWE in both dose- and time-dependent manners (p < 0.05 and 0.001 for 24 and 48 h, respectively). The half maximal inhibitory concentrations (IC 50 ) of 4βHWE in H1299 cells for 24 and 48 h were 0.6 and 0.71 μg/mL, respectively, suggesting it could be a potential therapeutic agent against lung cancer. In a flow cytometric analysis, 4βHWE produced cell cycle perturbation in the form of sub-G 1 accumulation and slight arrest at the G 2 /M phase with 1 μg/mL for 12 and 24 h, respectively. Using flow cytometric and annexin V/propidium iodide immunofluorescence double-staining techniques, these phenomena were proven to be apoptosis and complete G 2 /M arrest for H1299 cells treated with 5 μg/mL for 24 h. In this study, we demonstrated that golden berry-derived 4βHWE is a potential DNA-damaging and chemotherapeutic agent against lung cancer

  9. 4β-Hydroxywithanolide E from Physalis peruviana (golden berry) inhibits growth of human lung cancer cells through DNA damage, apoptosis and G2/M arrest

    Science.gov (United States)

    2010-01-01

    Background The crude extract of the fruit bearing plant, Physalis peruviana (golden berry), demonstrated anti-hepatoma and anti-inflammatory activities. However, the cellular mechanism involved in this process is still unknown. Methods Herein, we isolated the main pure compound, 4β-Hydroxywithanolide (4βHWE) derived from golden berries, and investigated its antiproliferative effect on a human lung cancer cell line (H1299) using survival, cell cycle, and apoptosis analyses. An alkaline comet-nuclear extract (NE) assay was used to evaluate the DNA damage due to the drug. Results It was shown that DNA damage was significantly induced by 1, 5, and 10 μg/mL 4βHWE for 2 h in a dose-dependent manner (p < 0.005). A trypan blue exclusion assay showed that the proliferation of cells was inhibited by 4βHWE in both dose- and time-dependent manners (p < 0.05 and 0.001 for 24 and 48 h, respectively). The half maximal inhibitory concentrations (IC50) of 4βHWE in H1299 cells for 24 and 48 h were 0.6 and 0.71 μg/mL, respectively, suggesting it could be a potential therapeutic agent against lung cancer. In a flow cytometric analysis, 4βHWE produced cell cycle perturbation in the form of sub-G1 accumulation and slight arrest at the G2/M phase with 1 μg/mL for 12 and 24 h, respectively. Using flow cytometric and annexin V/propidium iodide immunofluorescence double-staining techniques, these phenomena were proven to be apoptosis and complete G2/M arrest for H1299 cells treated with 5 μg/mL for 24 h. Conclusions In this study, we demonstrated that golden berry-derived 4βHWE is a potential DNA-damaging and chemotherapeutic agent against lung cancer. PMID:20167063

  10. p18(Hamlet) mediates different p53-dependent responses to DNA-damage inducing agents.

    Science.gov (United States)

    Lafarga, Vanesa; Cuadrado, Ana; Nebreda, Angel R

    2007-10-01

    Cells organize appropriate responses to environmental cues by activating specific signaling networks. Two proteins that play key roles in coordinating stress responses are the kinase p38alpha (MAPK14) and the transcription factor p53 (TP53). Depending on the nature and the extent of the stress-induced damage, cells may respond by arresting the cell cycle or by undergoing cell death, and these responses are usually associated with the phosphorylation of particular substrates by p38alpha as well as the activation of specific target genes by p53. We recently characterized a new p38alpha substrate, named p18(Hamlet) (ZNHIT1), which mediates p53-dependent responses to different genotoxic stresses. Thus, cisplatin or UV light induce stabilization of the p18(Hamlet) protein, which then enhances the ability of p53 to bind to and activate the promoters of pro-apoptotic genes such as NOXA and PUMA leading to apoptosis induction. In a similar way, we report here that p18(Hamlet) can also mediate the cell cycle arrest induced in response to gamma-irradiation, by participating in the p53-dependent upregulation of the cell cycle inhibitor p21(Cip1) (CDKN1A).

  11. Unscheduled DNA synthesis and elimination of DNA damage in liver cells of. gamma. -irradiated senescent mice

    Energy Technology Data Exchange (ETDEWEB)

    Gaziev, A.I.; Malakhova, L.V. (AN SSSR, Pushchino-na-Oke. Inst. Biologicheskoj Fiziki)

    1982-10-01

    The level of 'spontaneous' and ..gamma..-radiation-induced DNA synthesis which is not inhibited with hydroxyurea (unscheduled synthesis) is considerably lower in hepatocytes of 18-22-month-old mice than that of 1.5-2-month-old mice. The dose-dependent increase (10-300 Gy) of unscheduled DNA synthesis (UDS) in hepatocytes of senescent mice is higher than in young animals. The elimination of damage in DNA of ..gamma..-irradiated hepatocytes (100 Gy) was examined by using an enzyme system (M. luteus extract and DNA-polymerase I of E. coli). It was found that the rate of elimination of the DNA damage in hepatocytes of 20-month-old mice is lower than that of 2-month-old mice although the activities of DNA-polymerase ..beta.. and apurinic endonuclease remain equal in the liver of both senescent and young mice. However, the nucleoids from ..gamma..-irradiated liver nuclei of 2-month-old mice are relaxed to a greater extent (as judged by the criterion of ethidium-binding capacity) than those of 20-month-old mice. The results suggest that there are limitations in the functioning of repair enzymes and in their access to damaged DNA sites in the chromatin of senescent mouse liver cells.

  12. ATM Deficiency Generating Genomic Instability Sensitizes Pancreatic Ductal Adenocarcinoma Cells to Therapy-Induced DNA Damage.

    Science.gov (United States)

    Perkhofer, Lukas; Schmitt, Anna; Romero Carrasco, Maria Carolina; Ihle, Michaela; Hampp, Stephanie; Ruess, Dietrich Alexander; Hessmann, Elisabeth; Russell, Ronan; Lechel, André; Azoitei, Ninel; Lin, Qiong; Liebau, Stefan; Hohwieler, Meike; Bohnenberger, Hanibal; Lesina, Marina; Algül, Hana; Gieldon, Laura; Schröck, Evelin; Gaedcke, Jochen; Wagner, Martin; Wiesmüller, Lisa; Sipos, Bence; Seufferlein, Thomas; Reinhardt, Hans Christian; Frappart, Pierre-Olivier; Kleger, Alexander

    2017-10-15

    Pancreatic ductal adenocarcinomas (PDAC) harbor recurrent functional mutations of the master DNA damage response kinase ATM, which has been shown to accelerate tumorigenesis and epithelial-mesenchymal transition. To study how ATM deficiency affects genome integrity in this setting, we evaluated the molecular and functional effects of conditional Atm deletion in a mouse model of PDAC. ATM deficiency was associated with increased mitotic defects, recurrent genomic rearrangements, and deregulated DNA integrity checkpoints, reminiscent of human PDAC. We hypothesized that altered genome integrity might allow synthetic lethality-based options for targeted therapeutic intervention. Supporting this possibility, we found that the PARP inhibitor olaparib or ATR inhibitors reduced the viability of PDAC cells in vitro and in vivo associated with a genotype-selective increase in apoptosis. Overall, our results offered a preclinical mechanistic rationale for the use of PARP and ATR inhibitors to improve treatment of ATM-mutant PDAC. Cancer Res; 77(20); 5576-90. ©2017 AACR . ©2017 American Association for Cancer Research.

  13. Ultraviolet B (UVB) induced DNA damage affects alternative splicing in skin cells

    International Nuclear Information System (INIS)

    Munoz, M.J.; Nieto Moreno, N.; Kornblihtt, A.R.

    2010-01-01

    The ultraviolet (UV) radiation from the Sun that reaches the Earth's surface is a combination of low (UVA, 320-400 nm) and high (UVB, 290-320 nm) energy light. UVB light causes two types of mutagenic DNA lesions: thymine dimers and (6-4) photo-products. UVB mutagenesis is a critical step in the generation of different forms of skin cancer, which develops almost exclusively in sun exposed areas. We have previously shown that RNA polymerase II (pol II) hyperphosphorylation induced by UVC (254 nm) irradiation of non-skin cells inhibits pol II elongation rates which in turn affects alternative splicing (AS) patterns, altering the synthesis of pro- and anti-apoptotic isoforms of key proteins like Bcl-x or Caspase 9 (C9). Since the UVC radiation is fully filtered by the ozone layer and AS regulation in skin pathologies has been poorly studied, we decided to extend our studies to human keratinocytes in culture treated with UVB (302 nm) light. We observed that pol II hyperphosphorylation is increased upon UVB irradiation, being this modification necessary for the observed change in AS of a model cassette exon. Moreover, UVB irradiation induces the proapoptotic mRNA isoforms of Bcl-x and C9 consistently with a key role of AS in skin response to DNA damage. (authors)

  14. Dietary spices protect against hydrogen peroxide-induced DNA damage and inhibit nicotine-induced cancer cell migration.

    Science.gov (United States)

    Jayakumar, R; Kanthimathi, M S

    2012-10-01

    Spices are rich sources of antioxidants due to the presence of phenols and flavonoids. In this study, the DNA protecting activity and inhibition of nicotine-induced cancer cell migration of 9 spices were analysed. Murine fibroblasts (3T3-L1) and human breast cancer (MCF-7) cells were pre-treated with spice extracts and then exposed to H₂O₂ and nicotine. The comet assay was used to analyse the DNA damage. Among the 9 spices, ginger, at 50 μg/ml protected against 68% of DNA damage in 3T3-L1 cells. Caraway, cumin and fennel showed statistically significant (pspices reduced this migration. Pepper, long pepper and ginger exhibited a high rate of inhibition of cell migration. The results of this study prove that spices protect DNA and inhibit cancer cell migration. Copyright © 2012 Elsevier Ltd. All rights reserved.

  15. Evaluating In Vitro DNA Damage Using Comet Assay.

    Science.gov (United States)

    Lu, Yanxin; Liu, Yang; Yang, Chunzhang

    2017-10-11

    DNA damage is a common phenomenon for each cell during its lifespan, and is defined as an alteration of the chemical structure of genomic DNA. Cancer therapies, such as radio- and chemotherapy, introduce enormous amount of additional DNA damage, leading to cell cycle arrest and apoptosis to limit cancer progression. Quantitative assessment of DNA damage during experimental cancer therapy is a key step to justify the effectiveness of a genotoxic agent. In this study, we focus on a single cell electrophoresis assay, also known as the comet assay, which can quantify single and double-strand DNA breaks in vitro. The comet assay is a DNA damage quantification method that is efficient and easy to perform, and has low time/budget demands and high reproducibility. Here, we highlight the utility of the comet assay for a preclinical study by evaluating the genotoxic effect of olaparib/temozolomide combination therapy to U251 glioma cells.

  16. In Barrett's esophagus patients and Barrett's cell lines, ursodeoxycholic acid increases antioxidant expression and prevents DNA damage by bile acids.

    Science.gov (United States)

    Peng, Sui; Huo, Xiaofang; Rezaei, Davood; Zhang, Qiuyang; Zhang, Xi; Yu, Chunhua; Asanuma, Kiyotaka; Cheng, Edaire; Pham, Thai H; Wang, David H; Chen, Minhu; Souza, Rhonda F; Spechler, Stuart Jon

    2014-07-15

    Hydrophobic bile acids like deoxycholic acid (DCA), which cause oxidative DNA damage and activate NF-κB in Barrett's metaplasia, might contribute to carcinogenesis in Barrett's esophagus. We have explored mechanisms whereby ursodeoxycholic acid (UDCA, a hydrophilic bile acid) protects against DCA-induced injury in vivo in patients and in vitro using nonneoplastic, telomerase-immortalized Barrett's cell lines. We took biopsies of Barrett's esophagus from 21 patients before and after esophageal perfusion with DCA (250 μM) at baseline and after 8 wk of oral UDCA treatment. DNA damage was assessed by phospho-H2AX expression, neutral CometAssay, and phospho-H2AX nuclear foci formation. Quantitative PCR was performed for antioxidants including catalase and GPX1. Nrf2, catalase, and GPX1 were knocked down with siRNAs. Reporter assays were performed using a plasmid construct containing antioxidant responsive element. In patients, baseline esophageal perfusion with DCA significantly increased phospho-H2AX and phospho-p65 in Barrett's metaplasia. Oral UDCA increased GPX1 and catalase levels in Barrett's metaplasia and prevented DCA perfusion from inducing DNA damage and NF-κB activation. In cells, DCA-induced DNA damage and NF-κB activation was prevented by 24-h pretreatment with UDCA, but not by mixing UDCA with DCA. UDCA activated Nrf2 signaling to increase GPX1 and catalase expression, and protective effects of UDCA pretreatment were blocked by siRNA knockdown of these antioxidants. UDCA increases expression of antioxidants that prevent toxic bile acids from causing DNA damage and NF-κB activation in Barrett's metaplasia. Elucidation of this molecular pathway for UDCA protection provides rationale for clinical trials on UDCA for chemoprevention in Barrett's esophagus. Copyright © 2014 the American Physiological Society.

  17. DNA Damage, Mutagenesis and Cancer

    Directory of Open Access Journals (Sweden)

    Ashis K. Basu

    2018-03-01

    Full Text Available A large number of chemicals and several physical agents, such as UV light and γ-radiation, have been associated with the etiology of human cancer. Generation of DNA damage (also known as DNA adducts or lesions induced by these agents is an important first step in the process of carcinogenesis. Evolutionary processes gave rise to DNA repair tools that are efficient in repairing damaged DNA; yet replication of damaged DNA may take place prior to repair, particularly when they are induced at a high frequency. Damaged DNA replication may lead to gene mutations, which in turn may give rise to altered proteins. Mutations in an oncogene, a tumor-suppressor gene, or a gene that controls the cell cycle can generate a clonal cell population with a distinct advantage in proliferation. Many such events, broadly divided into the stages of initiation, promotion, and progression, which may occur over a long period of time and transpire in the context of chronic exposure to carcinogens, can lead to the induction of human cancer. This is exemplified in the long-term use of tobacco being responsible for an increased risk of lung cancer. This mini-review attempts to summarize this wide area that centers on DNA damage as it relates to the development of human cancer.

  18. 4β-Hydroxywithanolide E from Physalis peruviana (golden berry inhibits growth of human lung cancer cells through DNA damage, apoptosis and G2/M arrest

    Directory of Open Access Journals (Sweden)

    Guo Zong-Lun

    2010-02-01

    Full Text Available Abstract Background The crude extract of the fruit bearing plant, Physalis peruviana (golden berry, demonstrated anti-hepatoma and anti-inflammatory activities. However, the cellular mechanism involved in this process is still unknown. Methods Herein, we isolated the main pure compound, 4β-Hydroxywithanolide (4βHWE derived from golden berries, and investigated its antiproliferative effect on a human lung cancer cell line (H1299 using survival, cell cycle, and apoptosis analyses. An alkaline comet-nuclear extract (NE assay was used to evaluate the DNA damage due to the drug. Results It was shown that DNA damage was significantly induced by 1, 5, and 10 μg/mL 4βHWE for 2 h in a dose-dependent manner (p p 50 of 4βHWE in H1299 cells for 24 and 48 h were 0.6 and 0.71 μg/mL, respectively, suggesting it could be a potential therapeutic agent against lung cancer. In a flow cytometric analysis, 4βHWE produced cell cycle perturbation in the form of sub-G1 accumulation and slight arrest at the G2/M phase with 1 μg/mL for 12 and 24 h, respectively. Using flow cytometric and annexin V/propidium iodide immunofluorescence double-staining techniques, these phenomena were proven to be apoptosis and complete G2/M arrest for H1299 cells treated with 5 μg/mL for 24 h. Conclusions In this study, we demonstrated that golden berry-derived 4βHWE is a potential DNA-damaging and chemotherapeutic agent against lung cancer.

  19. The Fanconi Anemia Pathway: Repairing the Link Between DNA Damage and Squamous Cell Carcinoma

    Science.gov (United States)

    Romick-Rosendale, Lindsey E.; Lui, Vivian W. Y.; Grandis, Jennifer R.; Wells, Susanne I.

    2013-01-01

    Fanconi anemia (FA) is a rare inherited recessive disease caused by mutations in one of fifteen genes known to encode FA pathway components. In response to DNA damage, nuclear FA proteins associate into high molecular weight complexes through a cascade of post-translational modifications and physical interactions, followed by the repair of damaged DNA. Hematopoietic cells are particularly sensitive to the loss of these interactions, and bone marrow failure occurs almost universally in FA patients. FA as a disease is further characterized by cancer susceptibility, which highlights the importance of the FA pathway in tumor suppression, and will be the focus of this review. Acute myeloid leukemia is the most common cancer type, often subsequent to bone marrow failure. However, FA patients are also at an extreme risk of squamous cell carcinoma (SCC) of the head and neck and gynecological tract, with an even greater incidence in those individuals who have received a bone marrow transplant and recovered from hematopoietic disease. FA tumor suppression in hematopoietic versus epithelial compartments could be mechanistically similar or distinct. Definition of compartment specific FA activities is now critical to assess the effects of today’s bone marrow failure treatments on tomorrow’s solid tumor development. It is our hope that current therapies can then be optimized to decrease the risk of malignant transformation in both hematopoietic and epithelial cells. Here we review our current understanding of the mechanisms of action of the Fanconi anemia pathway as it contributes to stress responses, DNA repair and squamous cell carcinoma susceptibility. PMID:23333482

  20. The Fanconi anemia pathway: Repairing the link between DNA damage and squamous cell carcinoma

    Energy Technology Data Exchange (ETDEWEB)

    Romick-Rosendale, Lindsey E. [Division of Oncology, Cancer and Blood Diseases Institute, Cincinnati Children' s Hospital Medical Center, Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, OH 45229 (United States); Lui, Vivian W.Y.; Grandis, Jennifer R. [Department of Otolaryngology, University of Pittsburgh School of Medicine, University of Pittsburgh, Pittsburgh, PA 15213 (United States); Wells, Susanne I., E-mail: Susanne.Wells@cchmc.org [Division of Oncology, Cancer and Blood Diseases Institute, Cincinnati Children' s Hospital Medical Center, Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, OH 45229 (United States)

    2013-03-15

    Fanconi anemia (FA) is a rare inherited recessive disease caused by mutations in one of fifteen genes known to encode FA pathway components. In response to DNA damage, nuclear FA proteins associate into high molecular weight complexes through a cascade of post-translational modifications and physical interactions, followed by the repair of damaged DNA. Hematopoietic cells are particularly sensitive to the loss of these interactions, and bone marrow failure occurs almost universally in FA patients. FA as a disease is further characterized by cancer susceptibility, which highlights the importance of the FA pathway in tumor suppression, and will be the focus of this review. Acute myeloid leukemia is the most common cancer type, often subsequent to bone marrow failure. However, FA patients are also at an extreme risk of squamous cell carcinoma (SCC) of the head and neck and gynecological tract, with an even greater incidence in those individuals who have received a bone marrow transplant and recovered from hematopoietic disease. FA tumor suppression in hematopoietic versus epithelial compartments could be mechanistically similar or distinct. Definition of compartment specific FA activities is now critical to assess the effects of today's bone marrow failure treatments on tomorrow's solid tumor development. It is our hope that current therapies can then be optimized to decrease the risk of malignant transformation in both hematopoietic and epithelial cells. Here we review our current understanding of the mechanisms of action of the Fanconi anemia pathway as it contributes to stress responses, DNA repair and squamous cell carcinoma susceptibility.

  1. Hypersensitivity of hypoxia grown Mycobacterium smegmatis to DNA damaging agents: implications of the DNA repair deficiencies in attenuation of mycobacteria.

    Science.gov (United States)

    Rex, Kervin; Kurthkoti, Krishna; Varshney, Umesh

    2013-10-01

    Mycobacteria are an important group of pathogenic bacteria. We generated a series of DNA repair deficient strains of Mycobacterium smegmatis, a model organism, to understand the importance of various DNA repair proteins (UvrB, Ung, UdgB, MutY and Fpg) in survival of the pathogenic strains. Here, we compared tolerance of the M. smegmatis strains to genotoxic stress (ROS and RNI) under aerobic, hypoxic and recovery conditions of growth by monitoring their survival. We show an increased susceptibility of mycobacteria to genotoxic stress under hypoxia. UvrB deficiency led to high susceptibility of M. smegmatis to the DNA damaging agents. Ung was second in importance in strains with single deficiencies. Interestingly, we observed that while deficiency of UdgB had only a minor impact on the strain's susceptibility, its combination with Ung deficiency resulted in severe consequences on the strain's survival under genotoxic stress suggesting a strong interdependence of different DNA repair pathways in safeguarding genomic integrity. Our observations reinforce the possibility of targeting DNA repair processes in mycobacteria for therapeutic intervention during active growth and latency phase of the pathogen. High susceptibility of the UvrB, or the Ung/UdgB deficient strains to genotoxic stress may be exploited in generation of attenuated strains of mycobacteria. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  2. Detection of DNA damage induced in vivo by a cross-linking agent with a circular channel crucible oscillating viscometer.

    Science.gov (United States)

    Balbi, C; Abelmoschi, M L; Roner, R; Giaretti, W; Parodi, S; Santi, L

    1985-11-01

    DNA damage induced in vivo by the cross-linking agent mitomycin C (MMC) was investigated with a new oscillating crucible viscometer. Viscosity was measured by lysing rat liver nuclei in an alkaline lysing solution (pH 12.5; 25 degrees C). In control samples the viscosity increased very slowly with time, reaching a plateau only after 10-12 h. The process was accelerated and the maximum viscosity was decreased by alkaline single-stranded breaks arising from methylation and subsequent depurination of DNA in vitro with dimethylsulphate (DMS). MMC, when given alone, had no evident effect on the time needed for reaching plateau viscosity but it induced a small increase in maximum viscosity. When MMC was given in association with DMS, the time of disentanglement remained unchanged (accelerated) but maximum viscosity was increased in a dose dependent way. We conclude that these data clearly confirm that the slow steady increase of the viscosity of control DNA with time reflects mainly the process of unwinding of the two strands. The speed of this process seems to depend only from the number of unwinding points in DNA (breaks).

  3. Labeling Adipose-Derived Stem Cells with Hoechst 33342: Usability and Effects on Differentiation Potential and DNA Damage

    Directory of Open Access Journals (Sweden)

    P. Schendzielorz

    2016-01-01

    Full Text Available Adipose-derived stem cells (ASCs have been extensively studied in the field of stem cell research and possess numerous clinical applications. Cell labeling is an essential component of various experimental protocols and Hoechst 33342 (H33342 represents a cost-effective and easy methodology for live staining. The purpose of this study was to evaluate the labeling of rat ASCs with two different concentrations of H33342 (0.5 μg/mL and 5 μg/mL, with particular regard to usability, interference with cell properties, and potential DNA damage. Hoechst 33342 used at a low concentration of 0.5 μg/mL did not significantly affect cell proliferation, viability, or differentiation potential of the ASCs, nor did it cause any significant DNA damage as measured by the olive tail moment. High concentrations of 5 μg/mL H33342, however, impaired the proliferation and viability of the ASCs, and considerable DNA damage was observed. Undesirable colabeling of unlabeled cocultivated cells was seen in particular with higher concentrations of H33342, independent of varying washing procedures. Hence, H33342 labeling with lower concentrations represents a usable method, which does not affect the tested cell properties. However, the colabeling of adjacent cells is a drawback of the technique.

  4. 3-Nitrobenzanthrone and 3-aminobenzanthrone induce DNA damage and cell signalling in Hepa1c1c7 cells.

    Science.gov (United States)

    Landvik, N E; Arlt, V M; Nagy, E; Solhaug, A; Tekpli, X; Schmeiser, H H; Refsnes, M; Phillips, D H; Lagadic-Gossmann, D; Holme, J A

    2010-02-03

    3-Nitrobenzanthrone (3-NBA) is a mutagenic and carcinogenic environmental pollutant found in diesel exhaust and urban air pollution. In the present work we have characterised the effects of 3-NBA and its metabolite 3-aminobenzanthrone (3-ABA) on cell death and cytokine release in mouse hepatoma Hepa1c1c7 cells. These effects were related to induced DNA damage and changes in cell signalling pathways. 3-NBA resulted in cell death and caused most DNA damage as judged by the amount of DNA adducts ((32)P-postlabelling assay), single strand (ss)DNA breaks and oxidative DNA lesions (comet assay) detected. An increased phosphorylation of H2AX, chk1, chk2 and partly ATM was observed using flow cytometry and/or Western blotting. Both compounds increased phosphorylation of p53 and MAPKs (ERK, p38 and JNK). However, only 3-NBA caused an accumulation of p53 in the nucleus and a translocation of Bax to the mitochondria. The p53 inhibitor pifithrin-alpha inhibited 3-NBA-induced apoptosis, indicating that cell death was a result of the triggering of DNA signalling pathways. The highest phosphorylation of Akt and degradation of IkappaB-alpha (suggesting activation of NF-kappaB) were also seen after treatment with 3-NBA. In contrast 3-ABA increased IL-6 release, but caused little or no toxicity. Cytokine release was inhibited by PD98059 and curcumin, suggesting that ERK and NF-kappaB play a role in this process. In conclusion, 3-NBA seems to have a higher potency to induce DNA damage compatible with its cytotoxic effects, while 3-ABA seems to have a greater effect on the immune system. Copyright 2009 Elsevier B.V. All rights reserved.

  5. 3-Nitrobenzanthrone and 3-aminobenzanthrone induce DNA damage and cell signalling in Hepa1c1c7 cells

    Energy Technology Data Exchange (ETDEWEB)

    Landvik, N.E. [Division of Environmental Medicine, Norwegian Institute of Public Health, P.O. Box 404 Torshov N-4303 Oslo (Norway); Arlt, V.M.; Nagy, E. [Section of Molecular Carcinogenesis, Institute of Cancer Research, Brookes Lawley Building, Sutton, Surrey SM2 5NG (United Kingdom); Solhaug, A. [Section for Toxicology, Department of Feed and Food Safety, National Veterinary Institute Pb 750 Sentrum, N-0106 Oslo (Norway); Tekpli, X. [EA SeRAIC, Equipe labellisee Ligue contre le Cancer, IFR 140, Universite de Rennes 1, Rennes (France); Schmeiser, H.H. [Research Group Genetic Alteration in Carcinogenesis, German Cancer Research Center, Im Neuenheimer Feld 280, 69120 Heidelberg (Germany); Refsnes, M. [Division of Environmental Medicine, Norwegian Institute of Public Health, P.O. Box 404 Torshov N-4303 Oslo (Norway); Phillips, D.H. [Section of Molecular Carcinogenesis, Institute of Cancer Research, Brookes Lawley Building, Sutton, Surrey SM2 5NG (United Kingdom); Lagadic-Gossmann, D. [EA SeRAIC, Equipe labellisee Ligue contre le Cancer, IFR 140, Universite de Rennes 1, Rennes (France); Holme, J.A., E-mail: jorn.holme@fhi.no [Division of Environmental Medicine, Norwegian Institute of Public Health, P.O. Box 404 Torshov N-4303 Oslo (Norway)

    2010-02-03

    3-Nitrobenzanthrone (3-NBA) is a mutagenic and carcinogenic environmental pollutant found in diesel exhaust and urban air pollution. In the present work we have characterised the effects of 3-NBA and its metabolite 3-aminobenzanthrone (3-ABA) on cell death and cytokine release in mouse hepatoma Hepa1c1c7 cells. These effects were related to induced DNA damage and changes in cell signalling pathways. 3-NBA resulted in cell death and caused most DNA damage as judged by the amount of DNA adducts ({sup 32}P-postlabelling assay), single strand (ss)DNA breaks and oxidative DNA lesions (comet assay) detected. An increased phosphorylation of H2AX, chk1, chk2 and partly ATM was observed using flow cytometry and/or Western blotting. Both compounds increased phosphorylation of p53 and MAPKs (ERK, p38 and JNK). However, only 3-NBA caused an accumulation of p53 in the nucleus and a translocation of Bax to the mitochondria. The p53 inhibitor pifithrin-alpha inhibited 3-NBA-induced apoptosis, indicating that cell death was a result of the triggering of DNA signalling pathways. The highest phosphorylation of Akt and degradation of I{kappa}B-{alpha} (suggesting activation of NF-{kappa}B) were also seen after treatment with 3-NBA. In contrast 3-ABA increased IL-6 release, but caused little or no toxicity. Cytokine release was inhibited by PD98059 and curcumin, suggesting that ERK and NF-{kappa}B play a role in this process. In conclusion, 3-NBA seems to have a higher potency to induce DNA damage compatible with its cytotoxic effects, while 3-ABA seems to have a greater effect on the immune system.

  6. The Possible Crosstalk of MOB2 With NDR1/2 Kinases in Cell Cycle and DNA Damage Signaling.

    Science.gov (United States)

    Gundogdu, Ramazan; Hergovich, Alexander

    2016-09-06

    This article is the authors' opinion of the roles of the signal transducer Mps one binder 2 (MOB2) in the control of cell cycle progression and the DNA Damage Response (DDR). We recently found that endogenous MOB2 is required to prevent the accumulation of endogenous DNA damage in order to prevent the undesired, and possibly detrimental, activation of cell cycle checkpoints. In this regard, it is noteworthy that MOB2 has been linked biochemically to the regulation of the NDR1/2 (aka STK38/STK38L) protein kinases, which themselves have functions at different steps of the cell cycle. Therefore, we are speculating in this article about the possible connections of MOB2 with NDR1/2 kinases in cell cycle and DDR Signaling.

  7. Solar ultraviolet light potentiates stannous chloride effects as a DNA damaging agent: a spectrophotometrical study

    International Nuclear Information System (INIS)

    Mattos, J.C.P. de; Bernardo-Filho, M.; Leitao, A.C.; Caldeira-de-Araujo, A.; Lage, C.; Leitao, A.C.

    1997-01-01

    Full text. Stannous chloride (Sn Cl 2 ) is a reducing agent widely used to reduce 99m Tc in several radio pharmaceuticals compounds. In spite of being used in nuclear medicine, its genotoxic effects are under investigation in our laboratory. In E. coli, Sn Cl 2 has been shown to have lethal and mutagenic effects, which are thought to occur mainly via active oxygen species. In order to detect some possible direct influence of Sn Cl 2 on nucleic acid, DNA, nucleotides and isolated bases were allowed to react with S N Cl 2 in an in vitro system and the effects analyzed spectro photometrically. Since Sn Cl 2 absorbs light in the UV region, we expected that UV could modify the Sn Cl 2 effects on DNA. Our results indicate that: a. Sn Cl 2 or UV (312 nm, 10 5 J/m 2 ) alone caused only slight alterations in the 260-nm absorption peak of supercoiled plasmid DNA (p U C 9.1); b. Sn Cl 2 + UV (312 nm, 10 5 J/m 2 ) led DNA (p U C 9.1) to a complete loss of its characteristic absorption in the 260-nm region; and c. when reacting with isolated A T P or T T P, Sn Cl 2 + UV (312 nm, 5 x 10 4 J/m 2 ) caused a significant decrease in their 260-nm absorption peaks, as compared to Sn CL 2 alone. Put together, our results indicate that Sn Cl 2 effects are potentiated by the action of solar UV light

  8. Dynamic alteration in H3 serine 10 phosphorylation is G1-phase specific during ionization radiation induced DNA damage response in human cells

    International Nuclear Information System (INIS)

    Sharma, Ajit K.; Bhattacharya, Saikat; Khan, Shafqat A.; Khade, Bharat; Gupta, Sanjay

    2015-01-01

    Highlights: • Loss of H3S10P in response to DNA damage is a universal phenomenon from G1 cells. • The loss happens predominantly from histone H3.3, a transcription activation mark. • Compaction of chromatin occurs during repair stage of DDR. • The alteration of H3S10P shows an inverse correlation with γH2AX. - Abstract: Chromatin acts as a natural barrier in DNA-damage recognition and repair. Histones undergo differential post-translational modification(s) to facilitate DNA damage response (DDR). Importance of modifications like phosphorylation of histone variant H2A.X in DNA repair is very well understood, however, ambiguous results exist in literature regarding the levels of certain histone modifications and their possible role in repair. In the present study, we have investigated in depth the alteration in the level of the highly dynamic histone mark H3S10P as it plays a dual role in different phases of the cell cycle. We show here that H3S10P decreases specifically from irradiated G1-enriched cells irrespective of the damaging agent or the cell line used in the study. Interestingly, the loss occurs predominantly from H3.3 variant which is a transcription activation mark like H3S10P itself, suggesting that the alteration might be implicated in transcription repression. The decrease in other transcription marks like H3K9Ac, H3K14Ac, H3K56Ac and H3S28P along with the occurrence of chromatin condensation in response to DNA damage in G1 phase strengthens the hypothesis. In addition, the alteration in the level of H3S10P shows an inverse correlation with that of γH2AX in a dose-dependent manner and probably occurs from the same mononucleosome. We propose that the drop in the levels of histone H3S10 phosphorylation is a universal phenomenon in response to DNA damage and is a trigger to induce transcription repressive state to facilitate repair

  9. Dynamic alteration in H3 serine 10 phosphorylation is G1-phase specific during ionization radiation induced DNA damage response in human cells

    Energy Technology Data Exchange (ETDEWEB)

    Sharma, Ajit K.; Bhattacharya, Saikat; Khan, Shafqat A.; Khade, Bharat; Gupta, Sanjay, E-mail: sgupta@actrec.gov.in

    2015-03-15

    Highlights: • Loss of H3S10P in response to DNA damage is a universal phenomenon from G1 cells. • The loss happens predominantly from histone H3.3, a transcription activation mark. • Compaction of chromatin occurs during repair stage of DDR. • The alteration of H3S10P shows an inverse correlation with γH2AX. - Abstract: Chromatin acts as a natural barrier in DNA-damage recognition and repair. Histones undergo differential post-translational modification(s) to facilitate DNA damage response (DDR). Importance of modifications like phosphorylation of histone variant H2A.X in DNA repair is very well understood, however, ambiguous results exist in literature regarding the levels of certain histone modifications and their possible role in repair. In the present study, we have investigated in depth the alteration in the level of the highly dynamic histone mark H3S10P as it plays a dual role in different phases of the cell cycle. We show here that H3S10P decreases specifically from irradiated G1-enriched cells irrespective of the damaging agent or the cell line used in the study. Interestingly, the loss occurs predominantly from H3.3 variant which is a transcription activation mark like H3S10P itself, suggesting that the alteration might be implicated in transcription repression. The decrease in other transcription marks like H3K9Ac, H3K14Ac, H3K56Ac and H3S28P along with the occurrence of chromatin condensation in response to DNA damage in G1 phase strengthens the hypothesis. In addition, the alteration in the level of H3S10P shows an inverse correlation with that of γH2AX in a dose-dependent manner and probably occurs from the same mononucleosome. We propose that the drop in the levels of histone H3S10 phosphorylation is a universal phenomenon in response to DNA damage and is a trigger to induce transcription repressive state to facilitate repair.

  10. Implications of caspase-dependent proteolytic cleavage of cyclin A1 in DNA damage-induced cell death

    Energy Technology Data Exchange (ETDEWEB)

    Woo, Sang Hyeok; Seo, Sung-Keum [Division of Radiation Cancer Research, Korea Institute of Radiological and Medical Sciences, 215-4 Gongneung-dong, Nowon-gu, Seoul (Korea, Republic of); An, Sungkwan; Choe, Tae-Boo [Department of Microbiological Engineering, Kon-Kuk University, Gwangjin-gu, Seoul (Korea, Republic of); Hong, Seok-Il [Department of Laboratory Medicine, Korea Cancer Center Hospital, Korea Institute of Radiological and Medical Sciences, 215-4 Gongneung-dong, Nowon-gu, Seoul (Korea, Republic of); Lee, Yun-Han, E-mail: yhlee87@yuhs.ac [Department of Radiation Oncology, College of Medicine, Yonsei University, 250 Seongsan-no, Seodaemun-gu, Seoul (Korea, Republic of); Park, In-Chul, E-mail: parkic@kcch.re.kr [Division of Radiation Cancer Research, Korea Institute of Radiological and Medical Sciences, 215-4 Gongneung-dong, Nowon-gu, Seoul (Korea, Republic of)

    2014-10-24

    Highlights: • Caspase-1 mediates doxorubicin-induced downregulation of cyclin A1. • Active caspase-1 effectively cleaved cyclin A1 at D165. • Cyclin A1 expression is involved in DNA damage-induced cell death. - Abstract: Cyclin A1 is an A-type cyclin that directly binds to CDK2 to regulate cell-cycle progression. In the present study, we found that doxorubicin decreased the expression of cyclin A1 at the protein level in A549 lung cancer cells, while markedly downregulating its mRNA levels. Interestingly, doxorubicin upregulated caspase-1 in a concentration-dependent manner, and z-YAVD-fmk, a specific inhibitor of caspase-1, reversed the doxorubicin-induced decrease in cyclin A1 in A549 lung cancer and MCF7 breast cancer cells. Active caspase-1 effectively cleaved cyclin A1 at D165 into two fragments, which in vitro cleavage assays showed were further cleaved by caspase-3. Finally, we found that overexpression of cyclin A1 significantly reduced the cytotoxicity of doxorubicin, and knockdown of cyclin A1 by RNA interference enhanced the sensitivity of cells to ionizing radiation. Our data suggest a new mechanism for the downregulation of cyclin A1 by DNA-damaging stimuli that could be intimately involved in the cell death induced by DNA damage-inducing stimuli, including doxorubicin and ionizing radiation.

  11. Two dietary polyphenols, fisetin and luteolin, reduce inflammation but augment DNA damage-induced toxicity in human RPE cells.

    Science.gov (United States)

    Hytti, Maria; Szabó, Dora; Piippo, Niina; Korhonen, Eveliina; Honkakoski, Paavo; Kaarniranta, Kai; Petrovski, Goran; Kauppinen, Anu

    2017-04-01

    Plant-derived polyphenols are known to possess anti-inflammatory and antioxidant effects. In recent years, several studies have investigated their potential benefits for treating chronic diseases associated with prolonged inflammation and excessive oxidative stress, such as age-related macular degeneration (AMD). Previously, two polyphenols, fisetin and luteolin, have been reported to increase the survival of retinal pigment epithelial (RPE) cells suffering from oxidative stress as well as decreasing inflammation but the benefits of polyphenol therapy seem to depend on the model system used. Our aim was to analyze the effects of fisetin and luteolin on inflammation and cellular viability in a model of nonoxidative DNA damage-induced cell death in human RPE (hRPE) cells. Pretreatment of ARPE-19 or primary hRPE cells with the polyphenols augmented etoposide-induced cell death as measured by the lactate dehydrogenase and 3-(4,5-dimethyldiazol-2-yl)-2,5-diphenyltetrazolium bromide assays. However, the treatment was able to reduce the release of two proinflammatory cytokines, IL-6 and IL-8, which were determined by enzyme-linked Immunosorbent assay. Analyses of caspase 3 activity, p53 acetylation and SIRT1 protein levels revealed the apoptotic nature of etoposide-evoked cell death and that fisetin and luteolin augmented the etoposide-induced acetylation of p53 and decreased SIRT1 levels. Taken together, our findings suggest that the cytoprotective effects of fisetin and luteolin depend on the stressor they need to combat, whereas their anti-inflammatory potential is sustained over a variety of model systems. Careful consideration of disease pathways will be necessary before fisetin or luteolin can be recommended as therapeutic agents for inflammatory diseases in general and specifically AMD. Copyright © 2017 Elsevier Inc. All rights reserved.

  12. Alpha Particles and X Rays Interact in Inducing DNA Damage in U2OS Cells.

    Science.gov (United States)

    Sollazzo, Alice; Brzozowska, Beata; Cheng, Lei; Lundholm, Lovisa; Haghdoost, Siamak; Scherthan, Harry; Wojcik, Andrzej

    2017-10-01

    Survivors of the atomic bombings of Hiroshima and Nagasaki are monitored for health effects within the Life Span Study (LSS). The LSS results represent the most important source of data about cancer effects from ionizing radiation exposure, which forms the foundation for the radiation protection system. One uncertainty connected to deriving universal risk factors from these results is related to the problem of mixed radiation qualities. The A-bomb explosions generated a mixed beam of the sparsely ionizing gamma radiation and densely ionizing neutrons. However, until now the possible interaction of the two radiation types of inducing biological effects has not been taken into consideration. The existence of such interaction would suggest that the application of risk factors derived from the LSS to predict cancer effects after pure gamma-ray irradiation (such as in the Fukushima prefecture) leads to an overestimation of risk. To analyze the possible interaction of radiation types, a mixed-beam exposure facility was constructed where cells can be exposed to sparsely ionizing X rays and densely ionizing alpha particles. U2OS cells were used, which are stably transfected with a plasmid coding for the DNA repair gene 53BP1 coupled to a gene coding for the green fluorescent protein (GFP). The induction and repair of DNA damage, which are known to be related to cancer induction, were analyzed. The results suggest that alpha particles and X rays interact, leading to cellular and possibly cancer effects, which cannot be accurately predicted based on assuming simple additivity of the individual mixed-beam components.

  13. IFN-γ Induces Mimic Extracellular Trap Cell Death in Lung Epithelial Cells Through Autophagy-Regulated DNA Damage.

    Science.gov (United States)

    Lin, Chiou-Feng; Chien, Shun-Yi; Chen, Chia-Ling; Hsieh, Chia-Yuan; Tseng, Po-Chun; Wang, Yu-Chih

    2016-02-01

    Treatment of interferon-γ (IFN-γ) causes cell growth inhibition and cytotoxicity in lung epithelial malignancies. Regarding the induction of autophagy related to IFN-γ signaling, this study investigated the link between autophagy and IFN-γ cytotoxicity. In A549 human lung cancer cells, IFN-γ treatment induced concurrent apoptotic and nonapoptotic events. Unexpectedly, the nonapoptotic cells present mimic extracellular trap cell death (ETosis), which was regulated by caspase-3 and by autophagy induction through immunity-related GTPase family M protein 1 and activating transcription factor 6. Furthermore, IFN-γ signaling controlled mimic ETosis through a mechanism involving an autophagy- and Fas-associated protein with death domain-controlled caspase-8/-3 activation. Following caspase-mediated lamin degradation, IFN-γ caused DNA damage-associated ataxia telangiectasia and Rad3-related protein (ATR)/ataxia telangiectasia mutated (ATM)-regulated mimic ETosis. Upon ATR/ATM signaling, peptidyl arginine deiminase 4 (PAD4)-mediated histone 3 citrullination promoted mimic ETosis. Such IFN-γ-induced effects were defective in PC14PE6/AS2 human lung cancer cells, which were unsusceptible to IFN-γ-induced autophagy. Due to autophagy-based caspase cascade activation, IFN-γ triggers unconventional caspase-mediated DNA damage, followed by ATR/ATM-regulated PAD4-mediated histone citrullination during mimic ETosis in lung epithelial malignancy.

  14. Parental exposure to the herbicide diuron results in oxidative DNA damage to germinal cells of the Pacific oyster Crassostrea gigas.

    Science.gov (United States)

    Barranger, Audrey; Heude-Berthelin, Clothilde; Rouxel, Julien; Adeline, Béatrice; Benabdelmouna, Abdellah; Burgeot, Thierry; Akcha, Farida

    2016-02-01

    Chemical pollution by pesticides has been identified as a possible contributing factor to the massive mortality outbreaks observed in Crassostrea gigas for several years. A previous study demonstrated the vertical transmission of DNA damage by subjecting oyster genitors to the herbicide diuron at environmental concentrations during gametogenesis. This trans-generational effect occurs through damage to genitor-exposed gametes, as measured by the comet-assay. The presence of DNA damage in gametes could be linked to the formation of DNA damage in other germ cells. In order to explore this question, the levels and cell distribution of the oxidized base lesion 8-oxodGuo were studied in the gonads of exposed genitors. High-performance liquid chromatography coupled with UV and electrochemical detection analysis showed an increase in 8-oxodGuo levels in both male and female gonads after exposure to diuron. Immunohistochemistry analysis showed the presence of 8-oxodGuo at all stages of male germ cells, from early to mature stages. Conversely, the oxidized base was only present in early germ cell stages in female gonads. These results indicate that male and female genitors underwent oxidative stress following exposure to diuron, resulting in DNA oxidation in both early germ cells and gametes, such as spermatozoa, which could explain the transmission of diuron-induced DNA damage to offspring. Furthermore, immunostaining of early germ cells seems indicates that damages caused by exposure to diuron on germ line not only affect the current sexual cycle but also could affect future gametogenesis. Copyright © 2015 Elsevier Inc. All rights reserved.

  15. Oxidative DNA damage in bone marrow cells of patients with low-risk myelodysplastic syndrome

    Czech Academy of Sciences Publication Activity Database

    Novotná, Božena; Bagryantseva, Yana; Šišková, M.; Neuwirtová, R.

    2009-01-01

    Roč. 33, č. 2 (2009), s. 340-343 ISSN 0145-2126 R&D Projects: GA MZd NR8265 Institutional research plan: CEZ:AV0Z50390512 Keywords : Myelodysplastic syndrome * Refractory anemia * Oxidative DNA damage Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.358, year: 2009

  16. Arabidopsis RETINOBLASTOMA RELATED directly regulates DNA damage responses through functions beyond cell cycle control

    Czech Academy of Sciences Publication Activity Database

    Horvath, B.M.; Kourová, Hana; Nagy, S.; Nemeth, E.; Magyar, Z.; Papdi, C.; Ahmad, Z.; Sanchez-Perez, G.F.; Perilli, S.; Blilou, I.; Pettko-Szandtner, A.; Darula, Z.; Meszaros, T.; Binarová, Pavla; Bogre, L.; Scheres, B.

    2017-01-01

    Roč. 36, č. 9 (2017), s. 1261-1278 ISSN 0261-4189 R&D Projects: GA ČR GA15-11657S Institutional support: RVO:61388971 Keywords : Arabidopsis * BRCA1 * DNA damage response Subject RIV: EE - Microbiology, Virology OBOR OECD: Microbiology Impact factor: 9.792, year: 2016

  17. Radiation-induced DNA damage and repair in radiosensitive and radioresistant human tumour cells measured by field inversion gel electrophoresis

    International Nuclear Information System (INIS)

    Smeets, M.F.M.A.; Mooren, E.H.M.; Begg, A.C.

    1993-01-01

    Radiation-induced DNA damage induction and repair was measured in two human squamous carcinoma cell lines with differing radiosensitivities. Experiments were carried out with field inversion gel electrophoresis (FIGE), adapted to measure DNA double strand break (DSB) induction and repair in unlabelled cells. The sensitivity of the method was increased by introducing a hybridization membrane into the agarose gel. Damaged DNA accumulated on one spot on the membrane resulting in high local concentrations. This DNA was quantified using radioactively-labelled total human DNA as a probe. Radiosensitivity differences at physiological temperatures could not be explained by differences in either induction or repair of DNA damage as measured by pulsed field gel electrophoresis. (author)

  18. Radiobiological Effects of Alpha-Particles from Astatine-211: From DNA Damage to Cell Death

    Energy Technology Data Exchange (ETDEWEB)

    Claesson, Kristina

    2011-05-15

    In recent years, the use of high linear energy transfer (LET) radiation for radiotherapeutic applications has gained increased interest. Astatine-211 (211At) is an alpha-particle emitting radionuclide, promising for targeted radioimmunotherapy of isolated tumor cells and microscopic clusters. To improve development of safe radiotherapy using 211At it is important to increase our knowledge of the radiobiological effects in cells. During radiotherapy, both tumors and adjacent normal tissue will be irradiated and therefore, it is of importance to understand differences in the radio response between proliferating and resting cells. The aim of this thesis was to investigate effects in fibroblasts with different proliferation status after irradiation with alpha-particles from 211At or X-rays, from inflicted DNA damage, to cellular responses and biological consequences. Throughout this work, irradiation was performed with alpha-particles from 211A or X-rays. The induction and repair of double-strand breaks (DSBs) in human normal fibroblasts were investigated using pulsed-field gel electrophoresis and fragment analysis. The relative biological effectiveness (RBE) of 211At for DSB induction varied between 1.4 and 3.1. A small increase of DSBs was observed in cycling cells compared to stationary cells. The repair kinetics was slower after 211At and more residual damage was found after 24 h. Comparison between cells with different proliferation status showed that the repair was inefficient in cycling cells with more residual damage, regardless of radiation quality. Activation of cell cycle arrests was investigated using immunofluorescent labeling of the checkpoint kinase Chk2 and by measuring cell cycle distributions with flow cytometry analysis. After alpha-particle irradiation, the average number of Chk2-foci was larger and the cells had a more affected cell cycle progression for several weeks compared with X-irradiated cells, indicating a more powerful arrest after 211At

  19. Grape juice concentrate prevents oxidative DNA damage in peripheral blood cells of rats subjected to a high-cholesterol diet.

    Science.gov (United States)

    Aguiar, Odair; Gollücke, Andréa Pittelli Boiago; de Moraes, Bárbara Bueno; Pasquini, Gabriela; Catharino, Rodrigo Ramos; Riccio, Maria Francesca; Ihara, Silvia Saiuli Miki; Ribeiro, Daniel Araki

    2011-03-01

    The goal of the present study was to investigate whether subchronic treatment with grape juice concentrate is able to protect liver and peripheral blood cells against cholesterol-induced injury in rats. The effects of the grape juice concentrate treatment on histopathological changes, immunohistochemistry for cyclo-oxygenase-2 (COX-2), and basal and oxidative DNA damage induced by H2O2 using a single-cell gel (comet) assay were evaluated. Male Wistar rats (n 18) were divided into three groups: group 1--negative control; group 2--cholesterol at 1 % (w/w) in their diet, treated for 5 weeks; group 3--cholesterol at 1 % in their chow, treated for 5 weeks, and grape juice concentrate at 222 mg/d in their drinking-water in the final week only. The results indicated that the treatment with grape juice concentrate did not show remarkable differences regarding liver tissue in group 3 compared with group 2. However, grape juice concentrate was able to decrease oxidative DNA damage induced by H2O2 in peripheral blood cells, as depicted by the tail moment results. COX-2 expression in the liver did not show statistically significant differences (P>0·05) between groups. Taken together, the present results suggest that the administration of subchronic grape juice concentrate prevents oxidative DNA damage in peripheral blood cells.

  20. MAXIMIZATION OF DNA DAMAGE TO MGMT(+ EGFR(+ GBM CELLS USING OPTIMAL COMBINATION OF TEMOZOLOMIDE-ANTI EGFR MONOCLONAL ANTIBODY NIMOTUZUMAB

    Directory of Open Access Journals (Sweden)

    M. A. M. Inggas

    2015-09-01

    Full Text Available Background: Glioblastoma multiforme (GBM is the most aggressive primary brain tumor in adultswith dismal prognosis due to the unavailability of an effective therapy. Up to now, there had been no definitive studies published on EGFR inhibition therapy as a chemosensitizer for GBM therapy using Temozolomide (TMZ. This study aims to reveal the most effective method and timing to administer TMZ-anti EGFR targeted therapy which causes maximal DNA damage on GBM cells.Methods: Various regimens of anti EGFR monoclonal antibody Nimotuzumab (NMZ was administered in different combinations with TMZ, performed on U87MG MGMT(+ EGFR(+ cells. The effectiveness of the combinations were evaluated by measuring yH2AX levels which reflects the degree of DNA damage. One-way Anova and LSD tests were performed to determine the effects of each treatment with p<0.05. Results and discussion: the mean SD of yH2AX of each treatment was: 11,90±1,25 for the control group; 29.33±1.91 for NMZ alone; 28.13±1.58 for TMZ alone; 41.53±3.51 for concurrent use; 35.67 ±2.65 for NMZ after 24 hours TMZ; 31.87±2.94 for NMZ after 48 hours TMZ; 39.57±4.2 for TMZ after 24 hours NMZ; and 35.93 ±3.56 for TMZ after 48 hours NMZ. The administration of TMZ concurrent with or after 24 hours NMZ gives the highest amount of DNA damage to GBM cells. Conclusion: The administration of Nimotuzumab targeted therapy up to 24 hours before Temozolomide chemotherapy has been proven to be effective in maximizing the amount of DNA damage done to GBM cells in vitro. 

  1. Cytotoxic and DNA-damaging properties of glyphosate and Roundup in human-derived buccal epithelial cells.

    Science.gov (United States)

    Koller, Verena J; Fürhacker, Maria; Nersesyan, Armen; Mišík, Miroslav; Eisenbauer, Maria; Knasmueller, Siegfried

    2012-05-01

    Glyphosate (G) is the largest selling herbicide worldwide; the most common formulations (Roundup, R) contain polyoxyethyleneamine as main surfactant. Recent findings indicate that G exposure may cause DNA damage and cancer in humans. Aim of this investigation was to study the cytotoxic and genotoxic properties of G and R (UltraMax) in a buccal epithelial cell line (TR146), as workers are exposed via inhalation to the herbicide. R induced acute cytotoxic effects at concentrations > 40 mg/l after 20 min, which were due to membrane damage and impairment of mitochondrial functions. With G, increased release of extracellular lactate dehydrogenase indicative for membrane damage was observed at doses > 80 mg/l. Both G and R induced DNA migration in single-cell gel electrophoresis assays at doses > 20 mg/l. Furthermore, an increase of nuclear aberrations that reflect DNA damage was observed. The frequencies of micronuclei and nuclear buds were elevated after 20-min exposure to 10-20 mg/l, while nucleoplasmatic bridges were only enhanced by R at the highest dose (20 mg/l). R was under all conditions more active than its active principle (G). Comparisons with results of earlier studies with lymphocytes and cells from internal organs indicate that epithelial cells are more susceptible to the cytotoxic and DNA-damaging properties of the herbicide and its formulation. Since we found genotoxic effects after short exposure to concentrations that correspond to a 450-fold dilution of spraying used in agriculture, our findings indicate that inhalation may cause DNA damage in exposed individuals.

  2. Assessment of vinyl chloride-induced DNA damage in lymphocytes of plastic industry workers using a single-cell gel electrophoresis technique.

    Science.gov (United States)

    Awara, W M; El-Nabi, S H; El-Gohary, M

    1998-06-26

    DNA damage and the formation of stable carcinogen-DNA adducts are considered critical events in the initiation of the carcinogenic process. This study was carried out to assess whether exposure of plastics industry workers to the vinyl chloride monomer (VCM) for different periods of time would cause DNA damage, using the single-cell gel electrophoresis (SCGE) technique. Levels of DNA damage was assessed by both extent of DNA migration and numbers of DNA damaged spots in the peripheral blood lymphocytes from 32 plastics workers with different periods of exposure to VCM; they were evaluated by comparison with a group of non-exposed individuals. It was found that plastics workers who were exposed to VCM for different periods of time showed significantly increased levels of DNA damage compared with the non-exposed subjects. There was a significant correlation between the severity of DNA damage and duration of exposure. However, no significant correlation was found between the age of all subjects and DNA damage. Concentrations of VCM in the air inside the factory were found to be significantly higher than values in non-exposed areas, despite being lower than the threshold limit value (TLV). Our results encourage the application of SCGE as a sensitive, simple, fast and useful technique in the regular health screening of workers occupationally exposed to VCM (even at concentrations below the TLV) to assess the possibility of any DNA damage.

  3. Ionizing radiation-induced DNA damage and its repair in human cells. Final performance report, July 1992 - June 1995

    International Nuclear Information System (INIS)

    Dizdaroglu, M.

    1995-01-01

    The studies of DNA damage in living cells in vitro and in vivo were continued. A variety of systems including cultured mammalian cells, animals, and human tissues were used to conduct these studies. In addition, enzymatic repair of DNA base damage was studied using several DNA glycosylases. To this end, substrate specificities of these enzymes were examined in terms of a large number of base lesions in DNA. In the first phase of the studies, the author sought to introduce improvements to his methodologies for measurement of DNA damage using the technique of gas chromatography/mass spectrometry (GC/MS). In particular, the quantitative measurement of DNA base damage and DNA-protein crosslinks was improved by incorporation of isotope-dilution mass spectrometry into the methodologies. This is one of the most accurate techniques for quantification of organic compounds. Having improved the measurement technique, studies of DNA damage in living cells and DNA repair by repair enzymes were pursued. This report provides a summary of these studies with references to the original work

  4. Space Radiation Effects on Human Cells: Modeling DNA Breakage, DNA Damage Foci Distribution, Chromosomal Aberrations and Tissue Effects

    Science.gov (United States)

    Ponomarev, A. L.; Huff, J. L.; Cucinotta, F. A.

    2011-01-01

    Future long-tem space travel will face challenges from radiation concerns as the space environment poses health risk to humans in space from radiations with high biological efficiency and adverse post-flight long-term effects. Solar particles events may dramatically affect the crew performance, while Galactic Cosmic Rays will induce a chronic exposure to high-linear-energy-transfer (LET) particles. These types of radiation, not present on the ground level, can increase the probability of a fatal cancer later in astronaut life. No feasible shielding is possible from radiation in space, especially for the heavy ion component, as suggested solutions will require a dramatic increase in the mass of the mission. Our research group focuses on fundamental research and strategic analysis leading to better shielding design and to better understanding of the biological mechanisms of radiation damage. We present our recent effort to model DNA damage and tissue damage using computational models based on the physics of heavy ion radiation, DNA structure and DNA damage and repair in human cells. Our particular area of expertise include the clustered DNA damage from high-LET radiation, the visualization of DSBs (DNA double strand breaks) via DNA damage foci, image analysis and the statistics of the foci for different experimental situations, chromosomal aberration formation through DSB misrepair, the kinetics of DSB repair leading to a model-derived spectrum of chromosomal aberrations, and, finally, the simulation of human tissue and the pattern of apoptotic cell damage. This compendium of theoretical and experimental data sheds light on the complex nature of radiation interacting with human DNA, cells and tissues, which can lead to mutagenesis and carcinogenesis later in human life after the space mission.

  5. Activation of EGFR and ERBB2 by Helicobacter pylori Results in Survival of Gastric Epithelial Cells with DNA Damage

    Science.gov (United States)

    Chaturvedi, Rupesh; Asim, Mohammad; Piazuelo, M. Blanca; Yan, Fang; Barry, Daniel P.; Sierra, Johanna Carolina; Delgado, Alberto G.; Hill, Salisha; Casero, Robert A.; Bravo, Luis E.; Dominguez, Ricardo L.; Correa, Pelayo; Polk, D. Brent; Washington, M. Kay; Rose, Kristie L.; Schey, Kevin L.; Morgan, Douglas R.; Peek, Richard M.; Wilson, Keith T.

    2014-01-01

    BACKGROUND & AIMS The gastric cancer-causing pathogen Helicobacter pylori upregulates spermine oxidase (SMOX) in gastric epithelial cells, causing oxidative stress-induced apoptosis and DNA damage. A subpopulation of SMOXhigh cells are resistant to apoptosis, despite their high levels of DNA damage. Because epidermal growth factor receptor (EGFR) activation can regulate apoptosis, we determined its role in SMOX-mediated effects. METHODS SMOX, apoptosis, and DNA damage were measured in gastric epithelial cells from H pylori-infected Egfrwa5 mice (which have attenuated EGFR activity), Egfr wild-type mice, or in infected cells incubated with EGFR inhibitors or deficient in EGFR. Phosphoproteomic analysis was performed. Two independent tissue microarrays containing each stage of disease, from gastritis to carcinoma, and gastric biopsies from Colombian and Honduran cohorts were analyzed by immunohistochemistry. RESULTS SMOX expression and DNA damage were decreased, and apoptosis increased in H pylori-infected Egfrwa5 mice. H pylori-infected cells with deletion or inhibition of EGFR had reduced levels of SMOX, DNA damage, and DNA damagehigh apoptosislow cells. Phosphoproteomic analysis revealed increased EGFR and ERBB2 signaling. Immunoblot analysis demonstrated the presence of a phosphorylated (p)EGFR–ERBB2 heterodimer and pERBB2; knockdown of ErbB2 facilitated apoptosis of DNA damagehigh apoptosislow cells. SMOX was increased in all stages of gastric disease, peaking in tissues with intestinal metaplasia, whereas pEGFR, pEGFR–ERBB2, and pERBB2 were increased predominantly in tissues demonstrating gastritis or atrophic gastritis. Principal component analysis separated gastritis tissues from patients with cancer vs those without cancer. pEGFR, pEGFR–ERBB2, pERBB2, and SMOX were increased in gastric samples from patients whose disease progressed to intestinal metaplasia or dysplasia, compared with patients whose disease did not progress. CONCLUSIONS In an analysis

  6. Attenuation of radiation-induced DNA damage due to paracrine interactions between normal human epithelial and stromal cells

    International Nuclear Information System (INIS)

    Saenko, V.A.; Nakazawa, Yu.; Rogounovitch, T.I.; Suzuki, K.; Mitsutake, N.; Matsuse, M.; Yamashita, S.

    2007-01-01

    Complete text of publication follows. Objective: Developmentally, every tissue accommodates different types of cells, such as epitheliocytes and stromal cells in parenchymal organs. To better understand the complexity of radiation response, it is necessary to evaluate possible cross-talk between different tissue components. This work was set out to investigate reciprocal influence of normal human epithelial cells and fibroblasts on the extent of radiation-induced DNA damage. Methods: Model cultures of primary human thyrocytes (PT), normal diploid fibroblasts (BJ), PT/BJ cell co-culture and conditioned medium transfer were used to examine DNA damage in terms of γ-H2AX foci number per cell or by Comet assay after exposure to different doses of γ-rays. Results: In co-cultures, the kinetics of γ-H2AX foci number change was dose-dependent and similar to that in individual PT and BJ cultures. The number of γ-H2AX foci in co-cultures was significantly lower (∼25%) in both types of cells comparing to individual cultures. Reciprocal conditioned medium transfer to individual counterpart cells prior to irradiation resulted in approximately 35% reduction in the number γ-H2AX foci at 1 Gy and lower doses in both PT and BJ demonstrating the role of paracrine soluble factors. Comet assay corroborated the results of γ-H2AX foci counting in conditioned medium transfer experiments. In contrast to medium conditioned on PT cells, conditioned medium collected from several human thyroid cancer cell lines failed to establish DNA-protected state in BJ fibroblasts. In its turn, medium conditioned on BJ cells did not change the extent of radiation-induced DNA damage in cancer cell lines tested. Conclusion: The results imply the existence of a network of soluble factor-mediated paracrine interactions between normal epithelial and stromal cells that could be a part of natural mechanism by which cells protect DNA from genotoxic stress.

  7. DNA damage and methylation induced by glyphosate in human peripheral blood mononuclear cells (in vitro study).

    Science.gov (United States)

    Kwiatkowska, Marta; Reszka, Edyta; Woźniak, Katarzyna; Jabłońska, Ewa; Michałowicz, Jaromir; Bukowska, Bożena

    2017-07-01

    Glyphosate is a very important herbicide that is widely used in the agriculture, and thus the exposure of humans to this substance and its metabolites has been noted. The purpose of this study was to assess DNA damage (determination of single and double strand-breaks by the comet assay) as well as to evaluate DNA methylation (global DNA methylation and methylation of p16 (CDKN2A) and p53 (TP53) promoter regions) in human peripheral blood mononuclear cells (PBMCs) exposed to glyphosate. PBMCs were incubated with the compound studied at concentrations ranging from 0.1 to 10 mM for 24 h. The study has shown that glyphosate induced DNA lesions, which were effectively repaired. However, PBMCs were unable to repair completely DNA damage induced by glyphosate. We also observed a decrease in global DNA methylation level at 0.25 mM of glyphosate. Glyphosate at 0.25 mM and 0.5 mM increased p53 promoter methylation, while it did not induce statistically significant changes in methylation of p16 promoter. To sum up, we have shown for the first time that glyphosate (at high concentrations from 0.5 to 10 mM) may induce DNA damage in leucocytes such as PBMCs and cause DNA methylation in human cells. Copyright © 2017 Elsevier Ltd. All rights reserved.

  8. Kaempferol induces DNA damage and inhibits DNA repair associated protein expressions in human promyelocytic leukemia HL-60 cells.

    Science.gov (United States)

    Wu, Lung-Yuan; Lu, Hsu-Feng; Chou, Yu-Cheng; Shih, Yung-Luen; Bau, Da-Tian; Chen, Jaw-Chyun; Hsu, Shu-Chun; Chung, Jing-Gung

    2015-01-01

    Numerous evidences have shown that plant flavonoids (naturally occurring substances) have been reported to have chemopreventive activities and protect against experimental carcinogenesis. Kaempferol, one of the flavonoids, is widely distributed in fruits and vegetables, and may have cancer chemopreventive properties. However, the precise underlying mechanism regarding induced DNA damage and suppressed DNA repair system are poorly understood. In this study, we investigated whether kaempferol induced DNA damage and affected DNA repair associated protein expression in human leukemia HL-60 cells in vitro. Percentages of viable cells were measured via a flow cytometry assay. DNA damage was examined by Comet assay and DAPI staining. DNA fragmentation (ladder) was examined by DNA gel electrophoresis. The changes of protein levels associated with DNA repair were examined by Western blotting. Results showed that kaempferol dose-dependently decreased the viable cells. Comet assay indicated that kaempferol induced DNA damage (Comet tail) in a dose-dependent manner and DAPI staining also showed increased doses of kaempferol which led to increased DNA condensation, these effects are all of dose-dependent manners. Western blotting indicated that kaempferol-decreased protein expression associated with DNA repair system, such as phosphate-ataxia-telangiectasia mutated (p-ATM), phosphate-ataxia-telangiectasia and Rad3-related (p-ATR), 14-3-3 proteins sigma (14-3-3σ), DNA-dependent serine/threonine protein kinase (DNA-PK), O(6)-methylguanine-DNA methyltransferase (MGMT), p53 and MDC1 protein expressions, but increased the protein expression of p-p53 and p-H2AX. Protein translocation was examined by confocal laser microscopy, and we found that kaempferol increased the levels of p-H2AX and p-p53 in HL-60 cells. Taken together, in the present study, we found that kaempferol induced DNA damage and suppressed DNA repair and inhibited DNA repair associated protein expression in HL-60

  9. E-Cigarette Aerosol Exposure Induces Reactive Oxygen Species, DNA Damage, and Cell Death in Vascular Endothelial Cells.

    Science.gov (United States)

    Anderson, Chastain; Majeste, Andrew; Hanus, Jakub; Wang, Shusheng

    2016-12-01

    Cigarette smoking remains one of the leading causes of preventable death worldwide. Vascular cell death and dysfunction is a central or exacerbating component in the majority of cigarette smoking related pathologies. The recent development of the electronic nicotine delivery systems known as e-cigarettes provides an alternative to conventional cigarette smoking; however, the potential vascular health risks of e-cigarette use remain unclear. This study evaluates the effects of e-cigarette aerosol extract (EAE) and conventional cigarette smoke extract (CSE) on human umbilical vein endothelial cells (HUVECs). A laboratory apparatus was designed to produce extracts from e-cigarettes and conventional cigarettes according to established protocols for cigarette smoking. EAE or conventional CSE was applied to human vascular endothelial cells for 4-72 h, dependent on the assay. Treated cells were assayed for reactive oxygen species, DNA damage, cell viability, and markers of programmed cell death pathways. Additionally, the anti-oxidants α-tocopherol and n-acetyl-l-cysteine were used to attempt to rescue e-cigarette induced cell death. Our results indicate that e-cigarette aerosol is capable of inducing reactive oxygen species, causing DNA damage, and significantly reducing cell viability in a concentration dependent fashion. Immunofluorescent and flow cytometry analysis indicate that both the apoptosis and programmed necrosis pathways are triggered by e-cigarette aerosol treatment. Additionally, anti-oxidant treatment provides a partial rescue of the induced cell death, indicating that reactive oxygen species play a causal role in e-cigarette induced cytotoxicity. © The Author 2016. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  10. Involvement of stress-activated protein kinase in the cellular response to 1-beta-D-arabinofuranosylcytosine and other DNA-damaging agents.

    Science.gov (United States)

    Saleem, A; Datta, R; Yuan, Z M; Kharbanda, S; Kufe, D

    1995-12-01

    The cellular response to 1-beta-D-arabinofuranosylcytosine (ara-C) includes activation of Jun/AP-1, induction of c-jun transcription, and programmed cell death. The stress-activated protein (SAP) kinases stimulate the transactivation function of c-jun by amino terminal phosphorylation. The present work demonstrates that ara-C activates p54 SAP kinase. The finding that SAP kinase is also activated by alkylating agents (mitomycin C and cisplatinum) and the topoisomerase I inhibitor 9-amino-camptothecin supports DNA damage as an initial signal in this cascade. The results demonstrate that ara-C also induces binding of SAP kinase to the SH2/SH3-containing adapter protein Grb2. SAP kinase binds to the SH3 domains of Grb2, while interaction of the p85 alpha-subunit of phosphatidylinositol 3-kinase complex. The results also demonstrate that ara-C treatment is associated with inhibition of lipid and serine kinase activities of PI 3-kinase. The potential significance of the ara-C-induced interaction between SAP kinase and PI 3-kinase is further supported by the demonstration that Wortmannin, an inhibitor of PI 3-kinase, stimulates SAP kinase activity. The finding that Wortmannin treatment is also associated with internucleosomal DNA fragmentation may support a potential link between PI 3-kinase and regulation of both SAP kinase and programmed cell death.

  11. DNA damage evaluation of hydroxyapatite on fibroblast cell L929 using the single cell gel electrophoresis assay.

    Science.gov (United States)

    Rajab, N F; Yaakob, T A; Ong, B Y; Hamid, M; Ali, A M; Annuar, B O; Inayat-Hussain, S H

    2004-05-01

    Hydroxyapatite is the main component of the bone which is a potential biomaterial substance that can be applied in orthopaedics. In this study, the biocompatibility of this biomaterial was assessed using an in vitro technique. The cytotoxicity and genotoxicity effect of HA2 and HA3 against L929 fibroblast cell was evaluated using the MTT Assay and Alkaline Comet Assay respectively. Both HA2 and HA3 compound showed low cytotoxicity effect as determined using MTT Assay. Cells viability following 72 hours incubation at maximum concentration of both HA2 and HA3 (200 mg/ml) were 75.3 +/- 8.8% and 86.7 +/- 13.1% respectively. However, the cytotoxicity effect of ZnSO4.7H2O as a positive control showed an IC50 values of 46 mg/ml (160 microM). On the other hand, both HA2 and HA3 compound showed a slight genotoxicity effect as determined using the Alkaline Comet Assay following incubation at the concentration 200 mg/ml for 72 hours. This assay has been widely used in genetic toxicology to detect DNA strand breaks and alkali-labile site. The percentage of the cells with DNA damage for both substance was 27.7 +/- 1.3% and 15.6 +/- 1.0% for HA2 and HA3 respectively. Incubation of the cells for 24 hours with 38 microg/ml (IC25) of positive control showed an increase in percentage of cells with DNA damage (67.5 +/- 0.7%). In conclusion, our study indicated that both hydroxyapatite compounds showed a good biocompatibility in fibroblast cells.

  12. Effect of polyamine depletion on DNA damage and repair following UV irradiation of HeLa cells

    International Nuclear Information System (INIS)

    Snyder, R.D.; Sunkara, P.S.

    1990-01-01

    Treatment of HeLa cells with the polyamine biosynthesis inhibitors, methylglyoxal bis(guanylhydrazone) (MGBG), difluoromethylornithine (DFMO) or a combination of the two, resulted in reduction in cellular polyamine levels. Analysis of UV light-induced DNA damage and repair in these polyamine depleted cells revealed distinct differences in the repair process relative to that seen in cells possessing a normal polyamine complement. Observed patterns of differential polyamine depletion by DFMO and MGBG, and partial reversal of repair inhibition by polyamine supplementation, suggest that polyamine depletion per se, rather than some secondary effect of inhibitor treatment, is responsible for the inhibition of repair. (author)

  13. Effect of polyamine depletion on DNA damage and repair following UV irradiation of HeLa cells

    Energy Technology Data Exchange (ETDEWEB)

    Snyder, R.D.; Sunkara, P.S. (Merrell Dow Research Inst., Cincinnati, OH (USA))

    1990-09-01

    Treatment of HeLa cells with the polyamine biosynthesis inhibitors, methylglyoxal bis(guanylhydrazone) (MGBG), difluoromethylornithine (DFMO) or a combination of the two, resulted in reduction in cellular polyamine levels. Analysis of UV light-induced DNA damage and repair in these polyamine depleted cells revealed distinct differences in the repair process relative to that seen in cells possessing a normal polyamine complement. Observed patterns of differential polyamine depletion by DFMO and MGBG, and partial reversal of repair inhibition by polyamine supplementation, suggest that polyamine depletion per se, rather than some secondary effect of inhibitor treatment, is responsible for the inhibition of repair. (author).

  14. Inhibition of Hsp90 acts synergistically with topoisomerase II poisons to increase the apoptotic killing of cells due to an increase in topoisomerase II mediated DNA damage.

    Science.gov (United States)

    Barker, Catherine R; McNamara, Anne V; Rackstraw, Stephen A; Nelson, David E; White, Mike R; Watson, Alastair J M; Jenkins, John R

    2006-01-01

    Topoisomerase II plays a crucial role during chromosome condensation and segregation in mitosis and meiosis and is a highly attractive target for chemotherapeutic agents. We have identified previously topoisomerase II and heat shock protein 90 (Hsp90) as part of a complex. In this paper we demonstrate that drug combinations targeting these two enzymes cause a synergistic increase in apoptosis. The objective of our study was to identify the mode of cell killing and the mechanism behind the increase in topoisomerase II mediated DNA damage. Importantly we demonstrate that Hsp90 inhibition results in an increased topoiosmerase II activity but not degradation of topoisomerase II and it is this, in the presence of a topoisomerase II poison that causes the increase in cell death. Our results suggest a novel mechanism of action where the inhibition of Hsp90 disrupts the Hsp90-topoisomerase II interaction leading to an increase in and activation of unbound topoisomerase II, which, in the presence of a topoisomerase II poison leads to the formation of an increased number of cleavable complexes ultimately resulting in rise in DNA damage and a subsequent increase cell death.

  15. The DNA damage of high doses of X-ray on human peripheral blood nucleated cell's and sperm

    International Nuclear Information System (INIS)

    Wang Hui; Zoulian; Jiang Qisheng; Li Fengsheng; He Rui; Song Xiujun

    2011-01-01

    Objective: To detect the DNA damage of high doses of X-ray on human peripheral blood nucleated cell's and sperm by single cell gel electrophoresis (SCGE). Evaluation the level of DNA damage of human peripheral blood nucleated cell's and sperm after high doses of X-ray. Methods: Using human peripheral blood with normal blood routine and normal sperm,give the dose of 0 Gy, 2 Gy, 4 Gy, 6 Gy, 8 Gy, 10 Gy X-ray radiation with energy of 6MU. Detect the percentage of comet-like tail, tail length and content of DNA in tail of whole blood cell's DNA and sperm's DNA by SCGE technique in 1 hour. Results: The peripheral blood nucleated cell's and sperm's comet rate were 1.00±0.10%, 2.1±1.5%, respectively, have an evidently variance in 0 Gy group (υ=18, t=2.31>1.734, P 1.734, P 1.734, P<0.05). The peripheral blood nucleated cell's and sperm's comet rate were all 100%, 100%, have no-statistical significance in 8 Gy, 10 Gy group. Conclusion: The evidence is powerful enough. That the sperm's SCGE is more sensitive than peripheral blood nucleated cell's SCGE in reflect the X-ray damage in a certain extent (2-6 Gy). (authors)

  16. Lead-induced DNA damage in Vicia faba root cells: Potential involvement of oxidative stress

    OpenAIRE

    Pourrut, Bertrand; Jean, Séverine; Silvestre, Jérôme; Pinelli, Eric

    2011-01-01

    Genotoxic effects of lead (0–20 µM) were investigated in whole-plant roots of Vicia faba L., grown hydroponically under controlled conditions. Lead-induced DNA damage in V. faba roots was evaluated by use of the comet assay, which allowed the detection of DNA strand-breakage and with the V. faba micronucleus test, which revealed chromosome aberrations. The results clearly indicate that lead induced DNA fragmentation in a dose-dependant manner with a maximum effect at 10 µM. In addition, at th...

  17. DNA damage in gill cells of Corbicula japonica exposed to natural and anthropogenic stressors

    Directory of Open Access Journals (Sweden)

    Valentina Vladimirovna Slobodskova

    2015-06-01

    The results are presented as the percentage distribution of nuclei in the various damage classes and summarized in an index of DNA integrity or genetic damage index GDI (Cavas, Kohen, 2008: The results from our study showed significant level of DNA damage from the C. japonica which were collected from polluted sites. Unpolluted sites were described as with no or minimal DNA strand breaks. Mollusks collected at polluted areas (estuary Razdolnaya river, lagoon Tihaya showed high levels of DNA damage, GDI is equal to 3.22±0.2 and 3.11±0.7 in gills respectively. C. japonica obtained from a ‘clean’ areas (estuary Artemovka river, estuary Partizanskaya river demonstrate less high level of DNA damage destruction, GDI is equal to 0,6±0,08 and 0,71±0,12 in gills respectively. Lipid peroxidation level was assayed measurement of malondialdehyde (MDA, a decomposition product of polyunsaturated fatty acids hydro peroxides were determined by the TBA reaction. The absorbance was read at 532 nm after removal of substances (TBARS formed was calculated by using an extinction coefficient of 1.56*105 M-1 cm -1 formed per g dry weight. C. japonica sampled at Artemovka estuary, Partizanskaya estuary, Razdolnaya estuary, Tihaya lagoon showed LPX level (3.46±0.59, 5.62±0.82, 12.85±0.52, 15.32±1.13 nmol TBARS/g dry wt in gills respectively. In conclusion, it can be noted that in the course of the experiment we found a clear relationship between the amount of DNA damage and the level of peroxidation products (MDA in the gills of bivalve C. japonica, collected from sites with varying degrees of anthropogenic load. It should be emphasized that the shellfish that live in polluted areas are likely to be in a state of oxidative stress, which is one of the causes the degradation of DNA.

  18. Lung Basal Stem Cells Rapidly Repair DNA Damage Using the Error-Prone Nonhomologous End-Joining Pathway

    Science.gov (United States)

    Weeden, Clare E.; Chen, Yunshun; Ma, Stephen B.; Hu, Yifang; Ramm, Georg; Sutherland, Kate D.; Smyth, Gordon K.

    2017-01-01

    Lung squamous cell carcinoma (SqCC), the second most common subtype of lung cancer, is strongly associated with tobacco smoking and exhibits genomic instability. The cellular origins and molecular processes that contribute to SqCC formation are largely unexplored. Here we show that human basal stem cells (BSCs) isolated from heavy smokers proliferate extensively, whereas their alveolar progenitor cell counterparts have limited colony-forming capacity. We demonstrate that this difference arises in part because of the ability of BSCs to repair their DNA more efficiently than alveolar cells following ionizing radiation or chemical-induced DNA damage. Analysis of mice harbouring a mutation in the DNA-dependent protein kinase catalytic subunit (DNA-PKcs), a key enzyme in DNA damage repair by nonhomologous end joining (NHEJ), indicated that BSCs preferentially repair their DNA by this error-prone process. Interestingly, polyploidy, a phenomenon associated with genetically unstable cells, was only observed in the human BSC subset. Expression signature analysis indicated that BSCs are the likely cells of origin of human SqCC and that high levels of NHEJ genes in SqCC are correlated with increasing genomic instability. Hence, our results favour a model in which heavy smoking promotes proliferation of BSCs, and their predilection for error-prone NHEJ could lead to the high mutagenic burden that culminates in SqCC. Targeting DNA repair processes may therefore have a role in the prevention and therapy of SqCC. PMID:28125611

  19. Toxic effect of silica nanoparticles on endothelial cells through DNA damage response via Chk1-dependent G2/M checkpoint.

    Directory of Open Access Journals (Sweden)

    Junchao Duan

    Full Text Available Silica nanoparticles have become promising carriers for drug delivery or gene therapy. Endothelial cells could be directly exposed to silica nanoparticles by intravenous administration. However, the underlying toxic effect mechanisms of silica nanoparticles on endothelial cells are still poorly understood. In order to clarify the cytotoxicity of endothelial cells induced by silica nanoparticles and its mechanisms, cellular morphology, cell viability and lactate dehydrogenase (LDH release were observed in human umbilical vein endothelial cells (HUVECs as assessing cytotoxicity, resulted in a dose- and time- dependent manner. Silica nanoparticles-induced reactive oxygen species (ROS generation caused oxidative damage followed by the production of malondialdehyde (MDA as well as the inhibition of superoxide dismutase (SOD and glutathione peroxidase (GSH-Px. Both necrosis and apoptosis were increased significantly after 24 h exposure. The mitochondrial membrane potential (MMP decreased obviously in a dose-dependent manner. The degree of DNA damage including the percentage of tail DNA, tail length and Olive tail moment (OTM were markedly aggravated. Silica nanoparticles also induced G2/M arrest through the upregulation of Chk1 and the downregulation of Cdc25C, cyclin B1/Cdc2. In summary, our data indicated that the toxic effect mechanisms of silica nanoparticles on endothelial cells was through DNA damage response (DDR via Chk1-dependent G2/M checkpoint signaling pathway, suggesting that exposure to silica nanoparticles could be a potential hazards for the development of cardiovascular diseases.

  20. DNA damaging bystander signalling from stem cells, cancer cells and fibroblasts after Cr(VI) exposure and its dependence on telomerase

    Energy Technology Data Exchange (ETDEWEB)

    Cogan, Nicola [Bristol Implant Research Centre, University of Bristol, Bristol, BS10 5NB (United Kingdom); Baird, Duncan M. [Department of Pathology School of Medicine, Cardiff University, Henry Wellcome Building for Biomedical Research in Wales, Heath Park, Cardiff, CF14 4XN (United Kingdom); Phillips, Ryan [Bristol Implant Research Centre, University of Bristol, Bristol, BS10 5NB (United Kingdom); Crompton, Lucy A.; Caldwell, Maeve A. [Henry Wellcome Laboratories for Integrative Neuroscience and Endocrinology, University of Bristol, Bristol, BS1 3NY (United Kingdom); Rubio, Miguel A. [Center of Regenerative Medicine in Barcelona, CMRB Dr. Aiguader, 88, 7th Floor, 08003 Barcelona (Spain); Newson, Roger [Radiation and Environmental Science Centre, Focas Institute, Dublin Institute of Technology, Dublin 2 (Ireland); Lyng, Fiona [National Heart and Lung Institute, Imperial College London, London, SW7 2AZ (United Kingdom); Case, C. Patrick, E-mail: c.p.case@bristol.ac.uk [Bristol Implant Research Centre, University of Bristol, Bristol, BS10 5NB (United Kingdom)

    2010-01-05

    The bystander effect is a feature of low dose radiation exposure and is characterized by a signaling process from irradiated cells to non irradiated cells, which causes DNA and chromosome damage in these 'nearest neighbour' cells. Here we show that a low and short dose of Cr(VI) can induce stem cells, cancer cells and fibroblasts to chronically secrete bystander signals, which cause DNA damage in neighboring cells. The Cr(VI) induced bystander signaling depended on the telomerase status of either cell. Telomerase negative fibroblasts were able to receive DNA damaging signals from telomerase positive or negative fibroblasts or telomerase positive cancer cells. However telomerase positive fibroblasts were resistant to signals from Cr(VI) exposed telomerase positive fibroblasts or cancer cells. Human embryonic stem cells, with positive Oct4 staining as a marker of pluripotency, showed no significant increase of DNA damage from adjacent Cr and mitomycin C exposed fibroblasts whilst those cells that were negatively stained did. This selectivity of DNA damaging bystander signaling could be an important consideration in developing therapies against cancer and in the safety and effectiveness of tissue engineering and transplantation using stem cells.

  1. DNA damaging bystander signalling from stem cells, cancer cells and fibroblasts after Cr(VI) exposure and its dependence on telomerase

    International Nuclear Information System (INIS)

    Cogan, Nicola; Baird, Duncan M.; Phillips, Ryan; Crompton, Lucy A.; Caldwell, Maeve A.; Rubio, Miguel A.; Newson, Roger; Lyng, Fiona; Case, C. Patrick

    2010-01-01

    The bystander effect is a feature of low dose radiation exposure and is characterized by a signaling process from irradiated cells to non irradiated cells, which causes DNA and chromosome damage in these 'nearest neighbour' cells. Here we show that a low and short dose of Cr(VI) can induce stem cells, cancer cells and fibroblasts to chronically secrete bystander signals, which cause DNA damage in neighboring cells. The Cr(VI) induced bystander signaling depended on the telomerase status of either cell. Telomerase negative fibroblasts were able to receive DNA damaging signals from telomerase positive or negative fibroblasts or telomerase positive cancer cells. However telomerase positive fibroblasts were resistant to signals from Cr(VI) exposed telomerase positive fibroblasts or cancer cells. Human embryonic stem cells, with positive Oct4 staining as a marker of pluripotency, showed no significant increase of DNA damage from adjacent Cr and mitomycin C exposed fibroblasts whilst those cells that were negatively stained did. This selectivity of DNA damaging bystander signaling could be an important consideration in developing therapies against cancer and in the safety and effectiveness of tissue engineering and transplantation using stem cells.

  2. Cellular Responses to Cisplatin-Induced DNA Damage

    Directory of Open Access Journals (Sweden)

    Alakananda Basu

    2010-01-01

    Full Text Available Cisplatin is one of the most effective anticancer agents widely used in the treatment of solid tumors. It is generally considered as a cytotoxic drug which kills cancer cells by damaging DNA and inhibiting DNA synthesis. How cells respond to cisplatin-induced DNA damage plays a critical role in deciding cisplatin sensitivity. Cisplatin-induced DNA damage activates various signaling pathways to prevent or promote cell death. This paper summarizes our current understandings regarding the mechanisms by which cisplatin induces cell death and the bases of cisplatin resistance. We have discussed various steps, including the entry of cisplatin inside cells, DNA repair, drug detoxification, DNA damage response, and regulation of cisplatin-induced apoptosis by protein kinases. An understanding of how various signaling pathways regulate cisplatin-induced cell death should aid in the development of more effective therapeutic strategies for the treatment of cancer.

  3. Development of a Fish Cell Biosensor System for Genotoxicity Detection Based on DNA Damage-Induced Trans-Activation of p21 Gene Expression

    Directory of Open Access Journals (Sweden)

    Huarong Guo

    2012-09-01

    Full Text Available p21CIP1/WAF1 is a p53-target gene in response to cellular DNA damage. Here we report the development of a fish cell biosensor system for high throughput genotoxicity detection of new drugs, by stably integrating two reporter plasmids of pGL3-p21-luc (human p21 promoter linked to firefly luciferase and pRL-CMV-luc (CMV promoter linked to Renilla luciferase into marine flatfish flounder gill (FG cells, referred to as p21FGLuc. Initial validation of this genotoxicity biosensor system showed that p21FGLuc cells had a wild-type p53 signaling pathway and responded positively to the challenge of both directly acting genotoxic agents (bleomycin and mitomycin C and indirectly acting genotoxic agents (cyclophosphamide with metabolic activation, but negatively to cyclophosphamide without metabolic activation and the non-genotoxic agents ethanol and D-mannitol, thus confirming a high specificity and sensitivity, fast and stable response to genotoxic agents for this easily maintained fish cell biosensor system. This system was especially useful in the genotoxicity detection of Di(2-ethylhexyl phthalate (DEHP, a rodent carcinogen, but negatively reported in most non-mammalian in vitro mutation assays, by providing a strong indication of genotoxicity for DEHP. A limitation for this biosensor system was that it might give false positive results in response to sodium butyrate and any other agents, which can trans-activate the p21 gene in a p53-independent manner.

  4. DNA damage and repair in plants

    International Nuclear Information System (INIS)

    Britt, A.B.

    1996-01-01

    The biological impact of any DNA damaging agent is a combined function of the chemical nature of the induced lesions and the efficiency and accuracy of their repair. Although much has been learned frommicrobes and mammals about both the repair of DNA damage and the biological effects of the persistence of these lesions, much remains to be learned about the mechanism and tissue-specificity of repair in plants. This review focuses on recent work on the induction and repair of DNA damage in higher plants, with special emphasis on UV-induced DNA damage products. (author)

  5. Accumulation of DNA damage-induced chromatin alterations in tissue-specific stem cells: the driving force of aging?

    Directory of Open Access Journals (Sweden)

    Nadine Schuler

    Full Text Available Accumulation of DNA damage leading to stem cell exhaustion has been proposed to be a principal mechanism of aging. Using 53BP1-foci as a marker for DNA double-strand breaks (DSBs, hair follicle stem cells (HFSCs in mouse epidermis were analyzed for age-related DNA damage response (DDR. We observed increasing amounts of 53BP1-foci during the natural aging process independent of telomere shortening and after protracted low-dose radiation, suggesting substantial accumulation of DSBs in HFSCs. Electron microscopy combined with immunogold-labeling showed multiple small 53BP1 clusters diffusely distributed throughout the highly compacted heterochromatin of aged HFSCs, but single large 53BP1 clusters in irradiated HFSCs. These remaining 53BP1 clusters did not colocalize with core components of non-homologous end-joining, but with heterochromatic histone modifications. Based on these results we hypothesize that these lesions were not persistently unrepaired DSBs, but may reflect chromatin rearrangements caused by the repair or misrepair of DSBs. Flow cytometry showed increased activation of repair proteins and damage-induced chromatin modifications, triggering apoptosis and cellular senescence in irradiated, but not in aged HFSCs. These results suggest that accumulation of DNA damage-induced chromatin alterations, whose structural dimensions reflect the complexity of the initial genotoxic insult, may lead to different DDR events, ultimately determining the biological outcome of HFSCs. Collectively, our findings support the hypothesis that aging might be largely the remit of structural changes to chromatin potentially leading to epigenetically induced transcriptional deregulation.

  6. DNA damage in haemocytes and midgut gland cells of Steatoda grossa (Theridiidae) spiders exposed to food contaminated with cadmium.

    Science.gov (United States)

    Stalmach, Monika; Wilczek, Grażyna; Wilczek, Piotr; Skowronek, Magdalena; Mędrzak, Monika

    2015-03-01

    The aim of this study was to assess the genotoxic effects of Cd on haemocytes and midgut gland cells of web-building spiders, Steatoda grossa (Theridiidae), exposed to the metal under laboratory conditions. Analyzes were conducted on adult females and males, fed for four weeks with cadmium-contaminated Drosophila hydei flies, grown on a medium suplemented with 0.25 mM CdCl2. The comet assay, providing a quantitative measure of DNA strand breaks, was used to evaluate the DNA damage caused by the metal. Cadmium content was measured in whole spider bodies by the AAS method. Metal body burden was significantly lower in females (0.25 µgg(-1) dry weight) than in males (3.03 µgg(-1) dry weight), suggesting that females may have more effective mechanisms controlling the uptake of metal, via the digestive tract, or its elimination from the body. Irrespectively of sex, spiders fed prey contaminated with cadmium showed significantly higher values of comet parameters: tail DNA (TDNA), tail length (TL) and olive tail moment (OTM), in comparison with the control. In midgut gland cells, the level of DNA damage was higher for males than females, while in haemocytes the genotoxic effect of cadmium was greater in females. The obtained results indicate that in spiders cadmium displays strong genotoxic effects and may cause DNA damage even at low concentrations, however the severity of damage seems to be sex- and internal organ-dependent. The comet assay can be considered a sensitive tool for measuring the deleterious effect of cadmium on DNA integrity in spiders. Copyright © 2014 Elsevier Inc. All rights reserved.

  7. MTH1 deficiency selectively increases non-cytotoxic oxidative DNA damage in lung cancer cells: more bad news than good?

    Science.gov (United States)

    Abbas, Hussein H K; Alhamoudi, Kheloud M H; Evans, Mark D; Jones, George D D; Foster, Steven S

    2018-04-16

    Targeted therapies are based on exploiting cancer-cell-specific genetic features or phenotypic traits to selectively kill cancer cells while leaving normal cells unaffected. Oxidative stress is a cancer hallmark phenotype. Given that free nucleotide pools are particularly vulnerable to oxidation, the nucleotide pool sanitising enzyme, MTH1, is potentially conditionally essential in cancer cells. However, findings from previous MTH1 studies have been contradictory, meaning the relevance of MTH1 in cancer is still to be determined. Here we ascertained the role of MTH1 specifically in lung cancer cell maintenance, and the potential of MTH1 inhibition as a targeted therapy strategy to improve lung cancer treatments. Using siRNA-mediated knockdown or small-molecule inhibition, we tested the genotoxic and cytotoxic effects of MTH1 deficiency on H23 (p53-mutated), H522 (p53-mutated) and A549 (wildtype p53) non-small cell lung cancer cell lines relative to normal MRC-5 lung fibroblasts. We also assessed if MTH1 inhibition augments current therapies. MTH1 knockdown increased levels of oxidatively damaged DNA and DNA damage signaling alterations in all lung cancer cell lines but not normal fibroblasts, despite no detectable differences in reactive oxygen species levels between any cell lines. Furthermore, MTH1 knockdown reduced H23 cell proliferation. However, unexpectedly, it did not induce apoptosis in any cell line or enhance the effects of gemcitabine, cisplatin or radiation in combination treatments. Contrastingly, TH287 and TH588 MTH1 inhibitors induced apoptosis in H23 and H522 cells, but only increased oxidative DNA damage levels in H23, indicating that they kill cells independently of DNA oxidation and seemingly via MTH1-distinct mechanisms. MTH1 has a NSCLC-specific p53-independent role for suppressing DNA oxidation and genomic instability, though surprisingly the basis of this may not be reactive-oxygen-species-associated oxidative stress. Despite this, overall

  8. Emamectin benzoate induces ROS-mediated DNA damage and apoptosis in Trichoplusia Tn5B1-4 cells.

    Science.gov (United States)

    Luan, Shaorong; Yun, Xinming; Rao, Wenbing; Xiao, Ciying; Xu, Zhikang; Lang, Jialin; Huang, Qingchun

    2017-08-01

    Emamectin benzoate (EMB), a novel macrocyclic lactone insecticide, possesses high efficacy and beneficial selective toxicity in agriculture, but so far the EMB-induced cytotoxic action in arthropod insect remains unclear. The present studies were carried out to characterize the property of EMB on the induction of reactive oxygen species (ROS)-mediated DNA damage and apoptosis in Trichoplusia Tn5B1-4 cell model. Following the exposure to EMB at 2.5, 5, 10 or 15 μM, the cells changed to be round, suspended and aggregated, and the decline of cell proliferating ability and cell viability was positively related with the exposure time. Median inhibitory concentration (IC 50 ) of EMB on cell viability was 3.72 μM during 72 h exposure. Apoptosis was induced in 29.8% (24 h) and 39.5% (48 h) of the cells by EMB at 15 μM, showing chromatin condensation in nuclei. The content of ROS in the cells increased rapidly as the concentration of EMB increased, and the pre-incubation of the cells with vitamin E significantly reduced the ROS accumulation. In the treatment of 15 μM EMB, the migrated cell nucleus with DNA strand breaks appeared a teardrop, pear-shaped, or large fan-like tail, and 63.1% of γH2AX-positive cells contained more than four foci, accompanying with high expression level of caspase-3 in time-dependent manner, which consequently led to cell apoptotic death. These evidences in ROS-mediated DNA damage and cell apoptosis induced by EMB may be helpful for deep understanding the cytotoxic action of EMB based on cell model. Copyright © 2017 Elsevier B.V. All rights reserved.

  9. TopBP1 is required at mitosis to reduce transmission of DNA damage to G1 daughter cells

    Science.gov (United States)

    Pedersen, Rune Troelsgaard; Kruse, Thomas; Nilsson, Jakob

    2015-01-01

    Genome integrity is critically dependent on timely DNA replication and accurate chromosome segregation. Replication stress delays replication into G2/M, which in turn impairs proper chromosome segregation and inflicts DNA damage on the daughter cells. Here we show that TopBP1 forms foci upon mitotic entry. In early mitosis, TopBP1 marks sites of and promotes unscheduled DNA synthesis. Moreover, TopBP1 is required for focus formation of the structure-selective nuclease and scaffold protein SLX4 in mitosis. Persistent TopBP1 foci transition into 53BP1 nuclear bodies (NBs) in G1 and precise temporal depletion of TopBP1 just before mitotic entry induced formation of 53BP1 NBs in the next cell cycle, showing that TopBP1 acts to reduce transmission of DNA damage to G1 daughter cells. Based on these results, we propose that TopBP1 maintains genome integrity in mitosis by controlling chromatin recruitment of SLX4 and by facilitating unscheduled DNA synthesis. PMID:26283799

  10. Zinc protects HepG2 cells against the oxidative damage and DNA damage induced by ochratoxin A

    International Nuclear Information System (INIS)

    Zheng, Juanjuan; Zhang, Yu; Xu, Wentao; Luo, YunBo; Hao, Junran; Shen, Xiao Li; Yang, Xuan; Li, Xiaohong; Huang, Kunlun

    2013-01-01

    Oxidative stress and DNA damage are the most studied mechanisms by which ochratoxin A (OTA) induces its toxic effects, which include nephrotoxicity, hepatotoxicity, immunotoxicity and genotoxicity. Zinc, which is an essential trace element, is considered a potential antioxidant. The aim of this paper was to investigate whether zinc supplement could inhibit OTA-induced oxidative damage and DNA damage in HepG2 cells and the mechanism of inhibition. The results indicated that that exposure of OTA decreased the intracellular zinc concentration; zinc supplement significantly reduced the OTA-induced production of reactive oxygen species (ROS) and decrease in superoxide dismutase (SOD) activity but did not affect the OTA-induced decrease in the mitochondrial membrane potential (Δψ m ). Meanwhile, the addition of the zinc chelator N,N,N′,N′-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) strongly aggravated the OTA-induced oxidative damage. This study also demonstrated that zinc helped to maintain the integrity of DNA through the reduction of OTA-induced DNA strand breaks, 8-hydroxy-2′-deoxyguanosine (8-OHdG) formation and DNA hypomethylation. OTA increased the mRNA expression of metallothionein1-A (MT1A), metallothionein2-A (MT2A) and Cu/Zn superoxide dismutase (SOD1). Zinc supplement further enhanced the mRNA expression of MT1A and MT2A, but it had no effect on the mRNA expression of SOD1 and catalase (CAT). Zinc was for the first time proven to reduce the cytotoxicity of OTA through inhibiting the oxidative damage and DNA damage, and regulating the expression of zinc-associated genes. Thus, the addition of zinc can potentially be used to reduce the OTA toxicity of contaminated feeds. - Highlights: ► OTA decreased the intracellular zinc concentration. ► OTA induced the formation of 8-OHdG in HepG2 cells. ► It was testified for the first time that OTA induced DNA hypomethylation. ► Zinc protects against the oxidative damage and DNA damage induced by OTA in

  11. Zinc protects HepG2 cells against the oxidative damage and DNA damage induced by ochratoxin A

    Energy Technology Data Exchange (ETDEWEB)

    Zheng, Juanjuan; Zhang, Yu [Laboratory of Food Safety and Molecular Biology, College of Food Science and Nutritional Engineering, China Agricultural University, Beijing 100083 (China); Xu, Wentao, E-mail: xuwentaoboy@sina.com [Laboratory of Food Safety and Molecular Biology, College of Food Science and Nutritional Engineering, China Agricultural University, Beijing 100083 (China); The Supervision, Inspection and Testing Center of Genetically Modified Organisms, Ministry of Agriculture, Beijing 100083 (China); Luo, YunBo [Laboratory of Food Safety and Molecular Biology, College of Food Science and Nutritional Engineering, China Agricultural University, Beijing 100083 (China); The Supervision, Inspection and Testing Center of Genetically Modified Organisms, Ministry of Agriculture, Beijing 100083 (China); Hao, Junran [Laboratory of Food Safety and Molecular Biology, College of Food Science and Nutritional Engineering, China Agricultural University, Beijing 100083 (China); Shen, Xiao Li [The Supervision, Inspection and Testing Center of Genetically Modified Organisms, Ministry of Agriculture, Beijing 100083 (China); Yang, Xuan [Laboratory of Food Safety and Molecular Biology, College of Food Science and Nutritional Engineering, China Agricultural University, Beijing 100083 (China); Li, Xiaohong [The Supervision, Inspection and Testing Center of Genetically Modified Organisms, Ministry of Agriculture, Beijing 100083 (China); Huang, Kunlun, E-mail: hkl009@163.com [Laboratory of Food Safety and Molecular Biology, College of Food Science and Nutritional Engineering, China Agricultural University, Beijing 100083 (China); The Supervision, Inspection and Testing Center of Genetically Modified Organisms, Ministry of Agriculture, Beijing 100083 (China)

    2013-04-15

    Oxidative stress and DNA damage are the most studied mechanisms by which ochratoxin A (OTA) induces its toxic effects, which include nephrotoxicity, hepatotoxicity, immunotoxicity and genotoxicity. Zinc, which is an essential trace element, is considered a potential antioxidant. The aim of this paper was to investigate whether zinc supplement could inhibit OTA-induced oxidative damage and DNA damage in HepG2 cells and the mechanism of inhibition. The results indicated that that exposure of OTA decreased the intracellular zinc concentration; zinc supplement significantly reduced the OTA-induced production of reactive oxygen species (ROS) and decrease in superoxide dismutase (SOD) activity but did not affect the OTA-induced decrease in the mitochondrial membrane potential (Δψ{sub m}). Meanwhile, the addition of the zinc chelator N,N,N′,N′-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) strongly aggravated the OTA-induced oxidative damage. This study also demonstrated that zinc helped to maintain the integrity of DNA through the reduction of OTA-induced DNA strand breaks, 8-hydroxy-2′-deoxyguanosine (8-OHdG) formation and DNA hypomethylation. OTA increased the mRNA expression of metallothionein1-A (MT1A), metallothionein2-A (MT2A) and Cu/Zn superoxide dismutase (SOD1). Zinc supplement further enhanced the mRNA expression of MT1A and MT2A, but it had no effect on the mRNA expression of SOD1 and catalase (CAT). Zinc was for the first time proven to reduce the cytotoxicity of OTA through inhibiting the oxidative damage and DNA damage, and regulating the expression of zinc-associated genes. Thus, the addition of zinc can potentially be used to reduce the OTA toxicity of contaminated feeds. - Highlights: ► OTA decreased the intracellular zinc concentration. ► OTA induced the formation of 8-OHdG in HepG2 cells. ► It was testified for the first time that OTA induced DNA hypomethylation. ► Zinc protects against the oxidative damage and DNA damage induced by

  12. DNA damage responsive miR-33b-3p promoted lung cancer cells survival and cisplatin resistance by targeting p21WAF1/CIP1.

    Science.gov (United States)

    Xu, Shun; Huang, Haijiao; Chen, Yu-Ning; Deng, Yun-Ting; Zhang, Bing; Xiong, Xing-Dong; Yuan, Yuan; Zhu, Yanmei; Huang, Haiyong; Xie, Luoyijun; Liu, Xinguang

    2016-11-01

    Cisplatin is the most potent and widespread used chemotherapy drug for lung cancer treatment. However, the development of resistance to cisplatin is a major obstacle in clinical therapy. The principal mechanism of cisplatin is the induction of DNA damage, thus the capability of DNA damage response (DDR) is a key factor that influences the cisplatin sensitivity of cancer cells. Recent advances have demonstrated that miRNAs (microRNAs) exerted critical roles in DNA damage response; nonetheless, the association between DNA damage responsive miRNAs and cisplatin resistance and its underlying molecular mechanism still require further investigation. The present study has attempted to identify differentially expressed miRNAs in cisplatin induced DNA damage response in lung cancer cells, and probe into the effects of the misexpressed miRNAs on cisplatin sensitivity. Deep sequencing showed that miR-33b-3p was dramatically down-regulated in cisplatin-induced DNA damage response in A549 cells; and ectopic expression of miR-33b-3p endowed the lung cancer cells with enhanced survival and decreased γH2A.X expression level under cisplatin treatment. Consistently, silencing of miR-33b-3p in the cisplatin-resistant A549/DDP cells evidently sensitized the cells to cisplatin. Furthermore, we identified CDKN1A (p21) as a functional target of miR-33b-3p, a critical regulator of G1/S checkpoint, which potentially mediated the protection effects of miR-33b-3p against cisplatin. In aggregate, our results suggested that miR-33b-3p modulated the cisplatin sensitivity of cancer cells might probably through impairing the DNA damage response. And the knowledge of the drug resistance conferred by miR-33b-3p has great clinical implications for improving the efficacy of chemotherapies for treating lung cancers.

  13. Clustered DNA damages induced in isolated DNA and in human cells by low doses of ionizing radiation

    Science.gov (United States)

    Sutherland, B. M.; Bennett, P. V.; Sidorkina, O.; Laval, J.; Lowenstein, D. I. (Principal Investigator)

    2000-01-01

    Clustered DNA damages-two or more closely spaced damages (strand breaks, abasic sites, or oxidized bases) on opposing strands-are suspects as critical lesions producing lethal and mutagenic effects of ionizing radiation. However, as a result of the lack of methods for measuring damage clusters induced by ionizing radiation in genomic DNA, neither the frequencies of their production by physiological doses of radiation, nor their repairability, nor their biological effects are known. On the basis of methods that we developed for quantitating damages in large DNAs, we have devised and validated a way of measuring ionizing radiation-induced clustered lesions in genomic DNA, including DNA from human cells. DNA is treated with an endonuclease that induces a single-strand cleavage at an oxidized base or abasic site. If there are two closely spaced damages on opposing strands, such cleavage will reduce the size of the DNA on a nondenaturing gel. We show that ionizing radiation does induce clustered DNA damages containing abasic sites, oxidized purines, or oxidized pyrimidines. Further, the frequency of each of these cluster classes is comparable to that of frank double-strand breaks; among all complex damages induced by ionizing radiation, double-strand breaks are only about 20%, with other clustered damage constituting some 80%. We also show that even low doses (0.1-1 Gy) of high linear energy transfer ionizing radiation induce clustered damages in human cells.

  14. DNA damage and γH2AX expression in EJ cells induced by 60Co gamma-rays

    International Nuclear Information System (INIS)

    Pan Yan; Tian Mei; Liu Jianxiang; Ruan Jianlei; Su Xu

    2009-01-01

    Objective: To investigate 60 Co γ-rays induced DNA damage of human bladder cancer cell line EJ cells and the relationship between different doses of 60 Co γ-rays, γH2AX foci number and γH2AX expression level. Methods: EJ cells were exposed to different doses of 60 Co γ-rays and the oliver tail moment (TM) of EJ cells were analyzed with single cell gel electrophoresis (SCGE) . Immunofluorescent microscopy was used to analysis γH2AX foci formation in EJ cells after exposure to different doses of γ-ray irradiation and time-course after exposure to 2 Gy γ-ray irradiation. FACSAria was used to detect the changes of γH2AX protein expression in EJ cells. Results: The TMs of EJ cells were increased with the irradiation dose. The TM of 0 Gy group and 4 Gy group was 0.24 and 5.26, respectively. Immunofluorescent analysis demonstrated that γH2AX foci could be induced by γ-ray irradiation in dose-dependent and time-dependent manners. The foci number and size in nuclei of EJ cells was significantly increased after exposed to different doses of γ-ray irradiation and the foci remained detectable at 24 h after exposed to irradiation. The dose range in which foci could be clearly detected was from 0.1 to 4 Gy. FACSDiva showed that γH2AX protein expression was increased after exposure to different doses of γ-ray irradiation. γH2AX protein expression level of 0.1 Gy group and 4 Gy group was 7.4% and 29.2% , respectively. Conclusions: γH2AX foci could be the most sensitive indicator for DNA damage and repair in mammalian cells, and it might be a new biomarker for radiological emergency. (authors)

  15. Formation and repair of physically and chemically induced DNA damage in human cells. Final report, September 1, 1976-November 30, 1978

    International Nuclear Information System (INIS)

    Cerutti, P.A.

    1979-01-01

    The major topic was the study of the formation and repair of DNA damage by energy related physical and chemical agents in cultured human cells. Two pathways of damage production were distinguished: (1) indirect action, i.e., attack of DNA by active oxygen species which are formed by the reaction of the primary agent with a non-DNA target; and (2) direct action, i.e., reaction of the primary agent or a chemical derivative of the primary agent with DNA usually resulting in the formation of a covalent adduct. Near-ultraviolet light and ionizing radiation were studied as agents which operate at least in part via indirect action and benzo(a)pyrene as chemical carcinogen operating mostly by direct action. The formation of monomeric thymine damage of the 5,6-dihydroxy-dihydrothymine type by γ-rays and ultraviolet light was investigated. Indirect action of near-ultraviolet light is also responsible for the induction of DNA single strand breaks. Their formation and repair following exposure to 313 nm light was studied in skin fibroblasts from patients with the hereditary disease Xeroderma pigmentosum (XP). Excision repair of γ-ray induced 5,6-dihydroxy-dihydrothymine type lesions was studied in fibroblasts from Ataxia telangiectasia (AT) patients. The formation and repair of covalent purine adducts was studied in actively metabolizing rodent and human cells following treatment with the procarcinogen benzo(a)pyrene and with the ultimate metabolite benzo(a)pyrene-diol-epoxide I

  16. Enterococcus faecalis infection causes inflammation, intracellular oxphos-independent ROS production, and DNA damage in human gastric cancer cells.

    Directory of Open Access Journals (Sweden)

    Jesper A B Strickertsson

    Full Text Available BACKGROUND: Achlorhydria caused by e.g. atrophic gastritis allows for bacterial overgrowth, which induces chronic inflammation and damage to the mucosal cells of infected individuals driving gastric malignancies and cancer. Enterococcus faecalis (E. faecalis can colonize achlohydric stomachs and we therefore wanted to study the impact of E. faecalis infection on inflammatory response, reactive oxygen species (ROS formation, mitochondrial respiration, and mitochondrial genetic stability in gastric mucosal cells. METHODS: To separate the changes induced by bacteria from those of the inflammatory cells we established an in vitro E. faecalis infection model system using the gastric carcinoma cell line MKN74. Total ROS and superoxide was measured by fluorescence microscopy. Cellular oxygen consumption was characterized non-invasively using XF24 microplate based respirometry. Gene expression was examined by microarray, and response pathways were identified by Gene Set Analysis (GSA. Selected gene transcripts were verified by quantitative real-time polymerase chain reaction (qRT-PCR. Mitochondrial mutations were determined by sequencing. RESULTS: Infection of MKN74 cells with E. faecalis induced intracellular ROS production through a pathway independent of oxidative phosphorylation (oxphos. Furthermore, E. faecalis infection induced mitochondrial DNA instability. Following infection, genes coding for inflammatory response proteins were transcriptionally up-regulated while DNA damage repair and cell cycle control genes were down-regulated. Cell growth slowed down when infected with viable E. faecalis and responded in a dose dependent manner to E. faecalis lysate. CONCLUSIONS: Infection by E. faecalis induced an oxphos-independent intracellular ROS response and damaged the mitochondrial genome in gastric cell culture. Finally the bacteria induced an NF-κB inflammatory response as well as impaired DNA damage response and cell cycle control gene

  17. Enterococcus faecalis Infection Causes Inflammation, Intracellular Oxphos-Independent ROS Production, and DNA Damage in Human Gastric Cancer Cells

    DEFF Research Database (Denmark)

    Strickertsson, Jesper A. B; Desler, Claus; Martin-Bertelsen, Tomas

    2013-01-01

    therefore wanted to study the impact of E. faecalis infection on inflammatory response, reactive oxygen species (ROS) formation, mitochondrial respiration, and mitochondrial genetic stability in gastric mucosal cells. Methods To separate the changes induced by bacteria from those of the inflammatory cells...... intracellular ROS production through a pathway independent of oxidative phosphorylation (oxphos). Furthermore, E. faecalis infection induced mitochondrial DNA instability. Following infection, genes coding for inflammatory response proteins were transcriptionally up-regulated while DNA damage repair and cell...... cycle control genes were down-regulated. Cell growth slowed down when infected with viable E. faecalis and responded in a dose dependent manner to E. faecalis lysate. Conclusions Infection by E. faecalis induced an oxphos-independent intracellular ROS response and damaged the mitochondrial genome...

  18. Damage-recognition proteins as a potential indicator of DNA-damage-mediated sensitivity or resistance of human cells to ultraviolet radiation

    International Nuclear Information System (INIS)

    Chao, C.C.-K.

    1992-01-01

    The authors compared damage-recognition proteins in cells expressing different sensitivities to DNA damage. An increase in damage-recognition proteins and an enhancement of plasmid re-activation were detected in HeLa cells resistant to cisplatin and u.v. However, repair-defective cells derived from xeroderma-pigmentosum (a rare skin disease) patients did not express less cisplatin damage-recognition proteins than repair-competent cells, suggesting that damage-recognition-protein expression may not be related to DNA repair. By contrast, cells resistant to DNA damage consistently expressed high levels of u.v.-modified-DNA damage-recognition proteins. The results support the notion that u.v. damage-recognition proteins are different from those that bind to cisplatin. Findings also suggest that the damage-recognition proteins identified could be used as potential indicators of the sensitivity or resistance of cells to u.v. (author)

  19. Pim-3 contributes to radioresistance through regulation of the cell cycle and DNA damage repair in pancreatic cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Xiang-Yuan; Wang, Zhen [Cancer Research Institute, Fudan University Shanghai Cancer Center, Department of Oncology, Shanghai Medical College, Fudan University, Shanghai (China); Li, Bei [Department of Nuclear Medicine, Fudan University Shanghai Cancer Center, Department of Oncology, Shanghai Medical College, Fudan University, Shanghai (China); Zhang, Ying-Jian, E-mail: yjzhang111@aliyun.com [Department of Nuclear Medicine, Fudan University Shanghai Cancer Center, Department of Oncology, Shanghai Medical College, Fudan University, Shanghai (China); Li, Ying-Yi, E-mail: liyingyi@fudan.edu.cn [Cancer Research Institute, Fudan University Shanghai Cancer Center, Department of Oncology, Shanghai Medical College, Fudan University, Shanghai (China)

    2016-04-22

    Resistance of cancer cells to chemoradiotherapy is a major clinical problem in pancreatic cancer treatment. Therefore, understanding the molecular basis of cellular resistance and identifying novel targets are essential for improving treatment efficacy for pancreatic cancer patients. Previous studies have demonstrated a significant role for Pim-3 in pancreatic cancer survival against gemcitabine-induced genotoxic stress. Here, we observed that radiation treatment enhanced Pim-3 expression in human pancreatic cancer cells in vitro. Stable overexpression of Pim-3 in pancreatic cancer cells significantly protected cells against radiation treatment by attenuating G2/M phase cell cycle arrest and DNA damage response. Silencing of Pim-3 expression significantly elevated the phosphorylation of histone variant H2AX, a marker of DNA double strand breaks, and decreased the activation of ataxia-telangiectasia-mutated (ATM) kinase, along with its downstream targets, eventually enhancing the radiosensitivity of human pancreatic cancer cells in vitro and in vivo. Hence, we demonstrated a novel function for Pim-3 in human pancreatic cancer cell survival against radiation. Targeting Pim-3 may be a promising way to improve treatment efficacy in combination with radiotherapy in human pancreatic cancer. - Highlights: • This is first study to demonstrate that Pim-3 is endogenously induced by ionizing radiation in pancreatic cancer cells, and Pim-3 overexpression enhanced radioresistance of pancreatic cancer cells both in vitro and in vivo. • This is first study to provide evidence that radioresistance induced by Pim-3 is mainly attributed to Pim-3 induces activation of ATM, which subsequently activates checkpoint 1, leading to amplification of DNA repair through cell cycle arrest and DNA repair pathways. • This is first study to indicate that targeting Pim-3 may be a promising strategy to provide better treatment efficacy in combination with radiotherapy in human pancreatic

  20. Pim-3 contributes to radioresistance through regulation of the cell cycle and DNA damage repair in pancreatic cancer cells

    International Nuclear Information System (INIS)

    Chen, Xiang-Yuan; Wang, Zhen; Li, Bei; Zhang, Ying-Jian; Li, Ying-Yi

    2016-01-01

    Resistance of cancer cells to chemoradiotherapy is a major clinical problem in pancreatic cancer treatment. Therefore, understanding the molecular basis of cellular resistance and identifying novel targets are essential for improving treatment efficacy for pancreatic cancer patients. Previous studies have demonstrated a significant role for Pim-3 in pancreatic cancer survival against gemcitabine-induced genotoxic stress. Here, we observed that radiation treatment enhanced Pim-3 expression in human pancreatic cancer cells in vitro. Stable overexpression of Pim-3 in pancreatic cancer cells significantly protected cells against radiation treatment by attenuating G2/M phase cell cycle arrest and DNA damage response. Silencing of Pim-3 expression significantly elevated the phosphorylation of histone variant H2AX, a marker of DNA double strand breaks, and decreased the activation of ataxia-telangiectasia-mutated (ATM) kinase, along with its downstream targets, eventually enhancing the radiosensitivity of human pancreatic cancer cells in vitro and in vivo. Hence, we demonstrated a novel function for Pim-3 in human pancreatic cancer cell survival against radiation. Targeting Pim-3 may be a promising way to improve treatment efficacy in combination with radiotherapy in human pancreatic cancer. - Highlights: • This is first study to demonstrate that Pim-3 is endogenously induced by ionizing radiation in pancreatic cancer cells, and Pim-3 overexpression enhanced radioresistance of pancreatic cancer cells both in vitro and in vivo. • This is first study to provide evidence that radioresistance induced by Pim-3 is mainly attributed to Pim-3 induces activation of ATM, which subsequently activates checkpoint 1, leading to amplification of DNA repair through cell cycle arrest and DNA repair pathways. • This is first study to indicate that targeting Pim-3 may be a promising strategy to provide better treatment efficacy in combination with radiotherapy in human pancreatic

  1. Simple technique for quantitation of low levels of DNA damage in individual cells

    International Nuclear Information System (INIS)

    Singh, N.P.; McCoy, M.T.; Tice, R.R.; Schneider, E.L.

    1988-01-01

    Human lymphocytes were either exposed to X-irradiation (25 to 200 rads) or treated with H 2 O 2 (9.1 to 291 microM) at 4 degrees C and the extent of DNA migration was measured using a single-cell microgel electrophoresis technique under alkaline conditions. Both agents induced a significant increase in DNA migration, beginning at the lowest dose evaluated. Migration patterns were relatively homogeneous among cells exposed to X-rays but heterogeneous among cells treated with H 2 O 2 . An analysis of repair kinetics following exposure to 200 rads X-rays was conducted with lymphocytes obtained from three individuals. The bulk of the DNA repair occurred within the first 15 min, while all of the repair was essentially complete by 120 min after exposure. However, some cells demonstrated no repair during this incubation period while other cells demonstrated DNA migration patterns indicative of more damage than that induced by the initial irradiation with X-rays. This technique appears to be sensitive and useful for detecting damage and repair in single cells

  2. BEMER Electromagnetic Field Therapy Reduces Cancer Cell Radioresistance by Enhanced ROS Formation and Induced DNA Damage.

    Directory of Open Access Journals (Sweden)

    Katja Storch

    Full Text Available Each year more than 450,000 Germans are expected to be diagnosed with cancer subsequently receiving standard multimodal therapies including surgery, chemotherapy and radiotherapy. On top, molecular-targeted agents are increasingly administered. Owing to intrinsic and acquired resistance to these therapeutic approaches, both the better molecular understanding of tumor biology and the consideration of alternative and complementary therapeutic support are warranted and open up broader and novel possibilities for therapy personalization. Particularly the latter is underpinned by the increasing utilization of non-invasive complementary and alternative medicine by the population. One investigated approach is the application of low-dose electromagnetic fields (EMF to modulate cellular processes. A particular system is the BEMER therapy as a Physical Vascular Therapy for which a normalization of the microcirculation has been demonstrated by a low-frequency, pulsed EMF pattern. Open remains whether this EMF pattern impacts on cancer cell survival upon treatment with radiotherapy, chemotherapy and the molecular-targeted agent Cetuximab inhibiting the epidermal growth factor receptor. Using more physiological, three-dimensional, matrix-based cell culture models and cancer cell lines originating from lung, head and neck, colorectal and pancreas, we show significant changes in distinct intermediates of the glycolysis and tricarboxylic acid cycle pathways and enhanced cancer cell radiosensitization associated with increased DNA double strand break numbers and higher levels of reactive oxygen species upon BEMER treatment relative to controls. Intriguingly, exposure of cells to the BEMER EMF pattern failed to result in sensitization to chemotherapy and Cetuximab. Further studies are necessary to better understand the mechanisms underlying the cellular alterations induced by the BEMER EMF pattern and to clarify the application areas for human disease.

  3. Identification of BC005512 as a DNA damage responsive murine endogenous retrovirus of GLN family involved in cell growth regulation.

    Directory of Open Access Journals (Sweden)

    Yuanfeng Wu

    Full Text Available Genotoxicity assessment is of great significance in drug safety evaluation, and microarray is a useful tool widely used to identify genotoxic stress responsive genes. In the present work, by using oligonucleotide microarray in an in vivo model, we identified an unknown gene BC005512 (abbreviated as BC, official full name: cDNA sequence BC005512, whose expression in mouse liver was specifically induced by seven well-known genotoxins (GTXs, but not by non-genotoxins (NGTXs. Bioinformatics revealed that BC was a member of the GLN family of murine endogenous retrovirus (ERV. However, the relationship to genotoxicity and the cellular function of GLN are largely unknown. Using NIH/3T3 cells as an in vitro model system and quantitative real-time PCR, BC expression was specifically induced by another seven GTXs, covering diverse genotoxicity mechanisms. Additionally, dose-response and linear regression analysis showed that expression level of BC in NIH/3T3 cells strongly correlated with DNA damage, measured using the alkaline comet assay,. While in p53 deficient L5178Y cells, GTXs could not induce BC expression. Further functional studies using RNA interference revealed that down-regulation of BC expression induced G1/S phase arrest, inhibited cell proliferation and thus suppressed cell growth in NIH/3T3 cells. Together, our results provide the first evidence that BC005512, a member from GLN family of murine ERV, was responsive to DNA damage and involved in cell growth regulation. These findings could be of great value in genotoxicity predictions and contribute to a deeper understanding of GLN biological functions.

  4. Dihydrocoumarin, an HDAC Inhibitor, Increases DNA Damage Sensitivity by Inhibiting Rad52

    Directory of Open Access Journals (Sweden)

    Chin-Chuan Chen

    2017-12-01

    Full Text Available Effective DNA repair enables cancer cells to survive DNA damage induced by chemotherapeutic or radiotherapeutic treatments. Therefore, inhibiting DNA repair pathways is a promising therapeutic strategy for increasing the efficacy of such treatments. In this study, we found that dihydrocoumarin (DHC, a flavoring agent, causes deficiencies in double-stand break (DSB repair and prolonged DNA damage checkpoint recovery in yeast. Following DNA damage, Rad52 recombinase was revealed to be inhibited by DHC, which results in deficiencies in DSB repair and prolonged DNA damage checkpoint recovery. The deletion of RPD3, a class I histone deacetylase (HDAC, was found to mimic DHC-induced suppression of Rad52 expression, suggesting that the HDAC inhibitor activity of DHC is critical to DSB repair and DNA damage sensitivity. Overall, our findings delineate the regulatory mechanisms of DHC in DSB repair and suggest that it might potentially be used as an inhibitor of the DNA repair pathway in human cells.

  5. Sterigmatocystin-induced DNA damage triggers G2 arrest via an ATM/p53-related pathway in human gastric epithelium GES-1 cells in vitro.

    Directory of Open Access Journals (Sweden)

    Donghui Zhang

    Full Text Available Sterigmatocystin (ST, which is commonly detected in food and feed commodities, is a mutagenic and carcinogenic mycotoxin that has been recognized as a possible human carcinogen. Our previous study showed that ST can induce G2 phase arrest in GES-1 cells in vitro and that the MAPK and PI3K signaling pathways are involved in the ST-induced G2 arrest. It is now widely accepted that DNA damage plays a critical role in the regulation of cell cycle arrest and apoptosis. In response to DNA damage, a complex signaling network is activated in eukaryotic cells to trigger cell cycle arrest and facilitate DNA repair. To further explore the molecular mechanism through which ST induces G2 arrest, the current study was designed to precisely dissect the role of DNA damage and the DNA damage sensor ataxia telangiectasia-mutated (ATM/p53-dependent pathway in the ST-induced G2 arrest in GES-1 cells. Using the comet assay, we determined that ST induces DNA damage, as evidenced by the formation of DNA comet tails, in GES-1 cells. We also found that ST induces the activation of ATM and its downstream molecules, Chk2 and p53, in GES-1 cells. The ATM pharmacological inhibitor caffeine was found to effectively inhibit the activation of the ATM-dependent pathways and to rescue the ST-induced G2 arrest in GES-1 cells, which indicating its ATM-dependent characteristic. Moreover, the silencing of the p53 expression with siRNA effectively attenuated the ST-induced G2 arrest in GES-1 cells. We also found that ST induces apoptosis in GES-1 cells. Thus, our results show that the ST-induced DNA damage activates the ATM/53-dependent signaling pathway, which contributes to the induction of G2 arrest in GES-1 cells.

  6. Immunoassay of DNA damage

    International Nuclear Information System (INIS)

    Gasparro, F.P.; Santella, R.M.

    1988-01-01

    The direct photomodification of DNA by ultraviolet light or the photo-induced addition of exogenous compounds to DNA components results in alterations of DNA structure ranging from subtle to profound. There are two consequences of these conformational changes. First, cells in which the DNA has been damaged are capable of executing repair steps. Second, the DNA which is usually of very low immunogenicity now becomes highly antigenic. This latter property has allowed the production of a series of monoclonal antibodies that recognize photo-induced DNA damage. Monoclonal antibodies have been generated that recognize the 4',5'-monoadduct and the crosslink of 8-methoxypsoralen in DNA. In addition, another antibody has been prepared which recognizes the furan-side monoadduct of 6,4,4'-trimethylangelicin in DNA. These monoclonal antibodies have been characterized as to sensitivity and specificity using non-competitive and competitive enzyme-linked-immunosorbent assays (ELISA). (author)

  7. Immunoassay of DNA damage

    Energy Technology Data Exchange (ETDEWEB)

    Gasparro, F P; Santella, R M

    1988-09-01

    The direct photomodification of DNA by ultraviolet light or the photo-induced addition of exogenous compounds to DNA components results in alterations of DNA structure ranging from subtle to profound. There are two consequences of these conformational changes. First, cells in which the DNA has been damaged are capable of executing repair steps. Second, the DNA which is usually of very low immunogenicity now becomes highly antigenic. This latter property has allowed the production of a series of monoclonal antibodies that recognize photo-induced DNA damage. Monoclonal antibodies have been generated that recognize the 4',5'-monoadduct and the crosslink of 8-methoxypsoralen in DNA. In addition, another antibody has been prepared which recognizes the furan-side monoadduct of 6,4,4'-trimethylangelicin in DNA. These monoclonal antibodies have been characterized as to sensitivity and specificity using non-competitive and competitive enzyme-linked-immunosorbent assays (ELISA).

  8. Inflammation, oxidative DNA damage, and carcinogenesis

    International Nuclear Information System (INIS)

    Lewis, J.G.; Adams, D.O.

    1987-01-01

    Inflammation has long been associated with carcinogenesis, especially in the promotion phase. The mechanism of action of the potent inflammatory agent and skin promoter 12-tetradecanoyl phorbol-13-acetate (TPA) is unknown. It is though that TPA selectively enhances the growth of initiated cells, and during this process, initiated cells progress to the preneoplastic state and eventually to the malignant phenotype. The authors and others have proposed that TPA may work, in part, by inciting inflammation and stimulating inflammatory cells to release powerful oxidants which then induce DNA damage in epidermal cells. Macrophages cocultured with target cells and TPA induce oxidized thymine bases in the target cells. This process is inhibited by both catalase and inhibitors of lipoxygenases, suggesting the involvement of both H 2 O 2 and oxidized lipid products. In vivo studies demonstrated that SENCAR mice, which are sensitive to promotion by TPA, have a more intense inflammatory reaction in skin that C57LB/6 mice, which are resistant to promotion by TPA. In addition, macrophages from SENCAR mice release more H 2 O 2 and metabolites of AA, and induce more oxidative DNA damage in cocultured cells than macrophages from C57LB/6 mice. These data support the hypothesis that inflammation and the release of genotoxic oxidants may be one mechanism whereby initiated cells receive further genetic insults. They also further complicate risk assessment by suggesting that some environmental agents may work indirectly by subverting host systems to induce damage rather than maintaining homeostasis

  9. DNA damage and radical reactions: Mechanistic aspects, formation in cells and repair studies

    International Nuclear Information System (INIS)

    Cadet, J.; Ravanat, J.L.; Carell, T.; Cellai, L.; Chatgilialoglu, Ch.; Gimisis, Th.; Miranda, M.; O'Neill, P.; Robert, M.

    2008-01-01

    Several examples of oxidative and reductive reactions of DNA components that lead to single and tandem modifications are discussed in this review. These include nucleophilic addition reactions of the one-electron oxidation-mediated guanine radical cation and the one-electron reduced intermediate of 8-bromo-purine 2'-de-oxy-ribo-nucleosides that give rise to either an oxidizing guanine radical or related 5',8-cyclo-purine nucleosides. In addition, mechanistic insights into the reductive pathways involved in the photolyase induced reversal of cyclo-buta-cli-pyrimidine and pyrimidine (6-4) pyrimidone photoproducts are provided. Evidence for the occurrence and validation in cellular DNA of (OH) · radical degradation pathways of guanine that have been established in model systems has been gained from the accurate measurement of degradation products. Relevant information on biochemical aspects of the repair of single and clustered oxidatively generated damage to DNA has been gained from detailed investigations that rely on the synthesis of suitable modified probes. Thus the preparation of stable carbocyclic derivatives of purine nucleoside containing defined sequence oligonucleotides has allowed detailed crystallographic studies of the recognition step of the base damage by enzymes implicated in the base excision repair (BER) pathway. Detailed insights are provided on the BER processing of non-double strand break bi-stranded clustered damage that may consist of base lesions, a single strand break or abasic sites and represent one of the main deleterious classes of radiation-induced DNA damage. (authors)

  10. Cytochrome P450 2A13 enhances the sensitivity of human bronchial epithelial cells to aflatoxin B1-induced DNA damage

    Energy Technology Data Exchange (ETDEWEB)

    Yang, Xuejiao [Key Lab of Modern Toxicology of Ministry of Education, School of Public Health, Nanjing Medical University, 818 East Tiangyuan Rd., Nanjing 211166 (China); Jiaojiang District Center for Disease Control and Prevention, 518 Jingdong Rd., Taizhou 318000 (China); Zhang, Zhan; Wang, Xichen; Wang, Yun; Zhang, Xiaoming; Lu, Huiyuan [Key Lab of Modern Toxicology of Ministry of Education, School of Public Health, Nanjing Medical University, 818 East Tiangyuan Rd., Nanjing 211166 (China); Wang, Shou-Lin, E-mail: wangshl@njmu.edu.cn [Key Lab of Modern Toxicology of Ministry of Education, School of Public Health, Nanjing Medical University, 818 East Tiangyuan Rd., Nanjing 211166 (China)

    2013-07-15

    Cytochrome P450 2A13 (CYP2A13) mainly expresses in human respiratory system and mediates the metabolic activation of aflatoxin B1 (AFB1). Our previous study suggested that CYP2A13 could increase the cytotoxic and apoptotic effects of AFB1 in immortalized human bronchial epithelial cells (BEAS-2B). However, the role of CYP2A13 in AFB1-induced DNA damage is unclear. Using BEAS-2B cells that stably express CYP2A13 (B-2A13), CYP1A2 (B-1A2), and CYP2A6 (B-2A6), we compared their effects in AFB1-induced DNA adducts, DNA damage, and cell cycle changes. BEAS-2B cells that were transfected with vector (B-vector) were used as a control. The results showed that AFB1 (5–80 nM) dose- and time-dependently induced DNA damage in B-2A13 cells. AFB1 at 10 and 80 nM significantly augmented this effect in B-2A13 and B-1A2 cells, respectively. B-2A6 cells showed no obvious DNA damage, similar to B-vector cells and the vehicle control. Similarly, compared with B-vector, B-1A2 or B-2A6 cells, B-2A13 cells showed more sensitivity in AFB1-induced γH2AX expression, DNA adduct 8-hydroxy-deoxyguanosine formation, and S-phase cell-cycle arrest. Furthermore, AFB1 activated the proteins related to DNA damage responses, such as ATM, ATR, Chk2, p53, BRCA1, and H2AX, rather than the proteins related to DNA repair. These effects could be almost completely inhibited by 100 μM nicotine (a substrate of CYP2A13) or 1 μM 8-methoxypsoralen (8-MOP; an inhibitor of CYP enzyme). Collectively, these findings suggest that CYP2A13 plays an important role in low-concentration AFB1-induced DNA damage, possibly linking environmental airborne AFB1 to genetic injury in human respiratory system. - Highlights: • CYP2A13 plays a critical role in low concentration of AFB1-induced DNA damage. • B-2A13 cells were more sensitive to AFB1 than B-1A2 cells and B-2A6 cells. • AFB1 dose- and time-dependently induced DNA damage in B-2A13 cells • AFB1-induced DNA adducts and damage can be inhibited by nicotine and 8

  11. Cytochrome P450 2A13 enhances the sensitivity of human bronchial epithelial cells to aflatoxin B1-induced DNA damage

    International Nuclear Information System (INIS)

    Yang, Xuejiao; Zhang, Zhan; Wang, Xichen; Wang, Yun; Zhang, Xiaoming; Lu, Huiyuan; Wang, Shou-Lin

    2013-01-01

    Cytochrome P450 2A13 (CYP2A13) mainly expresses in human respiratory system and mediates the metabolic activation of aflatoxin B1 (AFB1). Our previous study suggested that CYP2A13 could increase the cytotoxic and apoptotic effects of AFB1 in immortalized human bronchial epithelial cells (BEAS-2B). However, the role of CYP2A13 in AFB1-induced DNA damage is unclear. Using BEAS-2B cells that stably express CYP2A13 (B-2A13), CYP1A2 (B-1A2), and CYP2A6 (B-2A6), we compared their effects in AFB1-induced DNA adducts, DNA damage, and cell cycle changes. BEAS-2B cells that were transfected with vector (B-vector) were used as a control. The results showed that AFB1 (5–80 nM) dose- and time-dependently induced DNA damage in B-2A13 cells. AFB1 at 10 and 80 nM significantly augmented this effect in B-2A13 and B-1A2 cells, respectively. B-2A6 cells showed no obvious DNA damage, similar to B-vector cells and the vehicle control. Similarly, compared with B-vector, B-1A2 or B-2A6 cells, B-2A13 cells showed more sensitivity in AFB1-induced γH2AX expression, DNA adduct 8-hydroxy-deoxyguanosine formation, and S-phase cell-cycle arrest. Furthermore, AFB1 activated the proteins related to DNA damage responses, such as ATM, ATR, Chk2, p53, BRCA1, and H2AX, rather than the proteins related to DNA repair. These effects could be almost completely inhibited by 100 μM nicotine (a substrate of CYP2A13) or 1 μM 8-methoxypsoralen (8-MOP; an inhibitor of CYP enzyme). Collectively, these findings suggest that CYP2A13 plays an important role in low-concentration AFB1-induced DNA damage, possibly linking environmental airborne AFB1 to genetic injury in human respiratory system. - Highlights: • CYP2A13 plays a critical role in low concentration of AFB1-induced DNA damage. • B-2A13 cells were more sensitive to AFB1 than B-1A2 cells and B-2A6 cells. • AFB1 dose- and time-dependently induced DNA damage in B-2A13 cells • AFB1-induced DNA adducts and damage can be inhibited by nicotine and 8

  12. Effects of a Mangifera indica L. stem bark extract and mangiferin on radiation-induced DNA damage in human lymphocytes and lymphoblastoid cells.

    Science.gov (United States)

    Rodeiro, I; Delgado, R; Garrido, G

    2014-02-01

    Mangifera indica L. (mango) stem bark aqueous extract (MSBE) that has antioxidant, anti-inflammatory and immunomodulatory properties, can be obtained in Cuba. It is rich in polyphenols, where mangiferin is the main component. In this study, we have tested DNA damage and protection effects of MSBE and mangiferin on primary human lymphocytes and lymphoblastoid cells. Cell suspensions were incubated with the products (50-1000 μg/ml) for experiments on damage induction, and evaluation of any potential protective effects (5-100 μg/ml) for 60 min at 37 °C. Irradiation was performed using a γ-ray source, absorbed dose 5 Gy. At the end of exposure, DNA damage, protection and repair processes were evaluated using the comet assay. MSBE (100-1000 μg/ml) induced DNA damage in a concentration dependent manner in both cell types tested, primary cells being more sensitive. Mangiferin (200 μg/ml) only induced light DNA damage at higher concentrations. DNA repair capacity was not affected after MSBE or mangiferin exposure. On the other hand, MSBE (25 and 50 μg/ml) and mangiferin (5-25 ug/ml) protected against gamma radiation-induced DNA damage. These results show MSBE has protector or harmful effects on DNA in vitro depending on the experimental conditions, which suggest that the extract could be acting as an antioxidant or pro-oxidant product. Mangiferin was involved in protective effects of the extract. © 2013 John Wiley & Sons Ltd.

  13. Epigenetic and genetic factors in the cellular response to radiations and DNA-damaging chemicals

    International Nuclear Information System (INIS)

    Williams, J.R.; D'Arpa, P.

    1981-01-01

    DNA-damaging agents are widely used as therapeutic tools for a variety of disease states. Many such agents are considered to produce detrimental side effects. Thus, it is important to evaluate both therapeutic efficacy and potential risk. DNA-damaging agents can be so evaluated by comparison to agents whose therapeutic benefit and potential hazards are better known. We propose a framework for such comparison, demonstrating that a simple transformation of cytotoxicity-dose response patterns permits a facile comparison of variation between cells exposed to a single DNA-damaging agent or to different cytotoxic agents. Further, by transforming data from experiments which compare responses of 2 cell populations to an effects ratio, different patterns for the changes in cytotoxicity produced by epigenetic and genetic factors were compared. Using these transformations, we found that there is a wide variation (a factor of 4) between laboratories for a single agent (UVC) and only a slightly larger variation (factor of 6) between normal cell response for different types of DNA-damaging agents (x-ray, UVC, alkylating agents, crosslinking agents). Epigenetic factors such as repair and recovery appear to be a factor only at higher dose levels. Comparison in the cytotoxic effect of a spectrum of DNA-damaging agents in xeroderma pigmentosum, ataxia telangiectasia, and Fanconi's anemia cells indicates significantly different patterns, implying that the effect, and perhaps the nature, of these genetic conditions are quite different

  14. Role of the Checkpoint Clamp in DNA Damage Response

    Directory of Open Access Journals (Sweden)

    Mihoko Kai

    2013-01-01

    Full Text Available DNA damage occurs during DNA replication, spontaneous chemical reactions, and assaults by external or metabolism-derived agents. Therefore, all living cells must constantly contend with DNA damage. Cells protect themselves from these genotoxic stresses by activating the DNA damage checkpoint and DNA repair pathways. Coordination of these pathways requires tight regulation in order to prevent genomic instability. The checkpoint clamp complex consists of Rad9, Rad1 and Hus1 proteins, and is often called the 9-1-1 complex. This PCNA (proliferating cell nuclear antigen-like donut-shaped protein complex is a checkpoint sensor protein that is recruited to DNA damage sites during the early stage of the response, and is required for checkpoint activation. As PCNA is required for multiple pathways of DNA metabolism, the checkpoint clamp has also been implicated in direct roles in DNA repair, as well as in coordination of the pathways. Here we discuss roles of the checkpoint clamp in DNA damage response (DDR.

  15. Study on effect of artemisinin combined with 60Co γ-ray on DNA damage in HeLa and SiHa cells

    International Nuclear Information System (INIS)

    Feng Yang; Zhou Yuanyuan; Yang Wei; Chen Qiu; Li Ming; Zhang Shuyu; Zhu Wei; Cao Jianping; Zhang Xuguang

    2011-01-01

    Objective: To investigate the effect of Artemisinin combined with 60 Co γ-ray on DNA damage in HeLa and SiHa cells of human cervical cancer. Methods: Cell growth kinetics was evaluated by MTT assay to determine the most appropriate drug concentration. Effects of Artemisinin combined with 60 Co γ-ray on DNA damage in HeLa and SiHa cells were detected by single cell gel electrophoresis. Results: With the concentration increased during the effect of Artemisinin, the HeLa and SiHa cells had higher inhibition on cell proliferation. The SCGE showed that:the comet cell analysis indexes (the comet cells ratio, Tail Length, Olive Tail Moment and Tail DNA%) there was no statistic difference in between the artemisinin group and the control group (P>0.05). With radiation in the same dose, the comet cell analysis indexes of Hela cells treated with both artermisinin and exposed to radiation were higher than that only exposed to radiation group(P 0.05). Conclusion: Artemisinin can not induce DNA damage in both HeLa and SiHa cells, but it can make irradiated HeLa cells DNA damage to be aggravated and enhance HeLa cells' radiation sensitivity. However, Artemisinin has no radiosensitizing effect on SiHa cells. (authors)

  16. Radiation induced bystander signals are independent of DNA damage and DNA repair capacity of the irradiated cells

    Energy Technology Data Exchange (ETDEWEB)

    Kashino, Genro [Gray Cancer Institute, P.O. Box 100, Mount Vernon Hospital, Northwood, Middlesex HA6 2JR (United Kingdom); Particle Radiation Oncology Research Center, Research Reactor Institute, Kyoto University, 2-1010 Asashiro-nishi, Kumatori-cho, Sennan-gun, Osaka 590-0494 (Japan); Suzuki, Keiji [Division of Radiation Biology, Department of Radiology and Radiation Biology, Course of Life Sciences and Radiation Research, Graduate School of Biomedical Sciences, Nagasaki University, 1-14 Bunkyo-machi, Nagasaki 852-8521 (Japan); Matsuda, Naoki [Division of Radiation Biology and Protection, Center for Frontier Life Sciences, Nagasaki University, Nagasaki 852-8102 (Japan); Kodama, Seiji [Radiation Biology Laboratory, Radiation Research Center, Frontier Science Innovation Center, Organization for University-Industry-Government Cooperation, Osaka Prefecture University, 1-2 Gakuen-cho, Sakai, Osaka 599-8570 (Japan); Ono, Koji [Particle Radiation Oncology Research Center, Research Reactor Institute, Kyoto University, 2-1010 Asashiro-nishi, Kumatori-cho, Sennan-gun, Osaka 590-0494 (Japan); Watanabe, Masami [Laboratory of Radiation Biology, Division of Radiation Life Science, Department of Radiation Life Science and Radiation Medical Science, Kyoto University Research Reactor Institute, 2-1010 Asashiro-nishi, Kumatori-cho, Sennan-gun, Osaka 590-0494 (Japan); Prise, Kevin M [Gray Cancer Institute, P.O. Box 100, Mount Vernon Hospital, Northwood, Middlesex HA6 2JR (United Kingdom) and Centre for Cancer Research and Cell Biology, Queen' s University Belfast, Lisburn Road, Belfast BT9 7AB (United Kingdom)]. E-mail: prise@gci.ac.uk

    2007-06-01

    Evidence is accumulating that irradiated cells produce signals, which interact with non-exposed cells in the same population. Here, we analysed the mechanism for bystander signal arising in wild-type CHO cells and repair deficient varients, focussing on the relationship between DNA repair capacity and bystander signal arising in irradiated cells. In order to investigate the bystander effect, we carried out medium transfer experiments after X-irradiation where micronuclei were scored in non-targeted DSB repair deficient xrs5 cells. When conditioned medium from irradiated cells was transferred to unirradiated xrs5 cells, the level of induction was independent of whether the medium came from irradiated wild-type, ssb or dsb repair deficient cells. This result suggests that the activation of a bystander signal is independent of the DNA repair capacity of the irradiated cells. Also, pre-treatment of the irradiated cells with 0.5% DMSO, which suppresses micronuclei induction in CHO but not in xrs5 cells, suppressed bystander effects completely in both conditioned media, suggesting that DMSO is effective for suppression of bystander signal arising independently of DNA damage in irradiated cells. Overall the work presented here adds to the understanding that it is the repair phenotype of the cells receiving bystander signals, which determines overall response rather than that of the cell producing the bystander signal.

  17. Umbelliferone arrest cell cycle at G0/G1 phase and induces apoptosis in human oral carcinoma (KB) cells possibly via oxidative DNA damage.

    Science.gov (United States)

    Vijayalakshmi, Annamalai; Sindhu, Ganapathy

    2017-08-01

    Umbelliferone (UMB) has widespread pharmacological activity, comprising anti-inflammatory, anti-oxidant, anti-genotoxic and anti-immunomodulatory but the anticancer activity remains unknown in human oral carcinoma (HOC) KB cells. MTT assay determinations was revealed that treatment of KB cells with UMB, prevent and reduce the cell proliferation with the IC 50 - 200μM as well as induces loss of cell viability, morphology change and internucleosomal DNA fragmentation in a concentration dependent manner. Acridine orange and ethidium bromide dual staining assay established that UMB induced apoptosis in KB cells in a dose dependent manner. Alkaline comet assay determination revealed UMB has the potential to increase oxidative DNA damage in KB cells through DNA tail formation significantly (pKB cells. Similarly, we observed increased DNA damage stimulated apoptotic morphological changes in UMB treated cells. Taken together, the present study suggests that UMB exhibits anticancer effect on KB cell line with the increased generation of intracellular ROS, triggered oxidative stress mediated depolarization of mitochondria, which contributes cell death via DNA damage as well as cell cycle arrest at G0/G1 phase. The results have also provided us insight in the pharmacological backgrounds for the potential use of UMB, to target divergent pathways of cell survival and cell death. To conclude UMB could develop as a novel candidate for cancer chemoprevention and therapy, which is our future focus and to develop a connectivity map between in vivo and in vitro activity. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

  18. DNA damage and cellular death in oral mucosa cells of children who have undergone panoramic dental radiography

    International Nuclear Information System (INIS)

    Angelieri, Fernanda; Oliveira, Gabriela R. de; Sannomiya, Eduardo K.; Ribeiro, Daniel A.

    2007-01-01

    Despite wide use as a diagnostic tool in medical and dental practice, radiography can induce cytotoxic effects and genetic damage. To evaluate DNA damage (micronucleus) and cellular death (pyknosis, karyolysis and karyorrhexis) in exfoliated buccal mucosa cells taken from healthy children following exposure to radiation during dental radiography. A total of 17 children who had undergone panoramic dental radiography were included. We found no statistically significant differences (P > 0.05) between micronucleated oral mucosa cells in children before and after exposure to radiation. On the other hand, radiation did cause other nuclear alterations closely related to cytotoxicity including karyorrhexis, pyknosis and karyolysis. Taken together, these results indicate that panoramic dental radiography might not induce chromosomal damage, but may be cytotoxic. Overall, the results reinforce the importance of evaluating the health side effects of radiography and contribute to the micronucleus database, which will improve our understanding and practice of this methodology in children. (orig.)

  19. DNA damage and cellular death in oral mucosa cells of children who have undergone panoramic dental radiography

    Energy Technology Data Exchange (ETDEWEB)

    Angelieri, Fernanda; Oliveira, Gabriela R. de [Sao Paulo Metodista University (UMESP), Department of Orthodontics, Sao Bernardo do Campo, Sao Paulo (Brazil); Sannomiya, Eduardo K. [Sao Paulo Metodista University (UMESP), Department of Dento-Maxillofacial Radiology, Sao Bernardo do Campo, Sao Paulo (Brazil); Ribeiro, Daniel A. [Federal University of Sao Paulo (UNIFESP), Department of Health Sciences, Santos, Sao Paulo (Brazil); Universidade Federal de Sao Paulo (UNIFESP), Departamento de Ciencias da Saude, Santos, Sao Paulo (Brazil)

    2007-06-15

    Despite wide use as a diagnostic tool in medical and dental practice, radiography can induce cytotoxic effects and genetic damage. To evaluate DNA damage (micronucleus) and cellular death (pyknosis, karyolysis and karyorrhexis) in exfoliated buccal mucosa cells taken from healthy children following exposure to radiation during dental radiography. A total of 17 children who had undergone panoramic dental radiography were included. We found no statistically significant differences (P > 0.05) between micronucleated oral mucosa cells in children before and after exposure to radiation. On the other hand, radiation did cause other nuclear alterations closely related to cytotoxicity including karyorrhexis, pyknosis and karyolysis. Taken together, these results indicate that panoramic dental radiography might not induce chromosomal damage, but may be cytotoxic. Overall, the results reinforce the importance of evaluating the health side effects of radiography and contribute to the micronucleus database, which will improve our understanding and practice of this methodology in children. (orig.)

  20. TopBP1 is required at mitosis to reduce transmission of DNA damage to G1 daughter cells

    DEFF Research Database (Denmark)

    Pedersen, Rune Troelsgaard; Kruse, Thomas; Nilsson, Jakob

    2015-01-01

    mitotic entry. In early mitosis, TopBP1 marks sites of and promotes unscheduled DNA synthesis. Moreover, TopBP1 is required for focus formation of the structure-selective nuclease and scaffold protein SLX4 in mitosis. Persistent TopBP1 foci transition into 53BP1 nuclear bodies (NBs) in G1 and precise...... temporal depletion of TopBP1 just before mitotic entry induced formation of 53BP1 NBs in the next cell cycle, showing that TopBP1 acts to reduce transmission of DNA damage to G1 daughter cells. Based on these results, we propose that TopBP1 maintains genome integrity in mitosis by controlling chromatin...

  1. Time-Lapse Monitoring of DNA Damage Colocalized With Particle Tracks in Single Living Cells

    Energy Technology Data Exchange (ETDEWEB)

    McFadden, Conor H. [Department of Radiation Physics, The University of Texas MD Anderson Cancer Center, Houston, Texas (United States); Hallacy, Timothy M. [Department of Radiation Physics, The University of Texas MD Anderson Cancer Center, Houston, Texas (United States); Department of Physics and Astronomy, Rice University, Houston, Texas (United States); Flint, David B. [Department of Radiation Physics, The University of Texas MD Anderson Cancer Center, Houston, Texas (United States); Graduate School of Biomedical Sciences, The University of Texas, Houston, Texas (United States); Granville, Dal A. [Department of Medical Physics, The Ottawa Hospital Cancer Centre, Ottawa, Ontario (Canada); Asaithamby, Aroumougame [Division of Molecular Radiation Biology, Department of Radiation Oncology, University of Texas Southwestern Medical Centre, Dallas, Texas (United States); Sahoo, Narayan [Department of Radiation Physics, The University of Texas MD Anderson Cancer Center, Houston, Texas (United States); Akselrod, Mark S. [Crystal Growth Division, Landauer, Inc, Stillwater, Oklahoma (United States); Sawakuchi, Gabriel O., E-mail: gsawakuchi@mdanderson.org [Department of Radiation Physics, The University of Texas MD Anderson Cancer Center, Houston, Texas (United States); Graduate School of Biomedical Sciences, The University of Texas, Houston, Texas (United States)

    2016-09-01

    Purpose: Understanding the DNA damage and repair induced by hadron therapy (HT) beams is crucial for developing novel strategies to maximize the use of HT beams to treat cancer patients. However, spatiotemporal studies of DNA damage and repair for beam energies relevant to HT have been challenging. We report a technique that enables spatiotemporal measurement of radiation-induced damage in live cells and colocalization of this damage with charged particle tracks over a broad range of clinically relevant beam energies. The technique uses novel fluorescence nuclear track detectors with fluorescence confocal laser scanning microscopy in the beam line to visualize particle track traversals within the subcellular compartments of live cells within seconds after injury. Methods and Materials: We designed and built a portable fluorescence confocal laser scanning microscope for use in the beam path, coated fluorescence nuclear track detectors with fluorescent-tagged live cells (HT1080 expressing enhanced green fluorescent protein tagged to XRCC1, a single-strand break repair protein), placed the entire assembly into a proton therapy beam line, and irradiated the cells with a fluence of ∼1 × 10{sup 6} protons/cm{sup 2}. Results: We successfully obtained confocal images of proton tracks and foci of DNA single-strand breaks immediately after irradiation. Conclusions: This technique represents an innovative method for analyzing biological responses in any HT beam line at energies and dose rates relevant to therapy. It allows precise determination of the number of tracks traversing a subcellular compartment and monitoring the cellular damage therein, and has the potential to measure the linear energy transfer of each track from therapeutic beams.

  2. Inactivating UBE2M impacts the DNA damage response and genome integrity involving multiple cullin ligases.

    Directory of Open Access Journals (Sweden)

    Scott Cukras

    Full Text Available Protein neddylation is involved in a wide variety of cellular processes. Here we show that the DNA damage response is perturbed in cells inactivated with an E2 Nedd8 conjugating enzyme UBE2M, measured by RAD51 foci formation kinetics and cell based DNA repair assays. UBE2M knockdown increases DNA breakages and cellular sensitivity to DNA damaging agents, further suggesting heightened genomic instability and defective DNA repair activity. Investigating the downstream Cullin targets of UBE2M revealed that silencing of Cullin 1, 2, and 4 ligases incurred significant DNA damage. In particular, UBE2M knockdown, or defective neddylation of Cullin 2, leads to a blockade in the G1 to S progression and is associated with delayed S-phase dependent DNA damage response. Cullin 4 inactivation leads to an aberrantly high DNA damage response that is associated with increased DNA breakages and sensitivity of cells to DNA damaging agents, suggesting a DNA repair defect is associated. siRNA interrogation of key Cullin substrates show that CDT1, p21, and Claspin are involved in elevated DNA damage in the UBE2M knockdown cells. Therefore, UBE2M is required to maintain genome integrity by activating multiple Cullin ligases throughout the cell cycle.

  3. Inactivating UBE2M impacts the DNA damage response and genome integrity involving multiple cullin ligases.

    Science.gov (United States)

    Cukras, Scott; Morffy, Nicholas; Ohn, Takbum; Kee, Younghoon

    2014-01-01

    Protein neddylation is involved in a wide variety of cellular processes. Here we show that the DNA damage response is perturbed in cells inactivated with an E2 Nedd8 conjugating enzyme UBE2M, measured by RAD51 foci formation kinetics and cell based DNA repair assays. UBE2M knockdown increases DNA breakages and cellular sensitivity to DNA damaging agents, further suggesting heightened genomic instability and defective DNA repair activity. Investigating the downstream Cullin targets of UBE2M revealed that silencing of Cullin 1, 2, and 4 ligases incurred significant DNA damage. In particular, UBE2M knockdown, or defective neddylation of Cullin 2, leads to a blockade in the G1 to S progression and is associated with delayed S-phase dependent DNA damage response. Cullin 4 inactivation leads to an aberrantly high DNA damage response that is associated with increased DNA breakages and sensitivity of cells to DNA damaging agents, suggesting a DNA repair defect is associated. siRNA interrogation of key Cullin substrates show that CDT1, p21, and Claspin are involved in elevated DNA damage in the UBE2M knockdown cells. Therefore, UBE2M is required to maintain genome integrity by activating multiple Cullin ligases throughout the cell cycle.

  4. Enhancement of chemically induced reactive oxygen species production and DNA damage in human SH-SY5Y neuroblastoma cells by 872 MHz radiofrequency radiation

    Energy Technology Data Exchange (ETDEWEB)

    Luukkonen, Jukka [Department of Environmental Science, University of Kuopio, Bioteknia 2, P.O. Box 1627, FI-70211 Kuopio (Finland)], E-mail: Jukka.Luukkonen@uku.fi; Hakulinen, Pasi; Maeki-Paakkanen, Jorma [Department of Environmental Health, National Public Health Institute, P.O. Box 95, FI-70701 Kuopio (Finland); Juutilainen, Jukka; Naarala, Jonne [Department of Environmental Science, University of Kuopio, Bioteknia 2, P.O. Box 1627, FI-70211 Kuopio (Finland)

    2009-03-09

    The objective of the study was to investigate effects of 872 MHz radiofrequency (RF) radiation on intracellular reactive oxygen species (ROS) production and DNA damage at a relatively high SAR value (5 W/kg). The experiments also involved combined exposure to RF radiation and menadione, a chemical inducing intracellular ROS production and DNA damage. The production of ROS was measured using the fluorescent probe dichlorofluorescein and DNA damage was evaluated by the Comet assay. Human SH-SY5Y neuroblastoma cells were exposed to RF radiation for 1 h with or without menadione. Control cultures were sham exposed. Both continuous waves (CW) and a pulsed signal similar to that used in global system for mobile communications (GSM) mobile phones were used. Exposure to the CW RF radiation increased DNA breakage (p < 0.01) in comparison to the cells exposed only to menadione. Comparison of the same groups also showed that ROS level was higher in cells exposed to CW RF radiation at 30 and 60 min after the end of exposure (p < 0.05 and p < 0.01, respectively). No effects of the GSM signal were seen on either ROS production or DNA damage. The results of the present study suggest that 872 MHz CW RF radiation at 5 W/kg might enhance chemically induced ROS production and thus cause secondary DNA damage. However, there is no known mechanism that would explain such effects from CW RF radiation but not from GSM modulated RF radiation at identical SAR.

  5. Enhancement of chemically induced reactive oxygen species production and DNA damage in human SH-SY5Y neuroblastoma cells by 872 MHz radiofrequency radiation

    International Nuclear Information System (INIS)

    Luukkonen, Jukka; Hakulinen, Pasi; Maeki-Paakkanen, Jorma; Juutilainen, Jukka; Naarala, Jonne

    2009-01-01

    The objective of the study was to investigate effects of 872 MHz radiofrequency (RF) radiation on intracellular reactive oxygen species (ROS) production and DNA damage at a relatively high SAR value (5 W/kg). The experiments also involved combined exposure to RF radiation and menadione, a chemical inducing intracellular ROS production and DNA damage. The production of ROS was measured using the fluorescent probe dichlorofluorescein and DNA damage was evaluated by the Comet assay. Human SH-SY5Y neuroblastoma cells were exposed to RF radiation for 1 h with or without menadione. Control cultures were sham exposed. Both continuous waves (CW) and a pulsed signal similar to that used in global system for mobile communications (GSM) mobile phones were used. Exposure to the CW RF radiation increased DNA breakage (p < 0.01) in comparison to the cells exposed only to menadione. Comparison of the same groups also showed that ROS level was higher in cells exposed to CW RF radiation at 30 and 60 min after the end of exposure (p < 0.05 and p < 0.01, respectively). No effects of the GSM signal were seen on either ROS production or DNA damage. The results of the present study suggest that 872 MHz CW RF radiation at 5 W/kg might enhance chemically induced ROS production and thus cause secondary DNA damage. However, there is no known mechanism that would explain such effects from CW RF radiation but not from GSM modulated RF radiation at identical SAR

  6. Exposure to non-ionizing electromagnetic fields emitted from mobile phones induced DNA damage in human ear canal hair follicle cells.

    Science.gov (United States)

    Akdag, Mehmet; Dasdag, Suleyman; Canturk, Fazile; Akdag, Mehmet Zulkuf

    2018-01-01

    The aim of this study was to investigate effect of radiofrequency radiation (RFR) emitted from mobile phones on DNA damage in follicle cells of hair in the ear canal. The study was carried out on 56 men (age range: 30-60 years old)in four treatment groups with n = 14 in each group. The groups were defined as follows: people who did not use a mobile phone (Control), people use mobile phones for 0-30 min/day (second group), people use mobile phones for 30-60 min/day (third group) and people use mobile phones for more than 60 min/day (fourth group). Ear canal hair follicle cells taken from the subjects were analyzed by the Comet Assay to determine DNA damages. The Comet Assay parameters measured were head length, tail length, comet length, percentage of head DNA, tail DNA percentage, tail moment, and Olive tail moment. Results of the study showed that DNA damage indicators were higher in the RFR exposure groups than in the control subjects. In addition, DNA damage increased with the daily duration of exposure. In conclusion, RFR emitted from mobile phones has a potential to produce DNA damage in follicle cells of hair in the ear canal. Therefore, mobile phone users have to pay more attention when using wireless phones.

  7. Hypothermia postpones DNA damage repair in irradiated cells and protects against cell killing

    International Nuclear Information System (INIS)

    Baird, Brandon J.; Dickey, Jennifer S.; Nakamura, Asako J.; Redon, Christophe E.; Parekh, Palak; Griko, Yuri V.; Aziz, Khaled; Georgakilas, Alexandros G.; Bonner, William M.; Martin, Olga A.

    2011-01-01

    Hibernation is an established strategy used by some homeothermic organisms to survive cold environments. In true hibernation, the core body temperature of an animal may drop to below 0 o C and metabolic activity almost cease. The phenomenon of hibernation in humans is receiving renewed interest since several cases of victims exhibiting core body temperatures as low as 13.7 o C have been revived with minimal lasting deficits. In addition, local cooling during radiotherapy has resulted in normal tissue protection. The experiments described in this paper were prompted by the results of a very limited pilot study, which showed a suppressed DNA repair response of mouse lymphocytes collected from animals subjected to 7-Gy total body irradiation under hypothermic (13 o C) conditions, compared to normothermic controls. Here we report that human BJ-hTERT cells exhibited a pronounced radioprotective effect on clonogenic survival when cooled to 13 o C during and 12 h after irradiation. Mild hypothermia at 20 and 30 o C also resulted in some radioprotection. The neutral comet assay revealed an apparent lack on double strand break (DSB) rejoining at 13 o C. Extension of the mouse lymphocyte study to ex vivo-irradiated human lymphocytes confirmed lower levels of induced phosphorylated H2AX (γ-H2AX) and persistence of the lesions at hypothermia compared to the normal temperature. Parallel studies of radiation-induced oxidatively clustered DNA lesions (OCDLs) revealed partial repair at 13 o C compared to the rapid repair at 37 o C. For both γ-H2AX foci and OCDLs, the return of lymphocytes to 37 o C resulted in the resumption of normal repair kinetics. These results, as well as observations made by others and reviewed in this study, have implications for understanding the radiobiology and protective mechanisms underlying hypothermia and potential opportunities for exploitation in terms of protecting normal tissues against radiation.

  8. DNA damage, metabolism and aging in pro-inflammatory T cells: Rheumatoid arthritis as a model system.

    Science.gov (United States)

    Li, Yinyin; Goronzy, Jörg J; Weyand, Cornelia M

    2018-05-01

    The aging process is the major driver of morbidity and mortality, steeply increasing the risk to succumb to cancer, cardiovascular disease, infection and neurodegeneration. Inflammation is a common denominator in age-related pathologies, identifying the immune system as a gatekeeper in aging overall. Among immune cells, T cells are long-lived and exposed to intense replication pressure, making them sensitive to aging-related abnormalities. In successful T cell aging, numbers of naïve cells, repertoire diversity and activation thresholds are preserved as long as possible; in maladaptive T cell aging, protective T cell functions decline and pro-inflammatory effector cells are enriched. Here, we review in the model system of rheumatoid arthritis (RA) how maladaptive T cell aging renders the host susceptible to chronic, tissue-damaging inflammation. In T cells from RA patients, known to be about 20years pre-aged, three interconnected functional domains are altered: DNA damage repair, metabolic activity generating energy and biosynthetic precursor molecules, and shaping of plasma membranes to promote T cell motility. In each of these domains, key molecules and pathways have now been identified, including the glycolytic enzymes PFKFB3 and G6PD; the DNA repair molecules ATM, DNA-PKcs and MRE11A; and the podosome marker protein TKS5. Some of these molecules may help in defining targetable pathways to slow the T cell aging process. Copyright © 2017 Elsevier Inc. All rights reserved.

  9. Chromatin modifications and the DNA damage response to ionizing radiation

    International Nuclear Information System (INIS)

    Kumar, Rakesh; Horikoshi, Nobuo; Singh, Mayank; Gupta, Arun; Misra, Hari S.; Albuquerque, Kevin; Hunt, Clayton R.; Pandita, Tej K.

    2013-01-01

    In order to survive, cells have evolved highly effective repair mechanisms to deal with the potentially lethal DNA damage produced by exposure to endogenous as well as exogenous agents. Ionizing radiation exposure induces highly lethal DNA damage, especially DNA double-strand breaks (DSBs), that is sensed by the cellular machinery and then subsequently repaired by either of two different DSB repair mechanisms: (1) non-homologous end joining, which re-ligates the broken ends of the DNA and (2) homologous recombination, that employs an undamaged identical DNA sequence as a template, to maintain the fidelity of DNA repair. Repair of DSBs must occur within the natural context of the cellular DNA which, along with specific proteins, is organized to form chromatin, the overall structure of which can impede DNA damage site access by repair proteins. The chromatin complex is a dynamic structure and is known to change as required for ongoing cellular processes such as gene transcription or DNA replication. Similarly, during the process of DNA damage sensing and repair, chromatin needs to undergo several changes in order to facilitate accessibility of the repair machinery. Cells utilize several factors to modify the chromatin in order to locally open up the structure to reveal the underlying DNA sequence but post-translational modification of the histone components is one of the primary mechanisms. In this review, we will summarize chromatin modifications by the respective chromatin modifying factors that occur during the DNA damage response.

  10. Hypothermia postpones DNA damage repair in irradiated cells and protects against cell killing

    Energy Technology Data Exchange (ETDEWEB)

    Baird, Brandon J.; Dickey, Jennifer S.; Nakamura, Asako J.; Redon, Christophe E.; Parekh, Palak [Laboratory of Molecular Pharmacology, CCR, NCI, Bethesda, MD 20892 (United States); Griko, Yuri V. [Radiation and Space Biotechnology Branch, NASA Ames Research Center, Moffett Field, CA 94035 (United States); Aziz, Khaled; Georgakilas, Alexandros G. [Biology Department, East Carolina University, Greenville, NC 27858 (United States); Bonner, William M. [Laboratory of Molecular Pharmacology, CCR, NCI, Bethesda, MD 20892 (United States); Martin, Olga A., E-mail: sedelnio@mail.nih.gov [Laboratory of Molecular Pharmacology, CCR, NCI, Bethesda, MD 20892 (United States)

    2011-06-03

    Hibernation is an established strategy used by some homeothermic organisms to survive cold environments. In true hibernation, the core body temperature of an animal may drop to below 0 {sup o}C and metabolic activity almost cease. The phenomenon of hibernation in humans is receiving renewed interest since several cases of victims exhibiting core body temperatures as low as 13.7 {sup o}C have been revived with minimal lasting deficits. In addition, local cooling during radiotherapy has resulted in normal tissue protection. The experiments described in this paper were prompted by the results of a very limited pilot study, which showed a suppressed DNA repair response of mouse lymphocytes collected from animals subjected to 7-Gy total body irradiation under hypothermic (13 {sup o}C) conditions, compared to normothermic controls. Here we report that human BJ-hTERT cells exhibited a pronounced radioprotective effect on clonogenic survival when cooled to 13 {sup o}C during and 12 h after irradiation. Mild hypothermia at 20 and 30 {sup o}C also resulted in some radioprotection. The neutral comet assay revealed an apparent lack on double strand break (DSB) rejoining at 13 {sup o}C. Extension of the mouse lymphocyte study to ex vivo-irradiated human lymphocytes confirmed lower levels of induced phosphorylated H2AX ({gamma}-H2AX) and persistence of the lesions at hypothermia compared to the normal temperature. Parallel studies of radiation-induced oxidatively clustered DNA lesions (OCDLs) revealed partial repair at 13 {sup o}C compared to the rapid repair at 37 {sup o}C. For both {gamma}-H2AX foci and OCDLs, the return of lymphocytes to 37 {sup o}C resulted in the resumption of normal repair kinetics. These results, as well as observations made by others and reviewed in this study, have implications for understanding the radiobiology and protective mechanisms underlying hypothermia and potential opportunities for exploitation in terms of protecting normal tissues against

  11. Mobile phone radiation induces mode-dependent DNA damage in a mouse spermatocyte-derived cell line: a protective role of melatonin.

    Science.gov (United States)

    Liu, Chuan; Gao, Peng; Xu, Shang-Cheng; Wang, Yuan; Chen, Chun-Hai; He, Min-Di; Yu, Zheng-Ping; Zhang, Lei; Zhou, Zhou

    2013-11-01

    To evaluate whether exposure to mobile phone radiation (MPR) can induce DNA damage in male germ cells. A mouse spermatocyte-derived GC-2 cell line was exposed to a commercial mobile phone handset once every 20 min in standby, listen, dialed or dialing modes for 24 h. DNA damage was determined using an alkaline comet assay. The levels of DNA damage were significantly increased following exposure to MPR in the listen, dialed and dialing modes. Moreover, there were significantly higher increases in the dialed and dialing modes than in the listen mode. Interestingly, these results were consistent with the radiation intensities of these modes. However, the DNA damage effects of MPR in the dialing mode were efficiently attenuated by melatonin pretreatment. These results regarding mode-dependent DNA damage have important implications for the safety of inappropriate mobile phone use by males of reproductive age and also suggest a simple preventive measure: Keeping mobile phones as far away from our body as possible, not only during conversations but during 'dialed' and 'dialing' operation modes. Since the 'dialed' mode is actually part of the standby mode, mobile phones should be kept at a safe distance from our body even during standby operation. Furthermore, the protective role of melatonin suggests that it may be a promising pharmacological candidate for preventing mobile phone use-related reproductive impairments.

  12. Detection on emamectin benzoate-induced apoptosis and DNA damage in Spodoptera frugiperda Sf-9 cell line.

    Science.gov (United States)

    Wu, Xiwei; Zhang, Lei; Yang, Chao; Zong, Mimi; Huang, Qingchun; Tao, Liming

    2016-01-01

    Emamectin benzoate (EMB), an important macrocyclic lactone insecticide that belongs to the avermectin family and possesses excellent potency in controlling pests, is non-carcinogenic and non-mutagenic conducted in rats and mice, but EMB-induced cytotoxicity and genotoxicity in arthropod insect have been seldom reported yet. In the present paper, we quantified the cytotoxicity of EMB through the detections on cell viability, DNA damage, and cell apoptosis in Spodoptera frugiperda Sf-9 cells in vitro. The results showed that EMB caused a concentration- and time-dependent reduction on the viability of Sf-9 cells, and the median inhibitory concentrations (IC50) were 3.34μM at 72h of exposure. The dual acridine orange/ethidium bromide staining showed that exposure to EMB induced a significant time- and concentration-dependent increase on cell apoptosis. The alkaline comet assay revealed that EMB induced significant increases on single-strand DNA breaks, and the percentage of γH2AX-positive cells represented a time- and concentration-dependent formation of DNA double-strand breaks in Sf-9 cells. Interestingly, the similar cytotoxic actions of EMB also went for the human cancerous HeLa cells as a control cell group. Data demonstrated the potential cytotoxic effect of EMB on Sf-9 cells that was significantly greater than the effect of hydrogen peroxide at the same concentrations. Copyright © 2015 Elsevier Inc. All rights reserved.

  13. A Benzothiazole Derivative (5g) Induces DNA Damage And Potent G2/M Arrest In Cancer Cells.

    Science.gov (United States)

    Hegde, Mahesh; Vartak, Supriya V; Kavitha, Chandagirikoppal V; Ananda, Hanumappa; Prasanna, Doddakunche S; Gopalakrishnan, Vidya; Choudhary, Bibha; Rangappa, Kanchugarakoppal S; Raghavan, Sathees C

    2017-05-31

    Chemically synthesized small molecules play important role in anticancer therapy. Several chemical compounds have been reported to damage the DNA, either directly or indirectly slowing down the cancer cell progression by causing a cell cycle arrest. Direct or indirect reactive oxygen species formation causes DNA damage leading to cell cycle arrest and subsequent cell death. Therefore, identification of chemically synthesized compounds with anticancer potential is important. Here we investigate the effect of benzothiazole derivative (5g) for its ability to inhibit cell proliferation in different cancer models. Interestingly, 5g interfered with cell proliferation in both, cell lines and tumor cells leading to significant G2/M arrest. 5g treatment resulted in elevated levels of ROS and subsequently, DNA double-strand breaks (DSBs) explaining observed G2/M arrest. Consistently, we observed deregulation of many cell cycle associated proteins such as CDK1, BCL2 and their phosphorylated form, CyclinB1, CDC25c etc. Besides, 5g treatment led to decreased levels of mitochondrial membrane potential and activation of apoptosis. Interestingly, 5g administration inhibited tumor growth in mice without significant side effects. Thus, our study identifies 5g as a potent biochemical inhibitor to induce G2/M phase arrest of the cell cycle, and demonstrates its anticancer properties both ex vivo and in vivo.

  14. DNA damage in buccal mucosa cells of pre-school children exposed to high levels of urban air pollutants.

    Directory of Open Access Journals (Sweden)

    Elisabetta Ceretti

    Full Text Available Air pollution has been recognized as a human carcinogen. Children living in urban areas are a high-risk group, because genetic damage occurring early in life is considered able to increase the risk of carcinogenesis in adulthood. This study aimed to investigate micronuclei (MN frequency, as a biomarker of DNA damage, in exfoliated buccal cells of pre-school children living in a town with high levels of air pollution. A sample of healthy 3-6-year-old children living in Brescia, Northern Italy, was investigated. A sample of the children's buccal mucosa cells was collected during the winter months in 2012 and 2013. DNA damage was investigated using the MN test. Children's exposure to urban air pollution was evaluated by means of a questionnaire filled in by their parents that included items on various possible sources of indoor and outdoor pollution, and the concentration of fine particulate matter (PM10, PM2.5 and NO2 in the 1-3 weeks preceding biological sample collection. 181 children (mean age ± SD: 4.3 ± 0.9 years were investigated. The mean ± SD MN frequency was 0.29 ± 0.13%. A weak, though statistically significant, association of MN with concentration of air pollutants (PM10, PM2.5 and NO2 was found, whereas no association was apparent between MN frequency and the indoor and outdoor exposure variables investigated via the questionnaire. This study showed a high MN frequency in children living in a town with heavy air pollution in winter, higher than usually found among children living in areas with low or medium-high levels of air pollution.

  15. Butachlor induced dissipation of mitochondrial membrane potential, oxidative DNA damage and necrosis in human peripheral blood mononuclear cells

    International Nuclear Information System (INIS)

    Dwivedi, Sourabh; Saquib, Quaiser; Al-Khedhairy, Abdulaziz A.; Musarrat, Javed

    2012-01-01

    cytometry analysis of annexin V − /7-AAD + stained cells demonstrated substantial reduction in live population due to complete loss of cell membrane integrity. Overall the data suggested the formation of butachlor–DNA complex, as an initiating event in butachlor-induced DNA damage. The results elucidated the oxidative role of butachlor in intracellular ROS production, and consequent mitochondrial dysfunction, oxidative DNA damage, and chromosomal breakage, which eventually triggers necrosis in human PBMN cells.

  16. DNA damage response and evasion from immunosurveillance in CLL: New options for NK cell-based immunotherpies.

    Directory of Open Access Journals (Sweden)

    Olga M. Shatnyeva

    2015-02-01

    Full Text Available Chronic lymphocytic leukemia (CLL is the most prominent B cell malignancy among adults in the Western world and characterized by a clonal expansion of B cells. The patients suffer from severe immune defects resulting in increased susceptibility to infections and failure to generate an antitumor immune response. Defects in both, DNA damage response (DDR pathway and crosstalk with the tissue microenvironment have been reported to play a crucial role for the survival of CLL cells, therapy resistance and impaired immune response. To this end, major advances over the past years have highlighted several T cell immune evasion mechanisms in CLL. Here, we discuss the consequences of an impaired DDR pathway for detection and elimination of CLL cells by Natural killer (NK cells. NK cells are considered to be a major component of the immunosurveillance in leukemia but NK cell activity is impaired in CLL. Restoration of NK cell activity using immunoligands and immunoconstructs in combination with the conventional chemotherapy may provide a future perspective for CLL treatment.

  17. The studies of effects of 60Co γ-rays on DNA damage and repair in tumor cells

    International Nuclear Information System (INIS)

    Su Liaoyuan; Huang Hanxian; Cen Jiannong

    1997-06-01

    The effects of 60 Co γ-rays and its combination with hyperthermia on DNA strand breaks and their repair in L 5178y cells were studied using alkaline elution technique. When the cells were heated at 43 degree C for 30 min, there was obvious inhibition of repair of DNA damage caused by γ-irradiation. The hyperthermia before irradiation produced better inhibiting effect than that after irradiation. The effects of radiation on DNA strand breaks and its repair in HL-60 cells and HL-60 (VCR) cells were analyzed using technique of hydroxyapatite chromatography and the results sowed that the extent of DNA strand breaks in HL-60 cells and HL-60 (VCR) cells induced by irradiation was not markedly different, but the power of repair of strand breaks and the radioresistance of function of DNA synthesis in HL-60 (VCR) cells were higher than those in HL-60 cells. The difference was obvious. The results suggest that the heterogeneity of radiosensitivity of leukemia cells is correlated with its drug resistance. (10 refs., 3 tabs.)

  18. Beclin 1 and UVRAG confer protection from radiation-induced DNA damage and maintain centrosome stability in colorectal cancer cells.

    Directory of Open Access Journals (Sweden)

    Jae Myung Park

    Full Text Available Beclin 1 interacts with UV-irradiation-resistance-associated gene (UVRAG to form core complexes that induce autophagy. While cells with defective autophagy are prone to genomic instability that contributes to tumorigenesis, it is unknown whether Beclin1 or UVRAG can regulate the DNA damage/repair response to cancer treatment in established tumor cells. We found that siRNA knockdown of Beclin 1 or UVRAG can increase radiation-induced DNA double strand breaks (DSBs, shown by pATM and γH2Ax, and promote colorectal cancer cell death. Furthermore, knockdown of Beclin 1, UVRAG or ATG5 increased the percentage of irradiated cells with nuclear foci expressing 53BP1, a marker of nonhomologous end joining but not RAD51 (homologous recombination, compared to control siRNA. Beclin 1 siRNA was shown to attenuate UVRAG expression. Cells with a UVRAG deletion mutant defective in Beclin 1 binding showed increased radiation-induced DSBs and cell death compared to cells with ectopic wild-type UVRAG. Knockdown of Beclin 1 or UVRAG, but not ATG5, resulted in a significant increase in centrosome number (γ-tubulin staining in irradiated cells compared to control siRNA. Taken together, these data indicate that Beclin 1 and UVRAG confer protection against radiation-induced DNA DSBs and may maintain centrosome stability in established tumor cells.

  19. Silencing of poly(ADP-ribose) glycohydrolase sensitizes lung cancer cells to radiation through the abrogation of DNA damage checkpoint

    Energy Technology Data Exchange (ETDEWEB)

    Nakadate, Yusuke [Shien-Lab, National Cancer Center Hospital, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045 (Japan); Department of Bioengineering, Graduate School of Engineering, Osaka City University, 3-3-138 Sugimoto, Sumiyoshi-ku, Osaka 558-8585 (Japan); Kodera, Yasuo; Kitamura, Yuka [Shien-Lab, National Cancer Center Hospital, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045 (Japan); Tachibana, Taro [Department of Bioengineering, Graduate School of Engineering, Osaka City University, 3-3-138 Sugimoto, Sumiyoshi-ku, Osaka 558-8585 (Japan); Tamura, Tomohide [Division of Thoracic Oncology, National Cancer Center Hospital, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045 (Japan); Koizumi, Fumiaki, E-mail: fkoizumi@ncc.go.jp [Division of Thoracic Oncology, National Cancer Center Hospital, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045 (Japan)

    2013-11-29

    Highlights: •Radiosensitization by PARG silencing was observed in multiple lung cancer cells. •PAR accumulation was enhanced by PARG silencing after DNA damage. •Radiation-induced G2/M arrest and checkpoint activation were impaired by PARG siRNA. -- Abstract: Poly(ADP-ribose) glycohydrolase (PARG) is a major enzyme that plays a role in the degradation of poly(ADP-ribose) (PAR). PARG deficiency reportedly sensitizes cells to the effects of radiation. In lung cancer, however, it has not been fully elucidated. Here, we investigated whether PARG siRNA contributes to an increased radiosensitivity using 8 lung cancer cell lines. Among them, the silencing of PARG induced a radiosensitizing effect in 5 cell lines. Radiation-induced G2/M arrest was largely suppressed by PARG siRNA in PC-14 and A427 cells, which exhibited significantly enhanced radiosensitivity in response to PARG knockdown. On the other hand, a similar effect was not observed in H520 cells, which did not exhibit a radiosensitizing effect. Consistent with a cell cycle analysis, radiation-induced checkpoint signals were not well activated in the PC-14 and A427 cells when treated with PARG siRNA. These results suggest that the increased sensitivity to radiation induced by PARG knockdown occurs through the abrogation of radiation-induced G2/M arrest and checkpoint activation in lung cancer cells. Our findings indicate that PARG could be a potential target for lung cancer treatments when used in combination with radiotherapy.

  20. GADD45a Regulates Olaquindox-Induced DNA Damage and S-Phase Arrest in Human Hepatoma G2 Cells via JNK/p38 Pathways

    Directory of Open Access Journals (Sweden)

    Daowen Li

    2017-01-01

    Full Text Available Olaquindox, a quinoxaline 1,4-dioxide derivative, is widely used as a feed additive in many countries. The potential genotoxicity of olaquindox, hence, is of concern. However, the proper mechanism of toxicity was unclear. The aim of the present study was to investigate the effect of growth arrest and DNA damage 45 alpha (GADD45a on olaquindox-induced DNA damage and cell cycle arrest in HepG2 cells. The results showed that olaquindox could induce reactive oxygen species (ROS-mediated DNA damage and S-phase arrest, where increases of GADD45a, cyclin A, Cdk 2, p21 and p53 protein expression, decrease of cyclin D1 and the activation of phosphorylation-c-Jun N-terminal kinases (p-JNK, phosphorylation-p38 (p-p38 and phosphorylation-extracellular signal-regulated kinases (p-ERK were involved. However, GADD45a knockdown cells treated with olaquindox could significantly decrease cell viability, exacerbate DNA damage and increase S-phase arrest, associated with the marked activation of p-JNK, p-p38, but not p-ERK. Furthermore, SP600125 and SB203580 aggravated olaquindox-induced DNA damage and S-phase arrest, suppressed the expression of GADD45a. Taken together, these findings revealed that GADD45a played a protective role in olaquindox treatment and JNK/p38 pathways may partly contribute to GADD45a regulated olaquindox-induced DNA damage and S-phase arrest. Our findings increase the understanding on the molecular mechanisms of olaquindox.

  1. The potential value of the neutral comet assay and the expression of genes associated with DNA damage in assessing the radiosensitivity of tumor cells.

    Science.gov (United States)

    Jayakumar, Sundarraj; Bhilwade, Hari N; Pandey, Badri N; Sandur, Santosh K; Chaubey, Ramesh C

    2012-10-09

    The assessment of tumor radiosensitivity would be particularly useful in optimizing the radiation dose during radiotherapy. Therefore, the degree of correlation between radiation-induced DNA damage, as measured by the alkaline and the neutral comet assays, and the clonogenic survival of different human tumor cells was studied. Further, tumor radiosensitivity was compared with the expression of genes associated with the cellular response to radiation damage. Five different human tumor cell lines were chosen and the radiosensitivity of these cells was established by clonogenic assay. Alkaline and neutral comet assays were performed in γ-irradiated cells (2-8Gy; either acute or fractionated). Quantitative PCR was performed to evaluate the expression of DNA damage response genes in control and irradiated cells. The relative radiosensitivity of the cell lines assessed by the extent of DNA damage (neutral comet assay) immediately after irradiation (4Gy or 6Gy) was in agreement with radiosensitivity pattern obtained by the clonogenic assay. The survival fraction of irradiated cells showed a better correlation with the magnitude of DNA damage measured by the neutral comet assay (r=-0.9; Pcomet assay (r=-0.73; Pcomet assay was better than alkaline comet assay for assessment of radiosensitivities of tumor cells after acute or fractionated doses of irradiation. © 2012 Elsevier B.V. All rights reserved.

  2. Mobile phone specific electromagnetic fields induce transient DNA damage and nucleotide excision repair in serum-deprived human glioblastoma cells.

    Science.gov (United States)

    Al-Serori, Halh; Ferk, Franziska; Kundi, Michael; Bileck, Andrea; Gerner, Christopher; Mišík, Miroslav; Nersesyan, Armen; Waldherr, Monika; Murbach, Manuel; Lah, Tamara T; Herold-Mende, Christel; Collins, Andrew R; Knasmüller, Siegfried

    2018-01-01

    Some epidemiological studies indicate that the use of mobile phones causes cancer in humans (in particular glioblastomas). It is known that DNA damage plays a key role in malignant transformation; therefore, we investigated the impact of the UMTS signal which is widely used in mobile telecommunications, on DNA stability in ten different human cell lines (six brain derived cell lines, lymphocytes, fibroblasts, liver and buccal tissue derived cells) under conditions relevant for users (SAR 0.25 to 1.00 W/kg). We found no evidence for induction of damage in single cell gel electrophoresis assays when the cells were cultivated with serum. However, clear positive effects were seen in a p53 proficient glioblastoma line (U87) when the cells were grown under serum free conditions, while no effects were found in p53 deficient glioblastoma cells (U251). Further experiments showed that the damage disappears rapidly in U87 and that exposure induced nucleotide excision repair (NER) and does not cause double strand breaks (DSBs). The observation of NER induction is supported by results of a proteome analysis indicating that several proteins involved in NER are up-regulated after exposure to UMTS; additionally, we found limited evidence for the activation of the γ-interferon pathway. The present findings show that the signal causes transient genetic instability in glioma derived cells and activates cellular defense systems.

  3. N-Acetyl-L-cysteine protects thyroid cells against DNA damage induced by external and internal irradiation.

    Science.gov (United States)

    Kurashige, Tomomi; Shimamura, Mika; Nagayama, Yuji

    2017-11-01

    We evaluated the effect of the antioxidant N-acetyl-L-cysteine (NAC) on the levels of reactive oxygen species (ROS), DNA double strand breaks (DSB) and micronuclei (MN) induced by internal and external irradiation using a rat thyroid cell line PCCL3. In internal irradiation experiments, ROS and DSB levels increased immediately after 131 I addition and then gradually declined, resulting in very high levels of MN at 24 and 48 h. NAC administration both pre- and also post- 131 I addition suppressed ROS, DSB and MN. In external irradiation experiments with a low dose (0.5 Gy), ROS and DSB increased shortly and could be prevented by NAC administration pre-, but not post-irradiation. In contrast, external irradiation with a high dose (5 Gy) increased ROS and DSB in a bimodal way: ROS and DSB levels increased immediately after irradiation, quickly returned to the basal levels and gradually rose again after >24 h. The second phase was in parallel with an increase in 4-hydroxy-2-nonenal. The number of MN induced by the second wave of ROS/DSB elevations was much higher than that by the first peak. In this situation, NAC administered pre- and post-irradiation comparably suppressed MN induced by a delayed ROS elevation. In conclusion, a prolonged ROS increase during internal irradiation and a delayed ROS increase after external irradiation with a high dose caused serious DNA damage, which were efficiently prevented by NAC. Thus, NAC administration even both after internal or external irradiation prevents ROS increase and eventual DNA damage.

  4. Typical signature of DNA damage in white blood cells: a pilot study on etheno adducts in Danish mother-newborn child pairs

    DEFF Research Database (Denmark)

    Arab, K; Pedersen, Marie; Nair, J

    2009-01-01

    The impact of DNA damage commonly thought to be involved in chronic degenerative disease causation is particularly detrimental during fetal development. Within a multicenter study, we analyzed 77 white blood cell (WBC) samples from mother-newborn child pairs to see if imprinting of DNA damage...... in mother and newborn shows a similar pattern. Two adducts 1,N(6)-ethenodeoxyadenosine (epsilondA) and 3,N(4)-ethenodeoxycytidine (epsilondC) were measured by our ultrasensitive immunoaffinity (32)P-post-labeling method. These miscoding etheno-DNA adducts are generated by the reaction of lipid peroxidation...... arising from endogenous reactive aldehydes in WBC of both mother and newborn can be reliably assessed by epsilondA and epsilondC as biomarkers. The high correlation of etheno adduct levels in mother and child WBC suggests that a typical signature of DNA damage is induced similarly in fetus and mother...

  5. Reactive oxygen species mediate soft corals-derived sinuleptolide-induced antiproliferation and DNA damage in oral cancer cells

    Directory of Open Access Journals (Sweden)

    Chang YT

    2017-07-01

    ROS, mitochondrial superoxide, apoptosis, and DNA damage of oral cancer cells (Ca9-22. After sinuleptolide treatment, antiproliferation, apoptosis-like morphology, ROS/mitochondrial superoxide generation, annexin V-based apoptosis, and γH2AX-based DNA damage were induced. All these changes were blocked by NAC pretreatment at 4 mM for 1 h. This showed that the cell-killing mechanism of oral cancer cells of sinuleptolide is ROS dependent. Keywords: soft corals, oral cancer, N-acetylcysteine, oxidative stress, γH2AX 

  6. Keratin23 (KRT23 knockdown decreases proliferation and affects the DNA damage response of colon cancer cells.

    Directory of Open Access Journals (Sweden)

    Karin Birkenkamp-Demtröder

    Full Text Available Keratin 23 (KRT23 is strongly expressed in colon adenocarcinomas but absent in normal colon mucosa. Array based methylation profiling of 40 colon samples showed that the promoter of KRT23 was methylated in normal colon mucosa, while hypomethylated in most adenocarcinomas. Promoter methylation correlated with absent expression, while increased KRT23 expression in tumor samples correlated with promoter hypomethylation, as confirmed by bisulfite sequencing. Demethylation induced KRT23 expression in vitro. Expression profiling of shRNA mediated stable KRT23 knockdown in colon cancer cell lines showed that KRT23 depletion affected molecules of the cell cycle and DNA replication, recombination and repair. In vitro analyses confirmed that KRT23 depletion significantly decreased the cellular proliferation of SW948 and LS1034 cells and markedly decreased the expression of genes involved in DNA damage response, mainly molecules of the double strand break repair homologous recombination pathway. KRT23 knockdown decreased the transcript and protein expression of key molecules as e.g. MRE11A, E2F1, RAD51 and BRCA1. Knockdown of KRT23 rendered colon cancer cells more sensitive to irradiation and reduced proliferation of the KRT23 depleted cells compared to irradiated control cells.

  7. Effects of highly ripened cheeses on HL-60 human leukemia cells: antiproliferative activity and induction of apoptotic DNA damage.

    Science.gov (United States)

    Yasuda, S; Ohkura, N; Suzuki, K; Yamasaki, M; Nishiyama, K; Kobayashi, H; Hoshi, Y; Kadooka, Y; Igoshi, K

    2010-04-01

    To establish cheese as a dairy product with health benefits, we examined the multifunctional role of cheeses. In this report, we clarify whether different types of commercial cheeses may possess antiproliferative activity using HL-60 human promyelocytic leukemia cell lines as a cancer model. Among 12 cheese extracts tested, 6 (Montagnard, Pont-l'Eveque, Brie, Camembert, Danablue, and Blue) revealed strong growth inhibition activity and induction of DNA fragmentation in HL-60 cells. Based on the quantification of nitrogen contents in different cheese samples, a positive correlation between the ripeness of various cheeses and their antiproliferative activity tested in HL-60 cells was displayed. Four varieties of Blue cheese ripened for 0, 1, 2, or 3 mo demonstrated that the Blue cheese ripened for a long term was capable of causing the strong suppression of the cell growth and the induction of apoptotic DNA damage as well as nucleic morphological change in HL-60 cells. Collectively, these results obtained suggest a potential role of highly ripened cheeses in the prevention of leukemic cell proliferation. Copyright (c) 2010 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

  8. Reduction of arsenite-enhanced ultraviolet radiation-induced DNA damage by supplemental zinc

    Energy Technology Data Exchange (ETDEWEB)

    Cooper, Karen L.; King, Brenee S.; Sandoval, Monica M.; Liu, Ke Jian; Hudson, Laurie G., E-mail: lhudson@salud.unm.edu

    2013-06-01

    Arsenic is a recognized human carcinogen and there is evidence that arsenic augments the carcinogenicity of DNA damaging agents such as ultraviolet radiation (UVR) thereby acting as a co-carcinogen. Inhibition of DNA repair is one proposed mechanism to account for the co-carcinogenic actions of arsenic. We and others find that arsenite interferes with the function of certain zinc finger DNA repair proteins. Furthermore, we reported that zinc reverses the effects of arsenite in cultured cells and a DNA repair target protein, poly (ADP-ribose) polymerase-1. In order to determine whether zinc ameliorates the effects of arsenite on UVR-induced DNA damage in human keratinocytes and in an in vivo model, normal human epidermal keratinocytes and SKH-1 hairless mice were exposed to arsenite, zinc or both before solar-simulated (ss) UVR exposure. Poly (ADP-ribose) polymerase activity, DNA damage and mutation frequencies at the Hprt locus were measured in each treatment group in normal human keratinocytes. DNA damage was assessed in vivo by immunohistochemical staining of skin sections isolated from SKH-1 hairless mice. Cell-based findings demonstrate that ssUVR-induced DNA damage and mutagenesis are enhanced by arsenite, and supplemental zinc partially reverses the arsenite effect. In vivo studies confirm that zinc supplementation decreases arsenite-enhanced DNA damage in response to ssUVR exposure. From these data we can conclude that zinc offsets the impact of arsenic on ssUVR-stimulated DNA damage in cells and in vivo suggesting that zinc supplementation may provide a strategy to improve DNA repair capacity in arsenic exposed human populations. - Highlights: • Low levels of arsenite enhance UV-induced DNA damage in human keratinocytes. • UV-initiated HPRT mutation frequency is enhanced by arsenite. • Zinc supplementation offsets DNA damage and mutation frequency enhanced by arsenite. • Zinc-dependent reduction of arsenite enhanced DNA damage is confirmed in vivo.

  9. Progress on clustered DNA damage in radiation research

    International Nuclear Information System (INIS)

    Yang Li'na; Zhang Hong; Di Cuixia; Zhang Qiuning; Wang Xiaohu

    2012-01-01

    Clustered DNA damage which caused by high LET heavy ion radiation can lead to mutation, tumorigenesis and apoptosis. Promoting apoptosis of cancer cells is always the basis of cancer treatment. Clustered DNA damage has been the hot topic in radiobiology. The detect method is diversity, but there is not a detail and complete protocol to analyze clustered DNA damage. In order to provide reference for clustered DNA damage in the radiotherapy study, the clustered DNA damage characteristics, the latest progresses on clustered DNA damage and the detecting methods are reviewed and discussed in detail in this paper. (authors)

  10. Important role of the nucleotide excision repair pathway in Mycobacterium smegmatis in conferring protection against commonly encountered DNA-damaging agents.

    Science.gov (United States)

    Kurthkoti, Krishna; Kumar, Pradeep; Jain, Ruchi; Varshney, Umesh

    2008-09-01

    Mycobacteria are an important group of human pathogens. Although the DNA repair mechanisms in mycobacteria are not well understood, these are vital for the pathogen's persistence in the host macrophages. In this study, we generated a null mutation in the uvrB gene of Mycobacterium smegmatis to allow us to compare the significance of the nucleotide excision repair (NER) pathway with two important base excision repair pathways, initiated by uracil DNA glycosylase (Ung) and formamidopyrimidine DNA glycosylase (Fpg or MutM), in an isogenic strain background. The strain deficient in NER was the most sensitive to commonly encountered DNA-damaging agents such as UV, low pH, reactive oxygen species, hypoxia, and was also sensitive to acidified nitrite. Taken together with previous observations on NER-deficient M. tuberculosis, these results suggest that NER is an important DNA repair pathway in mycobacteria.

  11. NSC666715 and Its Analogs Inhibit Strand-Displacement Activity of DNA Polymerase β and Potentiate Temozolomide-Induced DNA Damage, Senescence and Apoptosis in Colorectal Cancer Cells.

    Directory of Open Access Journals (Sweden)

    Aruna S Jaiswal

    Full Text Available Recently approved chemotherapeutic agents to treat colorectal cancer (CRC have made some impact; however, there is an urgent need for newer targeted agents and strategies to circumvent CRC growth and metastasis. CRC frequently exhibits natural resistance to chemotherapy and those who do respond initially later acquire drug resistance. A mechanism to potentially sensitize CRC cells is by blocking the DNA polymerase β (Pol-β activity. Temozolomide (TMZ, an alkylating agent, and other DNA-interacting agents exert DNA damage primarily repaired by a Pol-β-directed base excision repair (BER pathway. In previous studies, we used structure-based molecular docking of Pol-β and identified a potent small molecule inhibitor (NSC666715. In the present study, we have determined the mechanism by which NSC666715 and its analogs block Fen1-induced strand-displacement activity of Pol-β-directed LP-BER, cause apurinic/apyrimidinic (AP site accumulation and induce S-phase cell cycle arrest. Induction of S-phase cell cycle arrest leads to senescence and apoptosis of CRC cells through the p53/p21 pathway. Our initial findings also show a 10-fold reduction of the IC50 of TMZ when combined with NSC666715. These results provide a guide for the development of a target-defined strategy for CRC chemotherapy that will be based on the mechanisms of action of NSC666715 and TMZ. This combination strategy can be used as a framework to further reduce the TMZ dosages and resistance in CRC patients.

  12. The cAMP signaling system inhibits the repair of {gamma}-ray-induced DNA damage by promoting Epac1-mediated proteasomal degradation of XRCC1 protein in human lung cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Cho, Eun-Ah [Department of Biochemistry and Molecular Biology, Cancer Research Center, Seoul National University College of Medicine, Seoul 110-799 (Korea, Republic of); Juhnn, Yong-Sung, E-mail: juhnn@snu.ac.kr [Department of Biochemistry and Molecular Biology, Cancer Research Center, Seoul National University College of Medicine, Seoul 110-799 (Korea, Republic of)

    2012-06-01

    Highlights: Black-Right-Pointing-Pointer cAMP signaling system inhibits repair of {gamma}-ray-induced DNA damage. Black-Right-Pointing-Pointer cAMP signaling system inhibits DNA damage repair by decreasing XRCC1 expression. Black-Right-Pointing-Pointer cAMP signaling system decreases XRCC1 expression by promoting its proteasomal degradation. Black-Right-Pointing-Pointer The promotion of XRCC1 degradation by cAMP signaling system is mediated by Epac1. -- Abstract: Cyclic AMP is involved in the regulation of metabolism, gene expression, cellular growth and proliferation. Recently, the cAMP signaling system was found to modulate DNA-damaging agent-induced apoptosis by regulating the expression of Bcl-2 family proteins and inhibitors of apoptosis. Thus, we hypothesized that the cAMP signaling may modulate DNA repair activity, and we investigated the effects of the cAMP signaling system on {gamma}-ray-induced DNA damage repair in lung cancer cells. Transient expression of a constitutively active mutant of stimulatory G protein (G{alpha}sQL) or treatment with forskolin, an adenylyl cyclase activator, augmented radiation-induced DNA damage and inhibited repair of the damage in H1299 lung cancer cells. Expression of G{alpha}sQL or treatment with forskolin or isoproterenol inhibited the radiation-induced expression of the XRCC1 protein, and exogenous expression of XRCC1 abolished the DNA repair-inhibiting effect of forskolin. Forskolin treatment promoted the ubiquitin and proteasome-dependent degradation of the XRCC1 protein, resulting in a significant decrease in the half-life of the protein after {gamma}-ray irradiation. The effect of forskolin on XRCC1 expression was not inhibited by PKA inhibitor, but 8-pCPT-2 Prime -O-Me-cAMP, an Epac-selective cAMP analog, increased ubiquitination of XRCC1 protein and decreased XRCC1 expression. Knockdown of Epac1 abolished the effect of 8-pCPT-2 Prime -O-Me-cAMP and restored XRCC1 protein level following {gamma}-ray irradiation. From

  13. Surface modification of amorphous nanosilica particles suppresses nanosilica-induced cytotoxicity, ROS generation, and DNA damage in various mammalian cells

    International Nuclear Information System (INIS)

    Yoshida, Tokuyuki; Yoshioka, Yasuo; Matsuyama, Keigo; Nakazato, Yasutaro; Tochigi, Saeko; Hirai, Toshiro; Kondoh, Sayuri; Nagano, Kazuya; Abe, Yasuhiro; Kamada, Haruhiko; Tsunoda, Shin-ichi; Nabeshi, Hiromi; Yoshikawa, Tomoaki; Tsutsumi, Yasuo

    2012-01-01

    Highlights: ► There is increasing concern regarding the potential health risks of nanomaterials. ► We evaluated the effect of surface properties of nanomaterials on cellular responses. ► We showed that the surface properties play an important in determining its safety. ► These data provide useful information for producing safer nanomaterials. -- Abstract: Recently, nanomaterials have been utilized in various fields. In particular, amorphous nanosilica particles are increasingly being used in a range of applications, including cosmetics, food technology, and medical diagnostics. However, there is concern that the unique characteristics of nanomaterials might induce undesirable effects. The roles played by the physical characteristics of nanomaterials in cellular responses have not yet been elucidated precisely. Here, by using nanosilica particles (nSPs) with a diameter of 70 nm whose surface was either unmodified (nSP70) or modified with amine (nSP70-N) or carboxyl groups (nSP70-C), we examined the relationship between the surface properties of nSPs and cellular responses such as cytotoxicity, reactive oxygen species (ROS) generation, and DNA damage. To compare the cytotoxicity of nSP70, nSP70-N, or nSP70-C, we examined in vitro cell viability after nSP treatment. Although the susceptibility of each cell line to the nSPs was different, nSP70-C and nSP70-N showed lower cytotoxicity than nSP70 in all cell lines. Furthermore, the generation of ROS and induction of DNA damage in nSP70-C- and nSP70-N-treated cells were lower than those in nSP70-treated cells. These results suggest that the surface properties of nSP70 play an important role in determining its safety, and surface modification of nSP70 with amine or carboxyl groups may be useful for the development of safer nSPs. We hope that our results will contribute to the development of safer nanomaterials.

  14. Systems biology approach identifies the kinase Csnk1a1 as a regulator of the DNA damage response in embryonic stem cells.

    Science.gov (United States)

    Carreras Puigvert, Jordi; von Stechow, Louise; Siddappa, Ramakrishnaiah; Pines, Alex; Bahjat, Mahnoush; Haazen, Lizette C J M; Olsen, Jesper V; Vrieling, Harry; Meerman, John H N; Mullenders, Leon H F; van de Water, Bob; Danen, Erik H J

    2013-01-22

    In pluripotent stem cells, DNA damage triggers loss of pluripotency and apoptosis as a safeguard to exclude damaged DNA from the lineage. An intricate DNA damage response (DDR) signaling network ensures that the response is proportional to the severity of the damage. We combined an RNA interference screen targeting all kinases, phosphatases, and transcription factors with global transcriptomics and phosphoproteomics to map the DDR in mouse embryonic stem cells treated with the DNA cross-linker cisplatin. Networks derived from canonical pathways shared in all three data sets were implicated in DNA damage repair, cell cycle and survival, and differentiation. Experimental probing of these networks identified a mode of DNA damage-induced Wnt signaling that limited apoptosis. Silencing or deleting the p53 gene demonstrated that genotoxic stress elicited Wnt signaling in a p53-independent manner. Instead, this response occurred through reduced abundance of Csnk1a1 (CK1α), a kinase that inhibits β-catenin. Together, our findings reveal a balance between p53-mediated elimination of stem cells (through loss of pluripotency and apoptosis) and Wnt signaling that attenuates this response to tune the outcome of the DDR.

  15. APTO-253 Stabilizes G-quadruplex DNA, Inhibits MYC Expression, and Induces DNA Damage in Acute Myeloid Leukemia Cells.

    Science.gov (United States)

    Local, Andrea; Zhang, Hongying; Benbatoul, Khalid D; Folger, Peter; Sheng, Xia; Tsai, Cheng-Yu; Howell, Stephen B; Rice, William G

    2018-06-01

    APTO-253 is a phase I clinical stage small molecule that selectively induces CDKN1A (p21), promotes G 0 -G 1 cell-cycle arrest, and triggers apoptosis in acute myeloid leukemia (AML) cells without producing myelosuppression in various animal species and humans. Differential gene expression analysis identified a pharmacodynamic effect on MYC expression, as well as induction of DNA repair and stress response pathways. APTO-253 was found to elicit a concentration- and time-dependent reduction in MYC mRNA expression and protein levels. Gene ontogeny and structural informatic analyses suggested a mechanism involving G-quadruplex (G4) stabilization. Intracellular pharmacokinetic studies in AML cells revealed that APTO-253 is converted intracellularly from a monomer to a ferrous complex [Fe(253) 3 ]. FRET assays demonstrated that both monomeric APTO-253 and Fe(253) 3 stabilize G4 structures from telomeres, MYC, and KIT promoters but do not bind to non-G4 double-stranded DNA. Although APTO-253 exerts a host of mechanistic sequelae, the effect of APTO-253 on MYC expression and its downstream target genes, on cell-cycle arrest, DNA damage, and stress responses can be explained by the action of Fe(253) 3 and APTO-253 on G-quadruplex DNA motifs. Mol Cancer Ther; 17(6); 1177-86. ©2018 AACR . ©2018 American Association for Cancer Research.

  16. Fibroblasts Cultured on Nanowires Exhibit Low Motility, Impaired Cell Division, and DNA Damage

    DEFF Research Database (Denmark)

    Persson, H.; Købler, Carsten; Mølhave, Kristian

    2013-01-01

    Mouse fibroblasts cultured on 7-μm-long vertical nanowires are reported on page 4006 by C. N. Prinz and co-workers. Culturing cells on this kind of substrate interferes greatly with cell function, causing the cells to develop into widely different morphologies. The cells' division is impaired...

  17. Evaluation of radio-induced DNA damage and their repair in human lymphocytes by comet assay or single cell gel electrophoresis

    International Nuclear Information System (INIS)

    Nascimento, Patricia A. do; Suzuki, Miriam F.; Okazaki, Kayo

    1997-01-01

    The comet assay, also called single cell gel electrophoresis technique, permits to evaluate quantitatively DNA breakage induced by chemical and physical agents at the level of the single cell. The present paper refers to the construction of dose-response curves to DNA damage and repair studies in human peripheral lymphocytes, utilizing the comet assay for the radiosensitivity analysis. So, the blood samples were obtained from healthy donors (40-50 year old), irradiated in a 60 Co source (GAMMACEL 220) with doses of 0.17, 0.25, 0.57, 1.10, 2.12 and 4.22 Gy (0.59 Gy/min.) and processed 1 and 24 hours after the exposition. Results obtained showed a increase in the total lenght of comet (DNA migration) as a function of radiation dose in samples processed 1 and 24 hours after the treatment. The DNA lesion in irradiated lymphocytes with 4.22 Gy (means value of 101.4 μm) were 3.4 times higher than in the untreated lymphocytes (mean value of 30 μm) instead of 24 hours after the irradiation were 1.5 times higher (mean value of 46.3 μm). This reduction on DNA repair occurred in these cells. It was also possible visualized the presence of subpopulations of the cells with different sensitivity and repair capacity to ionizing radiation in these donors. (author). 8 refs., 3 figs

  18. Involvement of DNA-PK and ATM in radiation- and heat-induced DNA damage recognition and apoptotic cell death

    International Nuclear Information System (INIS)

    Tomita, Masanori

    2010-01-01

    Exposure to ionizing radiation and hyperthermia results in important biological consequences, e.g. cell death, chromosomal aberrations, mutations, and DNA strand breaks. There is good evidence that the nucleus, specifically cellular DNA, is the principal target for radiation-induced cell lethality. DNA double-strand breaks (DSBs) are considered to be the most serious type of DNA damage induced by ionizing radiation. On the other hand, verifiable mechanisms which can lead to heat-induced cell death are damage to the plasma membrane and/or inactivation of heat-labile proteins caused by protein denaturation and subsequent aggregation. Recently, several reports have suggested that DSBs can be induced after hyperthermia because heat-induced phosphorylated histone H2AX (γ-H2AX) foci formation can be observed in several mammalian cell lines. In mammalian cells, DSBs are repaired primarily through two distinct and complementary mechanisms: non-homologous end joining (NHEJ), and homologous recombination (HR) or homology-directed repair (HDR). DNA-dependent protein kinase (DNA-PK) and ataxia-telangiectasia mutated (ATM) are key players in the initiation of DSB repair and phosphorylate and/or activate many substrates, including themselves. These phosphorylated substrates have important roles in the functioning of cell cycle checkpoints and in cell death, as well as in DSB repair. Apoptotic cell death is a crucial cell suicide mechanism during development and in the defense of homeostasis. If DSBs are unrepaired or misrepaired, apoptosis is a very important system which can protect an organism against carcinogenesis. This paper reviews recently obtained results and current topics concerning the role of DNA-PK and ATM in heat- or radiation-induced apoptotic cell death. (author)

  19. Cobalt oxide nanoparticles aggravate DNA damage and cell death in eggplant via mitochondrial swelling and NO signaling pathway.

    Science.gov (United States)

    Faisal, Mohammad; Saquib, Quaiser; Alatar, Abdulrahman A; Al-Khedhairy, Abdulaziz A; Ahmed, Mukhtar; Ansari, Sabiha M; Alwathnani, Hend A; Dwivedi, Sourabh; Musarrat, Javed; Praveen, Shelly

    2016-03-18

    Despite manifold benefits of nanoparticles (NPs), less information on the risks of NPs to human health and environment has been studied. Cobalt oxide nanoparticles (Co3O4-NPs) have been reported to cause toxicity in several organisms. In this study, we have investigated the role of Co3O4-NPs in inducing phytotoxicity, cellular DNA damage and apoptosis in eggplant (Solanum melongena L. cv. Violetta lunga 2). To the best of our knowledge, this is the first report on Co3O4-NPs showing phytotoxicity in eggplant. The data revealed that eggplant seeds treated with Co3O4-NPs for 2 h at a concentration of 1.0 mg/ml retarded root length by 81.5 % upon 7 days incubation in a moist chamber. Ultrastructural analysis by transmission electron microscopy (TEM) demonstrated the uptake and translocation of Co3O4-NPs into the cytoplasm. Intracellular presence of Co3O4-NPs triggered subcellular changes such as degeneration of mitochondrial cristae, abundance of peroxisomes and excessive vacuolization. Flow cytometric analysis of Co3O4-NPs (1.0 mg/ml) treated root protoplasts revealed 157, 282 and 178 % increase in reactive oxygen species (ROS), membrane potential (ΔΨm) and nitric oxide (NO), respectively. Besides, the esterase activity in treated protoplasts was also found compromised. About 2.4-fold greater level of DNA damage, as compared to untreated control was observed in Comet assay, and 73.2 % of Co3O4-NPs treated cells appeared apoptotic in flow cytometry based cell cycle analysis. This study demonstrate the phytotoxic potential of Co3O4-NPs in terms of reduction in seed germination, root growth, greater level of DNA and mitochondrial damage, oxidative stress and cell death in eggplant. The data generated from this study will provide a strong background to draw attention on Co3O4-NPs environmental hazards to vegetable crops.

  20. Characterization of coal fly ash nanoparticles and induced oxidative DNA damage in human peripheral blood mononuclear cells

    International Nuclear Information System (INIS)

    Dwivedi, Sourabh; Saquib, Quaiser; Al-Khedhairy, Abdulaziz A.; Ali, Al-Yousef Sulaiman; Musarrat, Javed

    2012-01-01

    The nano-sized particles present in coal fly ash (CFA) were characterized through the X-ray diffraction (XRD), transmission and scanning electron microscopy (TEM, SEM), atomic force microscopy (AFM) and Fourier transform infrared spectroscopy (FTIR) analyses. The XRD data revealed the average crystallite size of the CFA nanoparticles (CFA-NPs) as 14 nm. TEM and SEM imaging demonstrated predominantly spherical and some polymorphic structures in the size range of 11 to 25 nm. The amount of heavy metal associated with CFA particles (μg/g) were determined as Fe (34160.0 ± 1.38), Ni (150.8 ± 0.78), Cu (99.3 ± 0.56) and Cr (64.0 ± 0.86). However, the bioavailability of heavy metals in terms of percent release was in the order as Cr > Ni > Cu > Fe in CFA-dimethyl sulfoxide (DMSO) extract. The comet and cytokinesis blocked micronucleus (CBMN) assays revealed substantial genomic DNA damage in peripheral blood mononuclear (PBMN) cells treated with CFA-NPs in Aq and DMSO extracts. About 1.8 and 3.6 strand breaks per unit of DNA were estimated through alkaline unwinding assay at 1:100 DNA nucleotide/CFA ppm ratios with the Aq and DMSO extracts, respectively. The DNA and mitochondrial damage was invariably greater with CFA-DMSO extract vis-à-vis -Aq extract. Generation of superoxide anions (O 2 • − ) and intracellular reactive oxygen species (ROS) through metal redox-cycling, alteration in mitochondrial potential and 8-oxodG production elucidated CFA-NPs induced oxidative stress as a plausible mechanism for CFA-induced genotoxicity. -- Highlights: ► CFA consists of spherical crystalline nanoparticles in size range of 11–25 nm. ► Alkaline unwinding assay revealed single-strandedness in CFA treated ctDNA. ► CFA nanoparticles exhibited the ability to induce ROS and oxidative DNA damage. ► Comet and CBMN assays revealed DNA and chromosomal breakage in PBMN cells. ► CFA-NPs resulted in mitochondrial membrane damage in PBMN cells.

  1. Characterization of coal fly ash nanoparticles and induced oxidative DNA damage in human peripheral blood mononuclear cells

    Energy Technology Data Exchange (ETDEWEB)

    Dwivedi, Sourabh; Saquib, Quaiser; Al-Khedhairy, Abdulaziz A. [Department of Zoology, College of Science, King Saud University, P.O. Box 2455, Riyadh 11451 (Saudi Arabia); Ali, Al-Yousef Sulaiman [Department of Medical Laboratory Sciences, College of Applied Medical Science, University of Dammam, P.O. Box 1683, Hafr Al Batin-31991 (Saudi Arabia); Musarrat, Javed, E-mail: musarratj1@yahoo.com [Department of Zoology, College of Science, King Saud University, P.O. Box 2455, Riyadh 11451 (Saudi Arabia); Department of Agricultural Microbiology, Faculty of Agricultural Sciences, AMU, Aligarh202002 (India)

    2012-10-15

    The nano-sized particles present in coal fly ash (CFA) were characterized through the X-ray diffraction (XRD), transmission and scanning electron microscopy (TEM, SEM), atomic force microscopy (AFM) and Fourier transform infrared spectroscopy (FTIR) analyses. The XRD data revealed the average crystallite size of the CFA nanoparticles (CFA-NPs) as 14 nm. TEM and SEM imaging demonstrated predominantly spherical and some polymorphic structures in the size range of 11 to 25 nm. The amount of heavy metal associated with CFA particles ({mu}g/g) were determined as Fe (34160.0 {+-} 1.38), Ni (150.8 {+-} 0.78), Cu (99.3 {+-} 0.56) and Cr (64.0 {+-} 0.86). However, the bioavailability of heavy metals in terms of percent release was in the order as Cr > Ni > Cu > Fe in CFA-dimethyl sulfoxide (DMSO) extract. The comet and cytokinesis blocked micronucleus (CBMN) assays revealed substantial genomic DNA damage in peripheral blood mononuclear (PBMN) cells treated with CFA-NPs in Aq and DMSO extracts. About 1.8 and 3.6 strand breaks per unit of DNA were estimated through alkaline unwinding assay at 1:100 DNA nucleotide/CFA ppm ratios with the Aq and DMSO extracts, respectively. The DNA and mitochondrial damage was invariably greater with CFA-DMSO extract vis-a-vis -Aq extract. Generation of superoxide anions (O{sub 2} Bullet {sup -}) and intracellular reactive oxygen species (ROS) through metal redox-cycling, alteration in mitochondrial potential and 8-oxodG production elucidated CFA-NPs induced oxidative stress as a plausible mechanism for CFA-induced genotoxicity. -- Highlights: Black-Right-Pointing-Pointer CFA consists of spherical crystalline nanoparticles in size range of 11-25 nm. Black-Right-Pointing-Pointer Alkaline unwinding assay revealed single-strandedness in CFA treated ctDNA. Black-Right-Pointing-Pointer CFA nanoparticles exhibited the ability to induce ROS and oxidative DNA damage. Black-Right-Pointing-Pointer Comet and CBMN assays revealed DNA and chromosomal

  2. Critical periods during the in situ repair of radiation-induced DNA damage in rat cerebellar neurons and 9L brain tumor cells

    International Nuclear Information System (INIS)

    Wierowski, J.V.; Thomas, R.R.; Ritter, P.; Wheeler, K.T.

    1982-01-01

    The consequences of delivering a second 1250-rad dose at various times during and after the repair of DNA damage produced by an initial 1250-rad dose were assessed in intracerebral 9L tumor cells and rat cerebellar neurons by measuring the sedimentation properties of their DNA through alkaline sucrose gradients in zonal rotors with slow gradient reorienting capabilities.In cerebellar neurons, separating the two doses by 15 min resulted in an accumulation of DNA damage as expressed by an increase in the amount of DNA sedimenting >250 S over that obtained from unirradiated controls. Although not statistically different from unirradiated controls, a slight increase in the amount of fast-sedimenting neuronal DNA also occurred when a 1-hr interval between the two doses was investigated. At intervals of 2 hr or more, no such increase in fast-sedimenting neuronal DNA was observed. None of the periods between doses resulted in an accumulation of DNA damage in intracerebral 9L tumor cells. The accumulation of this type of DNA damage in neurons but not in tumor cells suggests that avoidance of a critical period in neuronal DNA repair may someday be an important concept in the design of brain tumor therapy schedules

  3. F-box protein FBXO31 is a dedicated checkpoint protein to facilitate cell cycle arrest through activation of regulators in radiation induced DNA damage

    International Nuclear Information System (INIS)

    Santra, Manas Kumar

    2017-01-01

    In response to radiation-induced DNA damage, eukaryotic cells initiate a complex signalling pathway, termed the DNA damage response (DDR), which coordinates cell cycle arrest with DNA repair. Previous study showed that induction of G1 arrest in response to radiation induced DNA damage is minimally a two-step process: a fast p53-independent initiation of G1 arrest mediated by cyclin D1 proteolysis and a slower maintenance of arrest resulting from increased p53 stability. We elucidated the molecular mechanism of slow and fast response of radiation induced DDR. We showed that FBXO31, a member of F-box family proteins, plays important role in DDR induced by ionizing radiation. We show that FBXO31 is responsible for promoting MDM2 degradation following radiation. FBXO31 interacts with and directs the degradation of MDM2 in ATM dependent phosphorylation of MDM2. FBXO31-mediated loss of MDM2 leads to elevated levels of p53, resulting in growth arrest. In cells depleted of FBXO31, MDM2 is not degraded and p53 levels do not increase following genotoxic stress. Thus, FBXO31 is essential for the classic robust increase in p53 levels following DNA damage

  4. Reactive oxygen species-mediated DNA damage and apoptosis in human skin epidermal cells after exposure to nickel nanoparticles.

    Science.gov (United States)

    Alarifi, Saud; Ali, Daoud; Alakhtani, Saad; Al Suhaibani, Entissar S; Al-Qahtani, Ahmed A

    2014-01-01

    Nickel nanoparticles (NiNPs) are increasingly used in various applications due to their unique properties. However, there is little information concerning the toxicity of NiNPs in the human skin cell (A431). The present study was designed to investigate the cytotoxicity, apoptosis, and DNA damage due to NiNPs in A431 cells. A cellular proliferative capacity test showed that NiNPs induce significant cytotoxicity in a dose- and time-dependent manner. NiNPs were also found to induce oxidative stress evidenced by the generation of reactive oxygen species (ROS) and depletion of glutathione (GSH). Further, co-treatment with the antioxidant N-acetylcysteine (NAC) mitigated the ROS generation due to NiNPs, suggesting the potential mechanism of oxidative stress. NiNPs also induced significant elevation of lipid peroxidation, catalase, and superoxide dismutase and caspase-3 activity in A431 cells. In addition, NAC suppressed NiNP-induced caspase-3 activity. DNA fragmentation analysis using the comet assay showed that the NiNPs cause genotoxicity in a dose- and time-dependent manner. Therefore, the study points out the capability of the NiNPs to induce oxidative stress resulting in apoptosis and genotoxicity. This study warrants more careful assessment of NiNPs before their industrial applications.

  5. DNA damage response and role of shelterin complex in human peripheral blood mononuclear cells exposed to gamma radiation

    International Nuclear Information System (INIS)

    Saini, Divyalakshmi; Das, Birajalaxmi

    2013-01-01

    Telomeres are the DNA protein structures that cap the ends of linear DNA. It consists of short repetitive DNA sequences (TTAGGG)n and specialized telomere binding proteins. There are six telomeric proteins (TRF1, TRF2, TIN2, TERF2, PTOP and POT1) called as shelterin complex/telosome which maintains telomere integrity. The function of this 'telosome' is to protect the natural ends of the chromosomes from being recognized as artificial DNA breaks, thereby preventing chromosome end-to-end fusions. DNA Damage Response (DDR) induced by radiation and its interaction with telomeric protein complex is poorly understood in human PBMCs at G 0 stage. Alterations in either telomeric DNA or telomere binding proteins can impair the function of the telosome, which may lead to senescence or apoptosis. Ionizing radiation which induces a plethora of DNA lesions in human cell may also alter the expression of telomere associated proteins. In the present study, we have made an attempt to study the DNA damage response of telomere proteins in human peripheral blood mononuclear cells exposed to gamma radiation. Venous blood samples were collected from eight random healthy volunteers and PBMCs were separated. Dose response as well as time point kinetics study was carried out at transcription as well as protein level. PBMCs were irradiated at various doses between 10 cGy to 2.0 Gy at a dose rate of 1.0 Gy/min. Total RNA was isolated for gene expression analysis at 0 hour and 4 hours respectively. cDNA was prepared and transcriptional pattern as studied using real time q-PCR where Taqman probes were used. Time point kinetics of transcriptional pattern of TRF1, TRF2, TIN2, TERF2, PTOP and POT1 was carried out at 0 min, 15 min, 30 min, 60 min, and 120 min for two different doses (1.0 Gy and 2.0 Gy). Dose response and time point kinetics of TRF2 was studied at similar doses using confocal microscopy. Our results revealed that at 2.0 Gy there was a two fold increase at the level of transcription

  6. Detection of DNA damage in cells exposed to ionizing radiation by use of antisingle-stranded-DNA monoclonal antibody

    International Nuclear Information System (INIS)

    Schans, G.P. van der; Loon, A.A.W.M. van; Groenendijk, R.H.; Baan, R.A.

    1989-03-01

    An immunochemical method has been developed for quantitative detection of DNA damage in mammalian cells. The method is based on the binding of a monoclonal antibody to single-stranded DNA. The clone producing this antibody, D1B, was obtained as a by-product from fusion of mouse myeloma cells with spleen cells isolated from a mouse immunized with chemically modified DNA. The technique is based upon the determination of the percentage single-strandedness resulting from the partial umwinding of cellular DNA under alkaline conditions, a time-dependent process. Single-strand and double-strand DNA breaks, or lesions converted into such breaks in alkaline medium, form initiation points for the unwinding. The extent of unwinding under controlled conditions is a measure, therefore, of the amount of such sites. The method is rapid, does not require radioactive labelling of DNA or physical separation of single- from double-stranded molecules, is sufficiently sensitive to detect damage induced by 1 Gu of ionizing radiation and needs only small amounts of cells. The usefulness of the technique was demonstrated in a study on the induction of damage and its repair in unlabelled cultured Chinese hamster cells and in DNA-containing cells of human blood, both after exposure to 60 Co-γ-rays, and in white blood cells and bone marrow cells of X-irradiated mice. A dose-related degree of unwinding was observed and repair could be observed up to 60 min after irradiation. (author). 19 refs.; 3 figs.; 1 tab

  7. ETV6/RUNX1 Induces Reactive Oxygen Species and Drives the Accumulation of DNA Damage in B Cells

    Directory of Open Access Journals (Sweden)

    Hans-Peter Kantner

    2013-11-01

    Full Text Available The t(12;21(p13;q22 chromosomal translocation is the most frequent translocation in childhood B cell precursor-acute lymphoblastic leukemia and results in the expression of an ETV6/RUNX1 fusion protein. The frequency of ETV6/RUNX1 fusions in newborns clearly exceeds the leukemia rate revealing that additional events occur in ETV6/RUNX1-positive cells for leukemic transformation. Hitherto, the mechanisms triggering these second hits remain largely elusive. Thus, we generated a novel ETV6/RUNX1 transgenic mouse model where the expression of the fusion protein is restricted to CD19+ B cells. These animals harbor regular B cell development and lack gross abnormalities. We established stable pro-B cell lines carrying the ETV6/RUNX1 transgene that allowed us to investigate whether ETV6/RUNX1 itself favors the acquisition of second hits. Remarkably, these pro-B cell lines as well as primary bone marrow cells derived from ETV6/RUNX1 transgenic animals display elevated levels of reactive oxygen species (ROS as tested with ETV6/RUNX1 transgenic dihydroethidium staining. In line, intracellular phospho-histone H2AX flow cytometry and comet assay revealed increased DNA damage indicating that ETV6/RUNX1 expression enhances ROS. On the basis of our data, we propose the following model: the expression of ETV6/RUNX1 creates a preleukemic clone and leads to increased ROS levels. These elevated ROS favor the accumulation of secondary hits by increasing genetic instability and doublestrand breaks, thus allowing preleukemic clones to develop into fully transformed leukemic cells.

  8. Overexpression of DNA damage-induced 45 α gene contributes to esophageal squamous cell cancer by promoter hypomethylation

    Directory of Open Access Journals (Sweden)

    Wang Bao xiang

    2012-02-01

    Full Text Available Abstract Background Environmental factors-induced dysfunction of esophageal squamous epithelium, including genomic DNA impairment and apoptosis, play an important role in the pathogenesis of esophageal squamous cell cancer. DNA damage-induced 45α (GADD45α has been found promoting DNA repair and removing methylation marker, Therefore, in this study we will investigate whether GADD45α expression is induced and its mechanism in esophageal squamous cell cancer. Methods Two human esophageal squamous cell lines (ESCC, ECA109 and KYSE510 were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS. Lipofectamine 2000 was used to transfect cells. mRNA level of GADD45α was measured by reverse transcription-quantitive PCR (RT-qPCR, protein level of GADD45α was detected by western blot and Immunohistochemistry. Global DNA methylation of tissue sample was measured using the Methylamp Global DNA Methylation Quantification Ultra kit (Epigentek Group and promoter methylation was measured by bisulfite sequencing. Results GADD45a mRNA and protein levels were increased significantly in tumor tissue than that in adjacent normal tissue. Hypomethylation of global genomic DNA and GADD45α promoter were found in ESCC. The cell sensitivity to Cisplatin DDP was decreased significantly in Eca109 and Kyse510 cells, in which GADD45α expression was down-regulated by RNA interference (RNAi. In addition, silence of GADD45a expression in ESCC cells inhibited proliferation and promoted apoptosis. Conclusion Overexpression of GADD45α gene is due to DNA hypomethylation in ESCC. GADD45α may be a protective factor in DDP chemotherapy for esophageal squamous cell carcinoma.

  9. OGG1 Involvement in High Glucose-Mediated Enhancement of Bupivacaine-Induced Oxidative DNA Damage in SH-SY5Y Cells

    Science.gov (United States)

    Liu, Zhong-Jie; Zhao, Wei; Zhang, Qing-Guo; Li, Le; Lai, Lu-Ying; Jiang, Shan; Xu, Shi-Yuan

    2015-01-01

    Hyperglycemia can inhibit expression of the 8-oxoG-DNA glycosylase (OGG1) which is one of the key repair enzymes for DNA oxidative damage. The effect of hyperglycemia on OGG1 expression in response to local anesthetics-induced DNA damage is unknown. This study was designed to determine whether high glucose inhibits OGG1 expression and aggravates bupivacaine-induced DNA damage via reactive oxygen species (ROS). SH-SY5Y cells were cultured with or without 50 mM glucose for 8 days before they were treated with 1.5 mM bupivacaine for 24 h. OGG1 expression was measured by quantitative real-time polymerase chain reaction (qRT-PCR) and western blot. ROS was estimated using the redox-sensitive fluorescent dye DCFH-DA. DNA damage was investigated with immunostaining for 8-oxodG and comet assays. OGG1 expression was inhibited in cells exposed to high glucose with concomitant increase in ROS production and more severe DNA damage as compared to control culture conditions, and these changes were further exacerbated by bupivacaine. Treatment with the antioxidant N-acetyl-L-cysteine (NAC) prevented high glucose and bupivacaine mediated increase in ROS production and restored functional expression of OGG1, which lead to attenuated high glucose-mediated exacerbation of bupivacaine neurotoxicity. Our findings indicate that subjects with diabetes may experience more detrimental effects following bupivacaine use. PMID:26161242

  10. OGG1 Involvement in High Glucose-Mediated Enhancement of Bupivacaine-Induced Oxidative DNA Damage in SH-SY5Y Cells

    Directory of Open Access Journals (Sweden)

    Zhong-Jie Liu

    2015-01-01

    Full Text Available Hyperglycemia can inhibit expression of the 8-oxoG-DNA glycosylase (OGG1 which is one of the key repair enzymes for DNA oxidative damage. The effect of hyperglycemia on OGG1 expression in response to local anesthetics-induced DNA damage is unknown. This study was designed to determine whether high glucose inhibits OGG1 expression and aggravates bupivacaine-induced DNA damage via reactive oxygen species (ROS. SH-SY5Y cells were cultured with or without 50 mM glucose for 8 days before they were treated with 1.5 mM bupivacaine for 24 h. OGG1 expression was measured by quantitative real-time polymerase chain reaction (qRT-PCR and western blot. ROS was estimated using the redox-sensitive fluorescent dye DCFH-DA. DNA damage was investigated with immunostaining for 8-oxodG and comet assays. OGG1 expression was inhibited in cells exposed to high glucose with concomitant increase in ROS production and more severe DNA damage as compared to control culture conditions, and these changes were further exacerbated by bupivacaine. Treatment with the antioxidant N-acetyl-L-cysteine (NAC prevented high glucose and bupivacaine mediated increase in ROS production and restored functional expression of OGG1, which lead to attenuated high glucose-mediated exacerbation of bupivacaine neurotoxicity. Our findings indicate that subjects with diabetes may experience more detrimental effects following bupivacaine use.

  11. Thymidine kinase 1 deficient cells show increased survival rate after UV-induced DNA damage

    DEFF Research Database (Denmark)

    Skovgaard, T; Rasmussen, Lene Juel; Munch-Petersen, Birgitte

    2010-01-01

    Balanced deoxynucleotide pools are known to be important for correct DNA repair, and deficiency for some of the central enzymes in deoxynucleotide metabolism can cause imbalanced pools, which in turn can lead to mutagenesis and cell death. Here we show that cells deficient for the thymidine salva...

  12. DNA Damage and Pulmonary Hypertension

    Science.gov (United States)

    Ranchoux, Benoît; Meloche, Jolyane; Paulin, Roxane; Boucherat, Olivier; Provencher, Steeve; Bonnet, Sébastien

    2016-01-01

    Pulmonary hypertension (PH) is defined by a mean pulmonary arterial pressure over 25 mmHg at rest and is diagnosed by right heart catheterization. Among the different groups of PH, pulmonary arterial hypertension (PAH) is characterized by a progressive obstruction of distal pulmonary arteries, related to endothelial cell dysfunction and vascular cell proliferation, which leads to an increased pulmonary vascular resistance, right ventricular hypertrophy, and right heart failure. Although the primary trigger of PAH remains unknown, oxidative stress and inflammation have been shown to play a key role in the development and progression of vascular remodeling. These factors are known to increase DNA damage that might favor the emergence of the proliferative and apoptosis-resistant phenotype observed in PAH vascular cells. High levels of DNA damage were reported to occur in PAH lungs and remodeled arteries as well as in animal models of PH. Moreover, recent studies have demonstrated that impaired DNA-response mechanisms may lead to an increased mutagen sensitivity in PAH patients. Finally, PAH was linked with decreased breast cancer 1 protein (BRCA1) and DNA topoisomerase 2-binding protein 1 (TopBP1) expression, both involved in maintaining genome integrity. This review aims to provide an overview of recent evidence of DNA damage and DNA repair deficiency and their implication in PAH pathogenesis. PMID:27338373

  13. Study on the effect of artesunate combined with irradiation on DNA damage of HeLa and Siha cells of human cervical cancer

    International Nuclear Information System (INIS)

    Zhou Yuanyuan; Feng Yang; Zhu Wei; Ni Qianying; Geng Chong; Chen Guanglie; Luo Judong; Fan Sanjun; Cao Jianping; Zhang Xuguang

    2011-01-01

    In order to investigate the effect of artesunate combined with irradiation on DNA damage of HeLa and Siha cells of human cervical cancer, HeLa and Siha cells were cultured in vitro and exposed to different concentration of artesunate for 24 h and MTT assay was used to observe the inhibitory effect of different concentration of artesunate on the proliferation of HeLa and Siha cells. The cells were divided into 2 groups as the irradiated group and the union treatment group. Here it was set up four absorbed doses of 60 Co γ-irradiation in each group with 0, 2, 4 and 6 Gy, and the DNA damage were detected by single cell gel electrophoresis assay. MTT analysis showed that the inhibition of artesunate on HeLa and Siha cells of cervical cancer was in concentration-dependent manners. Single cell gel electrophoresis showed that the DNA damage of HeLa cells treated with artesunate was more serious than that treated only with irradiation (P<0.05), but had no such effect on Siha cells. Artesunate can increase the radio-sensitivity of HeLa cells cervical cancer with p53 mutant, but has no such effect on wide type p53 cells. (authors)

  14. DNA damage response signaling in lung adenocarcinoma A549 cells following gamma and carbon beam irradiation.

    Science.gov (United States)

    Ghosh, Somnath; Narang, Himanshi; Sarma, Asitikantha; Krishna, Malini

    2011-11-01

    Carbon beams (5.16MeV/u, LET=290keV/μm) are high linear energy transfer (LET) radiation characterized by higher relative biological effectiveness than low LET radiation. The aim of the current study was to determine the signaling differences between γ-rays and carbon ion-irradiation. A549 cells were irradiated with 1Gy carbon or γ-rays. Carbon beam was found to be three times more cytotoxic than γ-rays despite the fact that the numbers of γ-H2AX foci were same. Percentage of cells showing ATM/ATR foci were more with γ-rays however number of foci per cell were more in case of carbon irradiation. Large BRCA1 foci were found in all carbon irradiated cells unlike γ-rays irradiated cells and prosurvival ERK pathway was activated after γ-rays irradiation but not carbon. The noteworthy finding of this study is the early phase apoptosis induction by carbon ions. In the present study in A549 lung adenocarcinoma, authors conclude that despite activation of same repair molecules such as ATM and BRCA1, differences in low and high LET damage responses might be due to their distinct macromolecular complexes rather than their individual activation and the activation of cytoplasmic pathways such as ERK, whether it applies to all the cell lines need to be further explored. Copyright © 2011 Elsevier B.V. All rights reserved.

  15. Silver nanoparticle induced cytotoxicity, oxidative stress, and DNA damage in CHO cells

    Energy Technology Data Exchange (ETDEWEB)

    Awasthi, Kumud Kant [University of Rajasthan, Department of Zoology (India); Awasthi, Anjali; Kumar, Narender; Roy, Partha [Indian Institute of Technology Roorkee, Department of Biotechnology (India); Awasthi, Kamlendra, E-mail: kamlendra.awasthi@gmail.com [Malaviya National Institute of Technology, Department of Physics (India); John, P. J., E-mail: placheriljohn@yahoo.com [University of Rajasthan, Department of Zoology (India)

    2013-09-15

    Silver nanoparticles (Ag NPs) are being used increasingly in wound dressings, catheters, and in various household products due to their antimicrobial activity. The present study reports the toxicity evaluation of synthesized and well characterized Ag NPs using Chinese hamster ovary (CHO) cells. The UV-Vis spectroscopy reveals the formation of silver nanoparticles by exhibiting the typical surface plasmon absorption maxima at 408-410 nm. Transmission electron microscopy (TEM) reveals that the average diameter of silver nanoparticles is about 5.0 {+-} 1.0 nm and that they have spherical shape. Cell visibility and cell viability percentage show dose-dependent cellular toxicity of Ag NPs. The half maximal inhibitory concentration (IC{sub 50}) for CHO cells is 68.0 {+-} 2.65 {mu}g/ml after 24 h Ag NPs exposure. Toxicity evaluations, including cellular morphology, mitochondrial function (MTT assay), reactive oxygen species (ROS), and DNA fragmentation assay (Ladder pattern) were assessed in unexposed CHO cells (control) and the cells exposed to Ag NPs concentrations of 15, 30, and 60 {mu}g/ml for 24 h. The findings may assist in the designing of Ag NPs for various applications and provide insights into their toxicity.

  16. Cr(VI) induces DNA damage, cell cycle arrest and polyploidization: a flow cytometric and comet assay study in Pisum sativum.

    Science.gov (United States)

    Rodriguez, Eleazar; Azevedo, Raquel; Fernandes, Pedro; Santos, Conceição

    2011-07-18

    Chromium(VI) is recognized as the most toxic valency of Cr, but its genotoxicity and cytostaticity in plants is still poorly studied. In order to analyze Cr(VI) cyto- and gentotoxicity, Pisum sativum L. plants were grown in soil and watered with solutions with different concentrations of Cr up to 2000 mg/L. After 28 days of exposure, leaves showed no significant variations in either cell cycle dynamics or ploidy level. As for DNA damage, flow cytometric (FCM) histograms showed significant differences in full peak coefficient of variation (FPCV) values, suggesting clastogenicity. This is paralleled by the Comet assay results, showing an increase in DNA damage for 1000 and 2000 mg/L. In roots, exposure to 2000 mg/L resulted in cell cycle arrest at the G(2)/M checkpoint. It was also verified that under the same conditions 40% of the individuals analyzed suffered polyploidization having both 2C and 4C levels. DNA damage analysis by the Comet assay and FCM revealed dose-dependent increases in DNA damage and FPCV. Through this, we have unequivocally demonstrated for the first time in plants that Cr exposure can result in DNA damage, cell cycle arrest, and polyploidization. Moreover, we critically compare the validity of the Comet assay and FCM in evaluating cytogenetic toxicity tests in plants and demonstrate that the data provided by both techniques complement each other and present high correlation levels. In conclusion, the data presented provides new insight on Cr effects in plants in general and supports the use of the parameters tested in this study as reliable endpoints for this metal toxicity in plants. © 2011 American Chemical Society

  17. Gonadotropin-Releasing Hormone Regulates Expression of the DNA Damage Repair Gene, Fanconi anemia A, in Pituitary Gonadotroph Cells1

    Science.gov (United States)

    Larder, Rachel; Chang, Lynda; Clinton, Michael; Brown, Pamela

    2007-01-01

    Gonadal function is critically dependant on regulated secretion of the gonadotropin hormones from anterior pituitary gonadotroph cells. Gonadotropin biosynthesis and release is triggered by the binding of hypothalamic GnRH to GnRH receptor expressed on the gonadotroph cell surface. The repertoire of regulatory molecules involved in this process are still being defined. We used the mouse LβT2 gonadotroph cell line, which expresses both gonadotropin hormones, as a model to investigate GnRH regulation of gene expression and differential display reverse transcription-polymerase chain reaction (RT-PCR) to identify and isolate hormonally induced changes. This approach identified Fanconi anemia a (Fanca), a gene implicated in DNA damage repair, as a differentially expressed transcript. Mutations in Fanca account for the majority of cases of Fanconi anemia (FA), a recessively inherited disease identified by congenital defects, bone marrow failure, infertility, and cancer susceptibility. We confirmed expression and hormonal regulation of Fanca mRNA by quantitative RT-PCR, which showed that GnRH induced a rapid, transient increase in Fanca mRNA. Fanca protein was also acutely upregulated after GnRH treatment of LβT2 cells. In addition, Fanca gene expression was confined to mature pituitary gonadotrophs and adult mouse pituitary and was not expressed in the immature αT3-1 gonadotroph cell line. Thus, this study extends the expression profile of Fanca into a highly specialized endocrine cell and demonstrates hormonal regulation of expression of the Fanca locus. We suggest that this regulatory mechanism may have a crucial role in the GnRH-response mechanism of mature gonadotrophs and perhaps the etiology of FA. PMID:15128600

  18. Gonadotropin-releasing hormone regulates expression of the DNA damage repair gene, Fanconi anemia A, in pituitary gonadotroph cells.

    Science.gov (United States)

    Larder, Rachel; Chang, Lynda; Clinton, Michael; Brown, Pamela

    2004-09-01

    Gonadal function is critically dependant on regulated secretion of the gonadotropin hormones from anterior pituitary gonadotroph cells. Gonadotropin biosynthesis and release is triggered by the binding of hypothalamic GnRH to GnRH receptor expressed on the gonadotroph cell surface. The repertoire of regulatory molecules involved in this process are still being defined. We used the mouse L beta T2 gonadotroph cell line, which expresses both gonadotropin hormones, as a model to investigate GnRH regulation of gene expression and differential display reverse transcription-polymerase chain reaction (RT-PCR) to identify and isolate hormonally induced changes. This approach identified Fanconi anemia a (Fanca), a gene implicated in DNA damage repair, as a differentially expressed transcript. Mutations in Fanca account for the majority of cases of Fanconi anemia (FA), a recessively inherited disease identified by congenital defects, bone marrow failure, infertility, and cancer susceptibility. We confirmed expression and hormonal regulation of Fanca mRNA by quantitative RT-PCR, which showed that GnRH induced a rapid, transient increase in Fanca mRNA. Fanca protein was also acutely upregulated after GnRH treatment of L beta T2 cells. In addition, Fanca gene expression was confined to mature pituitary gonadotrophs and adult mouse pituitary and was not expressed in the immature alpha T3-1 gonadotroph cell line. Thus, this study extends the expression profile of Fanca into a highly specialized endocrine cell and demonstrates hormonal regulation of expression of the Fanca locus. We suggest that this regulatory mechanism may have a crucial role in the GnRH-response mechanism of mature gonadotrophs and perhaps the etiology of FA.

  19. DNA damage in oral cancer cells induced by nitrogen atmospheric pressure plasma jets

    Science.gov (United States)

    Han, Xu; Klas, Matej; Liu, Yueying; Stack, M. Sharon; Ptasinska, Sylwia

    2013-09-01

    The nitrogen atmospheric pressure plasma jet (APPJ) has been shown to effectively induce DNA double strand breaks in SCC-25 oral cancer cells. The APPJ source constructed in our laboratory consists of two external electrodes wrapping around a quartz tube and nitrogen as a feed gas and operates based on dielectric barrier gas discharge. Generally, it is more challenging to ignite plasma in N2 atmosphere than in noble gases. However, this design provides additional advantages such as lower costs compared to the noble gases for future clinical operation. Different parameters of the APPJ configuration were tested in order to determine radiation dosage. To explore the effects of delayed damage and cell self-repairing, various incubation times of cells after plasma treatment were also performed. Reactive species generated in plasma jet and in liquid environment are essential to be identified and quantified, with the aim of unfolding the mystery of detailed mechanisms for plasma-induced cell apoptosis. Moreover, from the comparison of plasma treatment effect on normal oral cells OKF6T, an insight to the selectivity for cancer treatment by APPJ can be explored. All of these studies are critical to better understand the damage responses of normal and abnormal cellular systems to plasma radiation, which are useful for the development of advanced plasma therapy for cancer treatment at a later stage.

  20. Bacterial mutagenicity and mammalian cell DNA damage by several substituted anilines.

    Science.gov (United States)

    Zimmer, D; Mazurek, J; Petzold, G; Bhuyan, B K

    1980-04-01

    Several substituted alkyl- and haloanilines were tested for their ability to mutate Salmonella typhimurium and to damage the DNA of mammalian (V79) cells. These results were correlated with their reported carcinogenicity. Of 9 suspected carcinogens, 4 were bacterial mutagens and 4 (out of 7 tested) damaged DNA of V79 cells. The following compounds were weakly mutagenic (less than 150 revertants/mumole): 4-fluoroaniline, 2,3-, 2,4-, 2,5- and 3,4-dimethylaniline, and 2-methyl-4-fluoroaniline. The following compounds were strong mutagens: 2,4,5-trimethylaniline, 2-methyl-4-chloro-, and 2-methyl-4-bromo-, 4-methyl-2-chloro-, 4-methyl-2-bromo- and 2-ethyl-4-chloroaniline. The compounds which damaged DNA in V79 cells were: 2 methyl-4-chloroaniline, 2-methyl-4-bromoaniline, 2,4,5- and 2,4,6-trimethylaniline.

  1. The insecticide buprofezin induces morphological transformation and kinetochore-positive micronuclei in cultured Syrian hamster embryo cells in the absence of detectable DNA damage.

    Science.gov (United States)

    Herrera, L A; Ostrosky-Wegman, P; Schiffmann, D; Chen, Q Y; Ziegler-Skylakakis, K; Andrae, U

    1993-11-01

    The insecticide buprofezin was examined for its genotoxicity in cultured Syrian hamster embryo cells in order to better understand the mechanisms underlying the genotoxicity of the compound in mammalian cells. Exposure to buprofezin concentrations of 12.5-100 microM did not significantly affect the colony-forming ability of the cells, but did result in increased frequencies of morphologically transformed colonies. Treatment with buprofezin did not cause a detectable induction of DNA repair synthesis, an indicator of DNA damage, but significantly increased the frequency of micronuclei. Immunostaining of the cells with antikinetochore antibody (CREST antibody) showed that essentially all of the buprofezin-induced micronuclei were kinetochore-positive. The results suggest that morphological transformation of Syrian hamster embryo cells by buprofezin results from an interaction of the compound or a metabolite of it with the mitotic apparatus rather than from DNA damage.

  2. DNA damage in oral cancer and normal cells induced by nitrogen atmospheric pressure plasma jets

    Science.gov (United States)

    Han, Xu; Kapaldo, James; Liu, Yueying; Stack, M. Sharon; Ptasinska, Sylwia

    2015-09-01

    Nitrogen atmospheric pressure plasma jets (APPJs) have been shown to effectively induce DNA double strand breaks in SCC25 oral cancer cells. The APPJ source constructed in our laboratory operates based on dielectric barrier discharge. It consists of two copper electrodes alternatively wrapping around a fused silica tube with nitrogen as a feed gas. It is generally more challenging to ignite plasma in N2 atmosphere than in noble gases. However, N2 provides additional advantages such as lower costs compared to noble gases, thus this design can be beneficial for the future long-term clinical use. To compare the effects of plasma on cancer cells (SCC25) and normal cells (OKF), the cells from both types were treated at the same experimental condition for various treatment times. The effective area with different damage levels after the treatment was visualized as 3D maps. The delayed damage effects were also explored by varying the incubation times after the treatment. All of these studies are critical for a better understanding of the damage responses of cellular systems exposed to the plasma radiation, thus are useful for the development of the advanced plasma cancer therapy. The research described herein was supported by the Division of Chemical Sciences, Geosciences and Biosciences, Basic Energy Sciences, Office of Science, United States Department of Energy through Grant No. DE-FC02-04ER15533.

  3. Problem-Solving Test: Analysis of DNA Damage Recognizing Proteins in Yeast and Human Cells

    Science.gov (United States)

    Szeberenyi, Jozsef

    2013-01-01

    The experiment described in this test was aimed at identifying DNA repair proteins in human and yeast cells. Terms to be familiar with before you start to solve the test: DNA repair, germline mutation, somatic mutation, inherited disease, cancer, restriction endonuclease, radioactive labeling, [alpha-[superscript 32]P]ATP, [gamma-[superscript…

  4. A high sensitivity, high throughput, automated single-cell gel electrophoresis ('Comet') DNA damage assay

    International Nuclear Information System (INIS)

    Vojnovic, B.; Barber, P.R.; Johnston, P.J.; Gregory, H.C.; Locke, R.J.

    2003-01-01

    A fully automated microscopy machine vision image capture and analysis system for the collection of data from slides of 'comets' has been developed. The novel image processing algorithms employed in delineating the 'comet head' from the 'comet tail' allow us to determine accurately very low levels of damage. In conjunction with calibrated and automated image capture methods, we are able to eliminate operator subjectivity and analyse large numbers of cells (>2500) in a short time (<1 hour). The image processing algorithm is designed to handle particularly difficult nuclei containing a high degree of structure, due to DNA clumping. We also present techniques used to extend the assay's dynamic range by removing interfering background fluorescence and to define a region of interest. If subtle biological variations are to be quantified (e.g. cell cycle dependant damage), then the use of large cell populations is dictated. Under those circumstances, the use of a fully automated system is particularly advantageous providing that the manner in which data is extracted does not introduce any inadvertent bias. In practice, it is essential that the image processing steps are geared towards the correct recognition of an acceptable cell nucleus, i.e. comet 'head'. We acknowledge the financial support of CRUK, Programme Grant C133/A1812 - SP 2195-01/02 and the US Department of Energy Low Dose Radiation Research Program grant DE-FG07-99ER62878

  5. Silencing of the transcription factor STAT3 sensitizes lung cancer cells to DNA damaging drugs, but not to TNFα- and NK cytotoxicity

    Energy Technology Data Exchange (ETDEWEB)

    Kulesza, Dorota W. [Laboratory of Transcription Regulation, Department of Cell Biology, The Nencki Institute of Experimental Biology, Warsaw (Poland); Postgraduate School of Molecular Medicine, Warsaw Medical University, Warsaw (Poland); Carré, Thibault; Chouaib, Salem [Unité INSERM U753, Institut de Cancérologie Gustave Roussy, Villejuif Cedex (France); Kaminska, Bozena, E-mail: bozenakk@nencki.gov.pl [Laboratory of Transcription Regulation, Department of Cell Biology, The Nencki Institute of Experimental Biology, Warsaw (Poland)

    2013-02-15

    Transcription factor STAT3 (Signal Transducers and Activators of Transcription 3) is persistently active in human tumors and may contribute to tumor progression. Inhibition of STAT3 expression/activity could be a good strategy to modulate tumor cell survival and responses to cancer chemotherapeutics or immune cytotoxicity. We silenced STAT3 expression in human A549 lung cancer cells to elucidate its role in cell survival and resistance to chemotherapeutics, TNFα and natural killer (NK)-mediated cytotoxicity. We demonstrate that STAT3 is not essential for basal survival and proliferation of A549 cancer cells. Stable silencing of STAT3 expression sensitized A549 cells to DNA damaging chemotherapeutics doxorubicin and cisplatin in a p53-independent manner. Sensitization to DNA damage-inducing chemotherapeutics could be due to down-regulation of the Bcl-xL expression in STAT3 depleted cells. In contrast, knockdown of STAT3 in cancer cells did not modulate responses to TNFα and NK-mediated cytotoxicity. We found that STAT3 depletion increased the NFκB activity likely providing the compensatory, pro-survival signal. The treatment with TNFα, but not doxorubicin, enhanced this effect. We conclude that STAT3 is not crucial for the control of basal cell proliferation and survival of lung carcinoma cells but modulates susceptibility to DNA damaging chemotherapeutics by regulation of intrinsic pro-survival pathways. - Highlights: ► STAT3 silencing is negligent for basal lung cancer cell viability and proliferation. ► STAT3 depletion sensitizes lung cancer cells to DNA damaging chemotherapeutics. ► STAT3 depletion has no effect on susceptibility to extrinsic apoptosis inducers. ► Increased pro-survival NFκB activity may compensate for STAT3 depletion.

  6. LET dependence of the production of oxidative DNA damage in mammalian cells

    International Nuclear Information System (INIS)

    Ito, Atsushi; Furuichi, Wataru; Inoguchi, Hiroki; Hirayama, Ryoichi; Murayama, Chieko; Furusawa, Yoshiya

    2006-01-01

    Production of 8-hydroxy-2'-deoxyguanosine (8-OHdG) in human leukemia HL-60 cells was examined upon irradiation with carbon, neon silicon ions. Cell suspension with the concentration of 1 x 10 7 /ml was irradiated tinder air-saturated condition. After irradiation cells were subjected to the DNA extraction using isopropanol, separation of DNA strands by heat treatment, digestion into nucleosides with nuclease P1 and alkaline phosphatase. A single peak of 8-OHdG on a chromatogram was observed using newly installed ECD detector (Coulochem III; ESA, Inc. U.S.A.). Reproducibility was also greatly improved with this detector. 8-OHdG yield was decreased with increasing linear energy transfer (LET) for carbon and silicon beam. These results are in good accordance with those of dG solution which was previously reported by us. Ion species dependence in 8-OHdG yield was not so apparent through the comparison of carbon and neon beam with an LET of 80 keV/μm and neon and silicon beam with an LET of 150 keV/μm. (author)

  7. DNA damage and repair in rabbit lens epithelial cells following UVA radiation

    International Nuclear Information System (INIS)

    Sidjanin, Duska; Zigman, Seymour; Reddan, John

    1993-01-01

    Since ultraviolet light may be a contributing factor to cataractogenesis, we investigated the response of the lens epithelium, a potential target for UV insult, to UVA radiation. Cell survival and the induction and repair of DNA single-strand breaks (SSBs) were measured in cultured rabbit lens epithelial cells following UVA exposure. A 30 min exposure to UVA (180 KJ/m 2 ) induced measurable SSBs. An increase in UVA fluenced measurable SSBs. An increase in UVA fluence brought about an increase in UVA fluence brought about an increase in the number of DNA SSBs. Rejoining of SSBs were measured after the cells were irradiated in Tyrode's for 2 hrs and allowed to repair in the dark for 4 hrs at 36 o C in MEM containing 10% serum. Eighty percent of the DNA SSBs were repaired within 4 hrs as determined by analysis of the alkaline elution profile. The repair kinetics were biphasic with an initial fast and subsequently slower component. The results indicate that UVA can induce SSBs in lens-induced SSBs, and that UVA treatment can be toxic to the epithelium. (Author)

  8. DUBbing Cancer: Deubiquitylating Enzymes Involved in Epigenetics, DNA Damage and the Cell Cycle As Therapeutic Targets.

    Science.gov (United States)

    Pinto-Fernandez, Adan; Kessler, Benedikt M

    2016-01-01

    Controlling cell proliferation is one of the hallmarks of cancer. A number of critical checkpoints ascertain progression through the different stages of the cell cycle, which can be aborted when perturbed, for instance by errors in DNA replication and repair. These molecular checkpoints are regulated by a number of proteins that need to be present at the right time and quantity. The ubiquitin system has emerged as a central player controlling the fate and function of such molecules such as cyclins, oncogenes and components of the DNA repair machinery. In particular, proteases that cleave ubiquitin chains, referred to as deubiquitylating enzymes (DUBs), have attracted recent attention due to their accessibility to modulation by small molecules. In this review, we describe recent evidence of the critical role of DUBs in aspects of cell cycle checkpoint control, associated DNA repair mechanisms and regulation of transcription, representing pathways altered in cancer. Therefore, DUBs involved in these processes emerge as potentially critical targets for the treatment of not only hematological, but potentially also solid tumors.

  9. DUBbing cancer: Deubiquitylating enzymes involved in epigenetics, DNA damage and the cell cycle as therapeutic targets

    Directory of Open Access Journals (Sweden)

    Benedikt M Kessler

    2016-07-01

    Full Text Available Controlling cell proliferation is one of the hallmarks of cancer. A number of critical checkpoints ascertain progression through the different stages of the cell cycle, which can be aborted when perturbed, for instance by errors in DNA replication and repair. These molecular checkpoints are regulated by a number of proteins that need to be present at the right time and quantity. The ubiquitin system has emerged as a central player controlling the fate and function of such molecules such as cyclins, oncogenes and components of the DNA repair machinery. In particular, proteases that cleave ubiquitin chains, referred to as deubiquitylating enzymes (DUBs, have attracted recent attention due to their accessibility to modulation by small molecules. In this review, we describe recent evidence of the critical role of DUBs in aspects of cell cycle checkpoint control, associated DNA repair mechanisms and regulation of transcription, representing pathways altered in cancer. Therefore, DUBs involved in these processes emerge as potentially critical targets for the treatment of not only hematological, but potentially also solid tumors.

  10. miR-520 promotes DNA-damage-induced trophoblast cell apoptosis by targeting PARP1 in recurrent spontaneous abortion (RSA).

    Science.gov (United States)

    Dong, Xiujuan; Yang, Long; Wang, Hui

    2017-04-01

    The establishment and maintenance of successful pregnancy mainly depends on trophoblast cells. Their dysfunction has been implicated in recurrent spontaneous abortion (RSA), a major complication of pregnancy. However, the underlying mechanisms of trophoblasts dysfunction remain unclear. DNA-damage-induced cell apoptosis has been reported to play a vital role in cell death. In this study, we identified a novel microRNA (miR-520) in RSA progression via regulating trophoblast cell apoptosis. Microarray analysis showed that miR-520 was highly expressed in villus of RSA patients. By using flow cytometry analysis, we observed miR-520 expression was correlated with human trophoblast cell apoptosis in vitro, along with decreased poly (ADP-ribose) polymerase-1 (PARP1) expression. With the analysis of clinic samples, we observed that miR-520 level was negatively correlated with PARP1 level in RSA villus. In addition, overexpression of PARP1 restored the miR-520-induced trophoblast cell apoptosis in vitro. The status of chromosome in trophoblast implied that miR-520-promoted DNA-damage-induced cell apoptosis to regulate RSA progression. These results indicated that the level of miR-520 might associate with RSA by prompting trophoblast cell apoptosis via PARP1 dependent DNA-damage pathway.

  11. Differences in inhibition by beta-arabinofuranosyladenine (araA) of radiation induced DNA damage repair in exponentially growing and plateau-phase CHO-cells

    International Nuclear Information System (INIS)

    Iliakis, G.; Seaner, R.

    1988-01-01

    The effect of beta-arabinofuranosyladenine (araA) on the repair of radiation induced DNA damage, as measured by the DNA unwinding technique, was studied in exponentially growing and plateau-phase CHO-cells after exposure to X-rays. Induction of DNA damage by radiation was found to be similar in exponentially growing and plateau-phase cells. In the absence of araA, repair of radiation induced DNA damage proceeded with similar kinetics in exponentially growing and plateau-phase cells. AraA at concentrations between 0-1500 μM inhibited DNA repair both in exponentially growing and in plateau-phase cells. However, the degree of inhibition was significantly higher (by a factor of 3) in plateau-phase cells. A similar degree of repair inhibition by araA was observed in plateau-phase cells treated in their conditioned medium, as well as in plateau-phase cells that were transferred in fresh growth medium just before treatment initiation. These results indicate the importance of biochemical parameters associated with alterations in the growth state of the cells for the inhibitory effect of araA and may help in the elucidation of the molecular mechanism(s) underlying repair inhibition by inhibitors of DNA replication. (orig.)

  12. Two familial ALS proteins function in prevention/repair of transcription-associated DNA damage.

    Science.gov (United States)

    Hill, Sarah J; Mordes, Daniel A; Cameron, Lisa A; Neuberg, Donna S; Landini, Serena; Eggan, Kevin; Livingston, David M

    2016-11-29

    Amyotrophic lateral sclerosis (ALS) is a progressive motor neuron dysfunction disease that leads to paralysis and death. There is currently no established molecular pathogenesis pathway. Multiple proteins involved in RNA processing are linked to ALS, including FUS and TDP43, and we propose a disease mechanism in which loss of function of at least one of these proteins leads to an accumulation of transcription-associated DNA damage contributing to motor neuron cell death and progressive neurological symptoms. In support of this hypothesis, we find that FUS or TDP43 depletion leads to increased sensitivity to a transcription-arresting agent due to increased DNA damage. Thus, these proteins normally contribute to the prevention or repair of transcription-associated DNA damage. In addition, both FUS and TDP43 colocalize with active RNA polymerase II at sites of DNA damage along with the DNA damage repair protein, BRCA1, and FUS and TDP43 participate in the prevention or repair of R loop-associated DNA damage, a manifestation of aberrant transcription and/or RNA processing. Gaining a better understanding of the role(s) that FUS and TDP43 play in transcription-associated DNA damage could shed light on the mechanisms underlying ALS pathogenesis.

  13. Effect of fenofibrate on oxidative DNA damage and on gene expression related to cell proliferation and apoptosis in rats.

    Science.gov (United States)

    Nishimura, Jihei; Dewa, Yasuaki; Muguruma, Masako; Kuroiwa, Yuichi; Yasuno, Hiroaki; Shima, Tomomi; Jin, Mailan; Takahashi, Miwa; Umemura, Takashi; Mitsumori, Kunitoshi

    2007-05-01

    To investigate the relationship between fenofibrate (FF) and oxidative stress, enzymatic, histopathological, and molecular biological analyses were performed in the liver of male F344 rats fed 2 doses of FF (Experiment 1; 0 and 6000 ppm) for 3 weeks and 3 doses (Experiment 2; 0, 3000, and 6000 ppm) for 9 weeks. FF treatment increased the activity of enzymes such as carnitine acetyltransferase, carnitine palmitoyltransferase, fatty acyl-CoA oxidizing system, and catalase in the liver. However, it decreased those of superoxide dismutase in the liver in both experiments. Increased 8-hydroxy-2'-deoxyguanosine levels in liver DNA and lipofuscin accumulation were observed in the treated rats of Experiment 2. In vitro measurement of reactive oxygen species (ROS) in rat liver microsomes revealed a dose-dependent increase due to FF treatment. Microarray (only Experiment 1) or real-time reverse transcription-polymerase chain reaction analyses revealed that the expression levels of metabolism and DNA repair-related genes such as Aco, Cyp4a1, Cat, Yc2, Gpx2, Apex1, Xrcc5, Mgmt, Mlh1, Gadd45a, and Nbn were increased in FF-treated rats. These results provide evidence of a direct or indirect relationship between oxidative stress and FF treatment. In addition, increases in the expression levels of cell cycle-related genes such as Chek1, Cdc25a, and Ccdn1; increases in the expression levels of cell proliferation-related genes such as Hdgfrp3 and Vegfb; and fluctuations in the expression levels of apoptosis-related genes such as Casp11 and Trp53inp1 were observed in these rats. This suggests that cell proliferation induction, apoptosis suppression, and DNA damage due to oxidative stresses are probably involved in the mechanism of hepatocarcinogenesis due to FF in rats.

  14. Molecular cloning and characterization of an Erwinia carotovora subsp. carotovora pectin lyase gene that responds to DNA-damaging agents.

    OpenAIRE

    McEvoy, J L; Murata, H; Chatterjee, A K

    1990-01-01

    recA-mediated production of pectin lyase (PNL) and the bacteriocin carotovoricin occurs in Erwinia carotovora subsp. carotovora 71 when this organism is subjected to agents that damage or inhibit the synthesis of DNA. The structural gene pnlA was isolated from a strain 71 cosmid gene library following mobilization of the cosmids into a moderate PNL producer, strain 193. The cosmid complemented pnl::Tn5 but not ctv::Tn5 mutations. A constitutive level of PNL activity was detected in RecA+ and ...

  15. Neocarzinostatin-mediated DNA damage and repair in wild-type and repair-deficient Chinese hamster ovary cells

    International Nuclear Information System (INIS)

    Kuo, W.L.; Meyn, R.E.; Haidle, C.W.

    1984-01-01

    The formation and repair of neocarzinostatin (NCS)-mediated DNA damage were examined in two strains of Chinese hamster ovary cells. The response in strain EM9, a mutant line selected for its sensitivity to ethyl methanesulfonate and shown to have a defect in the repair of X-ray-induced DNA breaks, was compared with that observed in the parental strain (AA8). The DNA strand breaks and their subsequent rejoining were measured using the method of elution of DNA from filters under either alkaline (for single-strand breaks), or nondenaturing conditions (for double-strand breaks). Colony survival assays showed that the mutant was more sensitive to the action of NCS than was the parental strain by a factor of approximately 1.5. Elution analyses showed that the DNA from both strains was damaged by NCS; the mutant displayed more damage than the parent under the same treatment conditions. Single-strand breaks were produced with a frequency of about 10 to 15 times the frequency of double-strand breaks. Both strains were able to rejoin both single-strand breaks and double-strand breaks induced by NCS treatment. The strand break data suggest that the difference in NCS-mediated cytotoxicity between EM9 and AA8 cells may be directly related to the enhanced production of DNA strand breaks in EM9. However, the fact that much higher doses of NCS were required in the DNA studies compared to the colony survival assays implies that either a small number of DNA breaks occur in a critical region of the genome, or that lesions other than DNA strand breaks are partly responsible for the observed cytotoxicity

  16. Assessment of DNA damages caused by exposure of bacterial cells and spores to the Mars surface environment

    Science.gov (United States)

    Fajardo-Cavazos, Patricia; Schuerger, Andrew; Robles-Martinez, Jose; Douki, Thierry; Nicholson, Wayne

    Joint NASA and ESA missions are planned for the next decade to investigate the possibility of present or past life on Mars [1]. Evidence of extraterrestrial life will likely rely on the de-tection of biomarkers, highlighting the importance of preventing forward contamination not only with viable microorganisms, but also with biomolecules that could compromise the valid-ity of life-detection experiments [2-4]. The designation of DNA as a high-priority biomarker makes it necessary to evaluate its persistence in extraterrestrial environments, and the effects of exposure on its biological activity. To accomplish this, we deposited naked DNA, cells and spores of Bacillus subtilis 168 or B. pumilus SAFR-032, or cells of Acinetobacter radioresistens 50v1 onto spacecraft-qualified aluminum coupons. Samples were exposed to a simulated Mars surface environment as described in detail previously [4, 5] for various periods of time, and DNA damage was assessed by a number of measurements. Double-and single-strand breaks were measured by neutral and alkaline agarose gel electrophoresis, and DNA bipyrimidine pho-toproducts were measured by HPLC-mass spectrometry, as described previously [6, 7]. Loss of functionality of DNA to serve as a template for replication by DNA polymerase was measured using a quantitative polymerase chain reaction (qPCR) assay [8]. In all cases, DNA damage was directly correlated with time of exposure to simulated martian solar radiation (UV, visible, and infrared wavelengths). Exposure of samples to Mars surface conditions, but shielded from solar radiation, did not result in appreciable damage over the time periods tested, relative to controls. DNA contained within cells or spores was much less susceptible to damage than was naked DNA. Using the qPCR assay, we found that inactivation of naked DNA or DNA extracted from exposed spores of B. subtilis followed a multiphasic dose-response, and that a fraction of DNA molecules retained functionality after

  17. Oxidative stress induction by T-2 toxin causes DNA damage and triggers apoptosis via caspase pathway in human cervical cancer cells

    International Nuclear Information System (INIS)

    Chaudhari, Manjari; Jayaraj, R.; Bhaskar, A.S.B.; Lakshmana Rao, P.V.

    2009-01-01

    T-2 toxin is the most toxic trichothecene and both humans and animals suffer from several pathological conditions after consumption of foodstuffs contaminated with trichothecenes. We investigated the molecular mechanism of T-2 toxin induced cytotoxicity and cell death in HeLa cells. T-2 toxin at LC50 of 10 ng/ml caused time dependent increase in cytotoxicity as assessed by dye uptake, lactatedehydrogenase leakage and MTT assay. The toxin caused generation of reactive oxygen species as early as 30 min followed by significant depletion of glutathione levels and increased lipid peroxidation. The results indicate oxidative stress as underlying mechanism of cytotoxicity. Single stranded DNA damage after T-2 treatment was observed as early as 2 and 4 h by DNA diffusion assay. The cells exhibited apoptotic morphology like condensed chromatin and nuclear fragmentation after 4 h of treatment. Downstream of T-2 induced oxidative stress and DNA damage a time dependent increase in expression level of p53 protein was observed. The increase in Bax/Bcl2 ratio indicated shift in response, in favour of apoptotic process in T-2 toxin treated cells. Western blot analysis showed increase in levels of mitochondrial apoptogenic factors Bax, Bcl-2, cytochrome-c followed by activation of caspases-9, -3 and -7 leading to DNA fragmentation and apoptosis. In addition to caspase-dependent pathway, our results showed involvement of caspase-independent AIF pathway in T-2 induced apoptosis. Broad spectrum caspase inhibitor z-VAD-fmk could partially protect the cells from DNA damage but could not inhibit AIF induced oligonucleosomal DNA fragmentation beyond 4 h. Results of the study clearly show that oxidative stress is the underlying mechanism by which T-2 toxin causes DNA damage and apoptosis.

  18. ANTIMUTAGENICITY OF CINNAMALDEHYDE AND VANILLIN IN HUMAN CELLS: GLOBAL GENE EXPRESSION AND POSSIBLE ROLE OF DNA DAMAGE AND REPAIR

    Science.gov (United States)

    This study investigated the possibility that chemicals identified as antimutagens may, in fact, operate through a mechanism involving DNA damage. We addressed this question by using two chemicals to which a large proportion of the population are exposed: vanillin and cinnemaldehy...

  19. Ultraviolet radiation-induced interleukin 6 release in HeLa cells is mediated via membrane events in a DNA damage-independent way.

    Science.gov (United States)

    Kulms, D; Pöppelmann, B; Schwarz, T

    2000-05-19

    Evidence exists that ultraviolet radiation (UV) affects molecular targets in the nucleus or at the cell membrane. UV-induced apoptosis was found to be mediated via DNA damage and activation of death receptors, suggesting that nuclear and membrane effects are not mutually exclusive. To determine whether participation of nuclear and membrane components is also essential for other UV responses, we studied the induction of interleukin-6 (IL-6) by UV. Exposing HeLa cells to UV at 4 degrees C, which inhibits activation of surface receptors, almost completely prevented IL-6 release. Enhanced repair of UV-mediated DNA damage by addition of the DNA repair enzyme photolyase did not affect UV-induced IL-6 production, suggesting that in this case membrane events predominant over nuclear effects. UV-induced IL-6 release is mediated via NFkappaB since the NFkappaB inhibitor MG132 or transfection of cells with a super-repressor form of the NFkappaB inhibitor IkappaB reduced IL-6 release. Transfection with a dominant negative mutant of the signaling protein TRAF-2 reduced IL-6 release upon exposure to UV, indicating that UV-induced IL-6 release is mediated by activation of the tumor necrosis factor receptor-1. These data demonstrate that UV can exert biological effects mainly by affecting cell surface receptors and that this is independent of its ability to induce nuclear DNA damage.

  20. Combustion products of 1,3-butadiene inhibit catalase activity and induce expression of oxidative DNA damage repair enzymes in human bronchial epithelial cells.

    Science.gov (United States)

    Kennedy, Christopher H; Catallo, W James; Wilson, Vincent L; Mitchell, James B

    2009-10-01

    1,3-Butadiene, an important petrochemical, is commonly burned off when excess amounts need to be destroyed. This combustion process produces butadiene soot (BDS), which is composed of a complex mixture of polycyclic aromatic hydrocarbons in particulates ranging in size from enzyme inactivation due to protein amino acid oxidation and (2) induce oxidative DNA damage in NHBE cells. Thus, our aims were to determine the effect of butadiene soot ethanol extract (BSEE) on both enzyme activity and the expression of proteins involved in the repair of oxidative DNA damage. Catalase was found to be sensitive to BDS as catalase activity was potently diminished in the presence of BSEE. Using Western analysis, both the alpha isoform of human 8-oxoguanine DNA glycosylase (alpha-hOGG1) and human apurinic/apyrimidinic endonuclease (APE-1) were shown to be significantly overexpressed as compared to untreated controls after exposure of NHBE cells to BSEE. Our results indicate that BSEE is capable of effectively inactivating the antioxidant enzyme catalase, presumably via oxidation of protein amino acids. The presence of oxidized biomolecules may partially explain the extranuclear fluorescence that is detected when NHBE cells are treated with an organic extract of BDS. Overexpression of both alpha-hOGG1 and APE-1 proteins following treatment of NHBE cells with BSEE suggests that this mixture causes oxidative DNA damage.

  1. Signal transduction and HIV transcriptional activation after exposure to ultraviolet light and other DNA-damaging agents

    International Nuclear Information System (INIS)

    Valerie, K.; Laster, W.S.; Luhua Cheng; Kirkham, J.C.; Reavey, Peter; Kuemmerle, N.B.

    1996-01-01

    Short wavelength (254 nm) ultraviolet light (UVC) radiation was much more potent in activating transcription of human immunodeficiency virus 1 (HIV) reporter genes stably integrated into the genomes of human and monkey cells than ionizing radiation (IR) from a 137 Cs source at similarly cytotoxic doses. A similar differential was also observed when c-jun transcription levels were examined. However, these transcription levels do not correlate with activation of nuclear factor (NF)-kB and AP-1 measured by band-shift assays, i.e. both types of radiation produce similar increases in NF-kB and AP-1 activity, suggesting existence of additional levels of regulation during these responses. Because of the well-established involvement of cytoplasmic signaling pathways in the cellular response to tumor necrosis factor-α (TNF-α), UVC, and IR using other types of assays, the role of TNF-α in the UVC response of HIV and c-jun was investigated in our cell system. We demonstrate that UVC and TNF-α activate HIV gene expression in a synergistic fashion, suggesting that it is unlikely that TNF-α is involved in UVC activation of HIV transcription in stably transfected HeLa cells. Moreover, maximum TNF-α stimulation resulted in one order of magnitude lower levels of HIV expression than that observed after UVC exposure. We also observed an additive effect of UVC and TNF-α on c-jun steady-state mRNA levels, suggestive of a partial overlap in activation mechanism of c-jun by UVC and TNF-α; yet these responses are distinct to some extent. Our results indicate that the HIV, and to some extent also the c-jun, transcriptional responses to UVC are not the result of TNF-α stimulation and subsequent downstream cytoplasmic signaling events in HeLa cells. In addition to the new data, this report also summarizes our current views regarding UVC-induced activations of HIV gene expression in stably transfected cells. (Author)

  2. The DNA damage response in mammalian oocytes

    Directory of Open Access Journals (Sweden)

    John eCarroll

    2013-06-01

    Full Text Available DNA damage is one of the most common insults that challenge all cells. To cope, an elaborate molecular and cellular response has evolved to sense, respond to and correct the damage. This allows the maintenance of DNA fidelity essential for normal cell viability and the prevention of genomic instability that can lead to tumour formation. In the context of oocytes, the impact of DNA damage is not one of tumour formation but of the maintenance of fertility. Mammalian oocytes are particularly vulnerable to DNA damage because physiologically they may lie dormant in the ovary for many years (>40 in humans until they receive the stimulus to grow and acquire the competence to become fertilized. The implication of this is that in some organisms, such as humans, oocytes face the danger of cumulative genetic damage for decades. Thus, the ability to detect and repair DNA damage is essential to maintain the supply of oocytes necessary for reproduction. Therefore, failure to confront DNA damage in oocytes could cause serious anomalies in the embryo that may be propagated in the form of mutations to the next generation allowing the appearance of hereditary disease. Despite the potential impact of DNA damage on reproductive capacity and genetic fidelity of embryos, the mechanisms available to the oocyte for monitoring and repairing such insults have remained largely unexplored until recently. Here, we review the different aspects of the response to DNA damage in mammalian oocytes. Specifically, we address the oocyte DNA damage response from embryonic life to adulthood and throughout oocyte development.

  3. The effect of 2-[(aminopropyl)amino] ethanethiol (WR 1065) on radiation-induced DNA damage and repair and cell progression in V79 cells

    International Nuclear Information System (INIS)

    Grdina, D.J.; Nagy, B.

    1986-01-01

    The radioprotector 2-[(aminopropyl)amino] ethanethiol (WR 1065) was investigated with respect to its ability to affect radiation-induced DNA damage and repair in V79 cells. At a concentration of 4mM, WR 1065 protected against the formation of single strand breaks (SSB), when present during irradiation. The protector appeared, however, to inhibit the subsequent postirradiation repair or rejoining of SSB. While repair was complete within 24h, the protector reduced the rate of repair by a factor of 3. This inhibitory effect on the rate of repair did not correlate with either measured differences in cell survival or mutagenesis. WR 1065 present in the growth medium inhibited the progression of cells through S-phase, and cell-doubling time following a 3h exposure to the protector was increased from 11 to 18h. These data are consistent with the property of thiols to inhibit DNA polymerase activity. It was concluded that, while the presence of WR 1065 during irradiation reduced SSB-DNA damage, its effect on the subsequent rejoining of these breaks could not be correlated with its observed effect on protecting against radiation-induced mutagenesis. (author)

  4. DNA Damage, Repair, and Cancer Metabolism

    Science.gov (United States)

    Turgeon, Marc-Olivier; Perry, Nicholas J. S.; Poulogiannis, George

    2018-01-01

    Although there has been a renewed interest in the field of cancer metabolism in the last decade, the link between metabolism and DNA damage/DNA repair in cancer has yet to be appreciably explored. In this review, we examine the evidence connecting DNA damage and repair mechanisms with cell metabolism through three principal links. (1) Regulation of methyl- and acetyl-group donors through different metabolic pathways can impact DNA folding and remodeling, an essential part of accurate double strand break repair. (2) Glutamine, aspartate, and other nutrients are essential for de novo nucleotide synthesis, which dictates the availability of the nucleotide pool, and thereby influences DNA repair and replication. (3) Reactive oxygen species, which can increase oxidative DNA damage and hence the load of the DNA-repair machinery, are regulated through different metabolic pathways. Interestingly, while metabolism affects DNA repair, DNA damage can also induce metabolic rewiring. Activation of the DNA damage response (DDR) triggers an increase in nucleotide synthesis and anabolic glucose metabolism, while also reducing glutamine anaplerosis. Furthermore, mutations in genes involved in the DDR and DNA repair also lead to metabolic rewiring. Links between cancer metabolism and DNA damage/DNA repair are increasingly apparent, yielding opportunities to investigate the mechanistic basis behind potential metabolic vulnerabilities of a substantial fraction of tumors. PMID:29459886

  5. Cloning and characterisation of the sagA gene of Aspergillus nidulans: a gene which affects sensitivity to DNA-damaging agents.

    Science.gov (United States)

    Jones, G W; Hooley, P; Farrington, S M; Shawcross, S G; Iwanejko, L A; Strike, P

    1999-03-01

    Mutations within the sagA gene of Aspergillus nidulans cause sensitisation to DNA-damaging chemicals but have no effect upon spontaneous or damage-induced mutation frequency. The sagA gene was cloned on a 19-kb cosmid-derived fragment by functional complementation of a sagA1 sagC3 double mutant; subsequently, a fragment of the gene was also isolated on a 3.9-kb genomic subclone. Initial sequencing of a small section of the 19-kb fragment allowed the design of primers that were subsequently used in RTPCR experiments to show that this DNA is transcribed. A 277-bp fragment derived from the transcribed region was used to screen an A. nidulans cDNA library, resulting in the isolation of a 1.4-kb partial cDNA clone which had sequence overlap with the genomic sagA fragment. This partial cDNA was incomplete but appeared to contain the whole coding region of sagA. The sagA1 mutant was shown to possess two mutations; a G-T transversion and a+ 1 frameshift due to insertion of a T. causing disruption to the C-terminal region of the SagA protein. Translation of the sagA cDNA predicts a protein of 378 amino acids, which has homology to the Saccharomyces cerevisiae End3 protein and also to certain mammalian proteins capable of causing cell transformation.

  6. Investigating the role of melanin in UVA/UVB- and hydrogen peroxide-induced cellular and mitochondrial ROS production and mitochondrial DNA damage in human melanoma cells.

    Science.gov (United States)

    Swalwell, Helen; Latimer, Jennifer; Haywood, Rachel M; Birch-Machin, Mark A

    2012-02-01

    Skin cancer incidence is dramatically increasing worldwide, with exposure to ultraviolet radiation (UVR) a predominant factor. The UVA component initiates oxidative stress in human skin, although its exact role in the initiation of skin cancer, particularly malignant melanoma, remains unclear and is controversial because there is evidence for a melanin-dependent mechanism in UVA-linked melanoma studies. Nonpigmented (CHL-1, A375), moderately pigmented (FM55, SKmel23), and highly pigmented (FM94, hyperpigmented FM55) human melanoma cell lines have been used to investigate UVA-induced production of reactive oxygen species using FACS analysis, at both the cellular (dihydrorhodamine-123) and the mitochondrial (MitoSOX) level, where most cellular stress is generated. For the first time, downstream mtDNA damage (utilizing a quantitative long-PCR assay) has been investigated. Using UVA, UVB, and H(2)O(2) as cellular stressors, we have explored the dual roles of melanin as a photoprotector and photosensitizer. The presence of melanin has no influence over cellular oxidative stress generation, whereas, in contrast, melanin protects against mitochondrial superoxide generation and mtDNA damage (one-way ANOVA with post hoc Tukey's analysis, Pmelanin binds directly to DNA, it acts as a direct photosensitizer of mtDNA damage during UVA irradiation (Pmelanin. Copyright © 2011 Elsevier Inc. All rights reserved.