Role of the DNA Damage Response in Human Papillomavirus RNA Splicing and Polyadenylation.

PubMed

Nilsson, Kersti; Wu, Chengjun; Schwartz, Stefan

2018-06-12

Human papillomaviruses (HPVs) have evolved to use the DNA repair machinery to replicate its DNA genome in differentiated cells. HPV activates the DNA damage response (DDR) in infected cells. Cellular DDR factors are recruited to the HPV DNA genome and position the cellular DNA polymerase on the HPV DNA and progeny genomes are synthesized. Following HPV DNA replication, HPV late gene expression is activated. Recent research has shown that the DDR factors also interact with RNA binding proteins and affects RNA processing. DDR factors activated by DNA damage and that associate with HPV DNA can recruit splicing factors and RNA binding proteins to the HPV DNA and induce HPV late gene expression. This induction is the result of altered alternative polyadenylation and splicing of HPV messenger RNA (mRNA). HPV uses the DDR machinery to replicate its DNA genome and to activate HPV late gene expression at the level of RNA processing.

Bioenergetic metabolites regulate base excision repair dependent cell death in response to DNA damage

PubMed Central

Tang, Jiang-bo; Goellner, Eva M.; Wang, Xiao-hong; Trivedi, Ram N.; Croix, Claudette M. St; Jelezcova, Elena; Svilar, David; Brown, Ashley R.; Sobol, Robert W.

2009-01-01

Base excision repair (BER) protein expression is important for resistance to DNA damage-induced cytotoxicity. Conversely, BER imbalance (Polß deficiency or repair inhibition) enhances cytotoxicity of radiation and chemotherapeutic DNA-damaging agents. Whereas inhibition of critical steps in the BER pathway result in the accumulation of cytotoxic DNA double-strand breaks, we report that DNA damage-induced cytotoxicity due to deficiency in the BER protein Polß triggers cell death dependent on PARP activation yet independent of poly(ADP-ribose) (PAR)-mediated AIF nuclear translocation or PARG, suggesting that cytotoxicity is not from PAR or PAR-catabolite signaling. Cell death is rescued by the NAD+ metabolite NMN and is synergistic with inhibition of NAD+ biosynthesis, demonstrating that DNA damage-induced cytotoxicity mediated via BER inhibition is primarily dependent on cellular metabolite bioavailability. We offer a mechanistic justification for the elevated alkylation-induced cytotoxicity of Polß deficient cells, suggesting a linkage between DNA repair, cell survival and cellular bioenergetics. PMID:20068071

11th International Conference of Radiation Research

DOE Office of Scientific and Technical Information (OSTI.GOV)

NONE

1999-07-18

Topics discussed in the conference included the following: Radiation Physics, Radiation Chemistry and modelling--Radiation physics and dosimetry; Electron transfer in biological media; Radiation chemistry; Biophysical and biochemical modelling; Mechanisms of DNA damage; Assays of DNA damage; Energy deposition in micro volumes; Photo-effects; Special techniques and technologies; Oxidative damage. Molecular and cellular effects-- Photobiology; Cell cycle effects; DNA damage: Strand breaks; DNA damage: Bases; DNA damage Non-targeted; DNA damage: other; Chromosome aberrations: clonal; Chromosomal aberrations: non-clonal; Interactions: Heat/Radiation/Drugs; Biochemical effects; Protein expression; Gene induction; Co-operative effects; ``Bystander'' effects; Oxidative stress effects; Recovery from radiation damage. DNA damage and repair -- DNAmore » repair genes; DNA repair deficient diseases; DNA repair enzymology; Epigenetic effects on repair; and Ataxia and ATM.« less

Contributions of DNA repair and damage response pathways to the non-linear genotoxic responses of alkylating agents

PubMed Central

Klapacz, Joanna; Pottenger, Lynn H.; Engelward, Bevin P.; Heinen, Christopher D.; Johnson, George E.; Clewell, Rebecca A.; Carmichael, Paul L.; Adeleye, Yeyejide; Andersen, Melvin E.

2016-01-01

From a risk assessment perspective, DNA-reactive agents are conventionally assumed to have genotoxic risks at all exposure levels, thus applying a linear extrapolation for low-dose responses. New approaches discussed here, including more diverse and sensitive methods for assessing DNA damage and DNA repair, strongly support the existence of measurable regions where genotoxic responses with increasing doses are insignificant relative to control. Model monofunctional alkylating agents have in vitro and in vivo datasets amenable to determination of points of departure (PoDs) for genotoxic effects. A session at the 2013 Society of Toxicology meeting provided an opportunity to survey the progress in understanding the biological basis of empirically-observed PoDs for DNA alkylating agents. Together with the literature published since, this review discusses cellular pathways activated by endogenous and exogenous alkylation DNA damage. Cells have evolved conserved processes that monitor and counteract a spontaneous steady-state level of DNA damage. The ubiquitous network of DNA repair pathways serves as the first line of defense for clearing of the DNA damage and preventing mutation. Other biological pathways discussed here that are activated by genotoxic stress include post-translational activation of cell cycle networks and transcriptional networks for apoptosis/cell death. The interactions of various DNA repair and DNA damage response pathways provide biological bases for the observed PoD behaviors seen with genotoxic compounds. Thus, after formation of DNA adducts, the activation of cellular pathways can lead to the avoidance a mutagenic outcome. The understanding of the cellular mechanisms acting within the low-dose region will serve to better characterize risks from exposures to DNA-reactive agents at environmentally-relevant concentrations. PMID:27036068

Contributions of DNA repair and damage response pathways to the non-linear genotoxic responses of alkylating agents.

PubMed

Klapacz, Joanna; Pottenger, Lynn H; Engelward, Bevin P; Heinen, Christopher D; Johnson, George E; Clewell, Rebecca A; Carmichael, Paul L; Adeleye, Yeyejide; Andersen, Melvin E

2016-01-01

From a risk assessment perspective, DNA-reactive agents are conventionally assumed to have genotoxic risks at all exposure levels, thus applying a linear extrapolation for low-dose responses. New approaches discussed here, including more diverse and sensitive methods for assessing DNA damage and DNA repair, strongly support the existence of measurable regions where genotoxic responses with increasing doses are insignificant relative to control. Model monofunctional alkylating agents have in vitro and in vivo datasets amenable to determination of points of departure (PoDs) for genotoxic effects. A session at the 2013 Society of Toxicology meeting provided an opportunity to survey the progress in understanding the biological basis of empirically-observed PoDs for DNA alkylating agents. Together with the literature published since, this review discusses cellular pathways activated by endogenous and exogenous alkylation DNA damage. Cells have evolved conserved processes that monitor and counteract a spontaneous steady-state level of DNA damage. The ubiquitous network of DNA repair pathways serves as the first line of defense for clearing of the DNA damage and preventing mutation. Other biological pathways discussed here that are activated by genotoxic stress include post-translational activation of cell cycle networks and transcriptional networks for apoptosis/cell death. The interactions of various DNA repair and DNA damage response pathways provide biological bases for the observed PoD behaviors seen with genotoxic compounds. Thus, after formation of DNA adducts, the activation of cellular pathways can lead to the avoidance of a mutagenic outcome. The understanding of the cellular mechanisms acting within the low-dose region will serve to better characterize risks from exposures to DNA-reactive agents at environmentally-relevant concentrations. Copyright © 2015 Elsevier B.V. All rights reserved.

The Translesion Polymerase Pol η Is Required for Efficient Epstein-Barr Virus Infectivity and Is Regulated by the Viral Deubiquitinating Enzyme BPLF1

PubMed Central

Dyson, Ossie F.; Pagano, Joseph S.

2017-01-01

ABSTRACT Epstein-Barr virus (EBV) infection and lytic replication are known to induce a cellular DNA damage response. We previously showed that the virally encoded BPLF1 protein interacts with and regulates several members of the translesion synthesis (TLS) pathway, a DNA damage tolerance pathway, and that these cellular factors enhance viral infectivity. BPLF1 is a late lytic cycle gene, but the protein is also packaged in the viral tegument, indicating that BPLF1 may function both early and late during infection. The BPLF1 protein expresses deubiquitinating activity that is strictly conserved across the Herpesviridae; mutation of the active site cysteine results in a loss of enzymatic activity. Infection with an EBV BPLF1 knockout virus results in decreased EBV infectivity. Polymerase eta (Pol η), a specialized DNA repair polymerase, functions in TLS and allows for DNA replication complexes to bypass lesions in DNA. Here we report that BPLF1 interacts with Pol η and that Pol η protein levels are increased in the presence of functional BPLF1. BPLF1 promotes a nuclear relocalization of Pol η molecules which are focus-like in appearance, consistent with the localization observed when Pol η is recruited to sites of DNA damage. Knockdown of Pol η resulted in decreased production of infectious virus, and further, Pol η was found to bind to EBV DNA, suggesting that it may allow for bypass of damaged viral DNA during its replication. The results suggest a mechanism by which EBV recruits cellular repair factors, such as Pol η, to sites of viral DNA damage via BPLF1, thereby allowing for efficient viral DNA replication. IMPORTANCE Epstein-Barr virus is the causative agent of infectious mononucleosis and infects approximately 90% of the world's population. It causes lymphomas in individuals with acquired and innate immune disorders and is strongly associated with Hodgkin's lymphoma, Burkitt's lymphoma, diffuse large B-cell lymphomas, nasopharyngeal carcinoma (NPC), and lymphomas that develop in organ transplant recipients. Cellular DNA damage is a major determinant in the establishment of oncogenic processes and is well studied, but there are few studies of endogenous repair of viral DNA. This work evaluates how EBV's BPLF1 protein and its conserved deubiquitinating activity regulate the cellular DNA repair enzyme polymerase eta and recruit it to potential sites of viral damage and replication, resulting in enhanced production of infectious virus. These findings help to establish how EBV enlists and manipulates cellular DNA repair factors during the viral lytic cycle, contributing to efficient infectious virion production. PMID:28724765

The Translesion Polymerase Pol η Is Required for Efficient Epstein-Barr Virus Infectivity and Is Regulated by the Viral Deubiquitinating Enzyme BPLF1.

PubMed

Dyson, Ossie F; Pagano, Joseph S; Whitehurst, Christopher B

2017-10-01

Epstein-Barr virus (EBV) infection and lytic replication are known to induce a cellular DNA damage response. We previously showed that the virally encoded BPLF1 protein interacts with and regulates several members of the translesion synthesis (TLS) pathway, a DNA damage tolerance pathway, and that these cellular factors enhance viral infectivity. BPLF1 is a late lytic cycle gene, but the protein is also packaged in the viral tegument, indicating that BPLF1 may function both early and late during infection. The BPLF1 protein expresses deubiquitinating activity that is strictly conserved across the Herpesviridae ; mutation of the active site cysteine results in a loss of enzymatic activity. Infection with an EBV BPLF1 knockout virus results in decreased EBV infectivity. Polymerase eta (Pol η), a specialized DNA repair polymerase, functions in TLS and allows for DNA replication complexes to bypass lesions in DNA. Here we report that BPLF1 interacts with Pol η and that Pol η protein levels are increased in the presence of functional BPLF1. BPLF1 promotes a nuclear relocalization of Pol η molecules which are focus-like in appearance, consistent with the localization observed when Pol η is recruited to sites of DNA damage. Knockdown of Pol η resulted in decreased production of infectious virus, and further, Pol η was found to bind to EBV DNA, suggesting that it may allow for bypass of damaged viral DNA during its replication. The results suggest a mechanism by which EBV recruits cellular repair factors, such as Pol η, to sites of viral DNA damage via BPLF1, thereby allowing for efficient viral DNA replication. IMPORTANCE Epstein-Barr virus is the causative agent of infectious mononucleosis and infects approximately 90% of the world's population. It causes lymphomas in individuals with acquired and innate immune disorders and is strongly associated with Hodgkin's lymphoma, Burkitt's lymphoma, diffuse large B-cell lymphomas, nasopharyngeal carcinoma (NPC), and lymphomas that develop in organ transplant recipients. Cellular DNA damage is a major determinant in the establishment of oncogenic processes and is well studied, but there are few studies of endogenous repair of viral DNA. This work evaluates how EBV's BPLF1 protein and its conserved deubiquitinating activity regulate the cellular DNA repair enzyme polymerase eta and recruit it to potential sites of viral damage and replication, resulting in enhanced production of infectious virus. These findings help to establish how EBV enlists and manipulates cellular DNA repair factors during the viral lytic cycle, contributing to efficient infectious virion production. Copyright © 2017 American Society for Microbiology.

Dynamic maps of UV damage formation and repair for the human genome

PubMed Central

Hu, Jinchuan; Adebali, Ogun; Adar, Sheera; Sancar, Aziz

2017-01-01

Formation and repair of UV-induced DNA damage in human cells are affected by cellular context. To study factors influencing damage formation and repair genome-wide, we developed a highly sensitive single-nucleotide resolution damage mapping method [high-sensitivity damage sequencing (HS–Damage-seq)]. Damage maps of both cyclobutane pyrimidine dimers (CPDs) and pyrimidine-pyrimidone (6-4) photoproducts [(6-4)PPs] from UV-irradiated cellular and naked DNA revealed that the effect of transcription factor binding on bulky adducts formation varies, depending on the specific transcription factor, damage type, and strand. We also generated time-resolved UV damage maps of both CPDs and (6-4)PPs by HS–Damage-seq and compared them to the complementary repair maps of the human genome obtained by excision repair sequencing to gain insight into factors that affect UV-induced DNA damage and repair and ultimately UV carcinogenesis. The combination of the two methods revealed that, whereas UV-induced damage is virtually uniform throughout the genome, repair is affected by chromatin states, transcription, and transcription factor binding, in a manner that depends on the type of DNA damage. PMID:28607063

Dynamic maps of UV damage formation and repair for the human genome.

PubMed

Hu, Jinchuan; Adebali, Ogun; Adar, Sheera; Sancar, Aziz

2017-06-27

Formation and repair of UV-induced DNA damage in human cells are affected by cellular context. To study factors influencing damage formation and repair genome-wide, we developed a highly sensitive single-nucleotide resolution damage mapping method [high-sensitivity damage sequencing (HS-Damage-seq)]. Damage maps of both cyclobutane pyrimidine dimers (CPDs) and pyrimidine-pyrimidone (6-4) photoproducts [(6-4)PPs] from UV-irradiated cellular and naked DNA revealed that the effect of transcription factor binding on bulky adducts formation varies, depending on the specific transcription factor, damage type, and strand. We also generated time-resolved UV damage maps of both CPDs and (6-4)PPs by HS-Damage-seq and compared them to the complementary repair maps of the human genome obtained by excision repair sequencing to gain insight into factors that affect UV-induced DNA damage and repair and ultimately UV carcinogenesis. The combination of the two methods revealed that, whereas UV-induced damage is virtually uniform throughout the genome, repair is affected by chromatin states, transcription, and transcription factor binding, in a manner that depends on the type of DNA damage.

Adenovirus Core Protein VII Downregulates the DNA Damage Response on the Host Genome

PubMed Central

Avgousti, Daphne C.; Della Fera, Ashley N.; Otter, Clayton J.; Herrmann, Christin; Pancholi, Neha J.

2017-01-01

ABSTRACT Viral manipulation of cellular proteins allows viruses to suppress host defenses and generate infectious progeny. Due to the linear double-stranded DNA nature of the adenovirus genome, the cellular DNA damage response (DDR) is considered a barrier to successful infection. The adenovirus genome is packaged with protein VII, a virally encoded histone-like core protein that is suggested to protect incoming viral genomes from detection by the cellular DNA damage machinery. We showed that protein VII localizes to host chromatin during infection, leading us to hypothesize that protein VII may affect DNA damage responses on the cellular genome. Here we show that protein VII at cellular chromatin results in a significant decrease in accumulation of phosphorylated H2AX (γH2AX) following irradiation, indicating that protein VII inhibits DDR signaling. The oncoprotein SET was recently suggested to modulate the DDR by affecting access of repair proteins to chromatin. Since protein VII binds SET, we investigated a role for SET in DDR inhibition by protein VII. We show that knockdown of SET partially rescues the protein VII-induced decrease in γH2AX accumulation on the host genome, suggesting that SET is required for inhibition. Finally, we show that knockdown of SET also allows ATM to localize to incoming viral genomes bound by protein VII during infection with a mutant lacking early region E4. Together, our data suggest that the protein VII-SET interaction contributes to DDR evasion by adenovirus. Our results provide an additional example of a strategy used by adenovirus to abrogate the host DDR and show how viruses can modify cellular processes through manipulation of host chromatin. IMPORTANCE The DNA damage response (DDR) is a cellular network that is crucial for maintaining genome integrity. DNA viruses replicating in the nucleus challenge the resident genome and must overcome cellular responses, including the DDR. Adenoviruses are prevalent human pathogens that can cause a multitude of diseases, such as respiratory infections and conjunctivitis. Here we describe how a small adenovirus core protein that localizes to host chromatin during infection can globally downregulate the DDR. Our study focuses on key players in the damage signaling pathway and highlights how viral manipulation of chromatin may influence access of DDR proteins to the host genome. PMID:28794020

HTLV-1 Tax Oncoprotein Subverts the Cellular DNA Damage Response via Binding to DNA-dependent Protein Kinase*S⃞

PubMed Central

Durkin, Sarah S.; Guo, Xin; Fryrear, Kimberly A.; Mihaylova, Valia T.; Gupta, Saurabh K.; Belgnaoui, S. Mehdi; Haoudi, Abdelali; Kupfer, Gary M.; Semmes, O. John

2008-01-01

Human T-cell leukemia virus type-1 is the causative agent for adult T-cell leukemia. Previous research has established that the viral oncoprotein Tax mediates the transformation process by impairing cell cycle control and cellular response to DNA damage. We showed previously that Tax sequesters huChk2 within chromatin and impairs the response to ionizing radiation. Here we demonstrate that DNA-dependent protein kinase (DNA-PK) is a member of the Tax·Chk2 nuclear complex. The catalytic subunit, DNA-PKcs, and the regulatory subunit, Ku70, were present. Tax-containing nuclear extracts showed increased DNA-PK activity, and specific inhibition of DNA-PK prevented Tax-induced activation of Chk2 kinase activity. Expression of Tax induced foci formation and phosphorylation of H2AX. However, Tax-induced constitutive signaling of the DNA-PK pathway impaired cellular response to new damage, as reflected in suppression of ionizing radiation-induced DNA-PK phosphorylation and γH2AX stabilization. Tax co-localized with phospho-DNA-PK into nuclear speckles and a nuclear excluded Tax mutant sequestered endogenous phospho-DNA-PK into the cytoplasm, suggesting that Tax interaction with DNA-PK is an initiating event. We also describe a novel interaction between DNA-PK and Chk2 that requires Tax. We propose that Tax binds to and stabilizes a protein complex with DNA-PK and Chk2, resulting in a saturation of DNA-PK-mediated damage repair response. PMID:18957425

Induction of a Cellular DNA Damage Response by Porcine Circovirus Type 2 Facilitates Viral Replication and Mediates Apoptotic Responses

PubMed Central

Wei, Li; Zhu, Shanshan; Wang, Jing; Quan, Rong; Yan, Xu; Li, Zixue; Hou, Lei; Wang, Naidong; Yang, Yi; Jiang, Haijun; Liu, Jue

2016-01-01

Cellular DNA damage response (DDR) triggered by infection of DNA viruses mediate cell cycle checkpoint activation, DNA repair, or apoptosis induction. In the present study, infection of porcine circovirus type 2 (PCV2), which serves as a major etiological agent of PCV2-associated diseases (PCVAD), was found to elicit a DNA damage response (DDR) as observed by the phosphorylation of H2AX and RPA32 following infection. The response requires active viral replication, and all the ATM (ataxia telangiectasia-mutated kinase), ATR (ATM- and Rad3-related kinase), and DNA-PK (DNA-dependent protein kinase) are the transducers of the DDR signaling events in the PCV2-infected cells as demonstrated by the phosphorylation of ATM, ATR, and DNA-PK signalings as well as reductions in their activations after treatment with specific kinase inhibitors. Inhibitions of ATM, ATR, and DNA-PK activations block viral replication and prevent apoptotic responses as observed by decreases in cleaved poly-ADP ribose polymerase (PARP) and caspase-3 as well as fragmented DNA following PCV2 infection. These results reveal that PCV2 is able to exploit the cellular DNA damage response machinery for its own efficient replication and for apoptosis induction, further extending our understanding for the molecular mechanism of PCV2 infection. PMID:27982097

The Role of the Transcriptional Response to DNA Replication Stress

PubMed Central

Herlihy, Anna E.; de Bruin, Robertus A.M.

2017-01-01

During DNA replication many factors can result in DNA replication stress. The DNA replication stress checkpoint prevents the accumulation of replication stress-induced DNA damage and the potential ensuing genome instability. A critical role for post-translational modifications, such as phosphorylation, in the replication stress checkpoint response has been well established. However, recent work has revealed an important role for transcription in the cellular response to DNA replication stress. In this review, we will provide an overview of current knowledge of the cellular response to DNA replication stress with a specific focus on the DNA replication stress checkpoint transcriptional response and its role in the prevention of replication stress-induced DNA damage. PMID:28257104

The Role of the Transcriptional Response to DNA Replication Stress.

PubMed

Herlihy, Anna E; de Bruin, Robertus A M

2017-03-02

During DNA replication many factors can result in DNA replication stress. The DNA replication stress checkpoint prevents the accumulation of replication stress-induced DNA damage and the potential ensuing genome instability. A critical role for post-translational modifications, such as phosphorylation, in the replication stress checkpoint response has been well established. However, recent work has revealed an important role for transcription in the cellular response to DNA replication stress. In this review, we will provide an overview of current knowledge of the cellular response to DNA replication stress with a specific focus on the DNA replication stress checkpoint transcriptional response and its role in the prevention of replication stress-induced DNA damage.

Lifestyle impacts on the aging associated expression of biomarkers of DNA damage and telomere dysfunction in human blood

PubMed Central

Song, Zhangfa; von Figura, Guido; Liu, Yan; Kraus, Johann M.; Torrice, Chad; Dillon, Patric; Rudolph-Watabe, Masami; Ju, Zhenyu; Kestler, Hans A.; Sanoff, Hanna; Rudolph, K. Lenhard

2010-01-01

Summary Cellular aging is characterised by telomere shortening, which can lead to uncapping of chromosome ends (telomere dysfunction) and that activation of DNA damage responses. There is some evidence the DNA damage accumulates during human aging and that lifestyle factors contribute to the accumulation of DNA damage. Recent studies have identified a set of serum markers that are induced by telomere dysfunction and DNA damage and these markers showed an increased expression in blood during human aging. Here, we investigated the influence of lifestyle factors (such as exercise, smoking, body mass) on the aging associated expression of serum markers of DNA damage (CRAMP, EF-1α, Stathmin, n-acetyl-glucosaminidase, and chitinase) in comparison to other described markers of cellular aging (p16INK4a upregulation and telomere shortening) in human peripheral blood. The study shows that lifestyle factors have an age-independent impact on the expression level of biomarkers of DNA damage. Smoking and increased body mass indices were associated with elevated levels of biomarkers of DNA damage independent of the age of the individuals. In contrast, exercise was associated with an age-independent reduction in the expression of biomarkers of DNA damage in human blood. The expression of biomarkers of DNA damage correlated positively with p16INK4a expression and negatively with telomere length in peripheral blood T-lymphocytes. Together, these data provide experimental evidence that both aging and lifestyle impact on the accumulation of DNA damage during human aging. PMID:20560902

A selective USP1-UAF1 inhibitor links deubiquitination to DNA damage responses

PubMed Central

Liang, Qin; Dexheimer, Thomas S; Zhang, Ping; Rosenthal, Andrew S; Villamil, Mark A; You, Changjun; Zhang, Qiuting; Chen, Junjun; Ott, Christine A; Sun, Hongmao; Luci, Diane K; Yuan, Bifeng; Simeonov, Anton; Jadhav, Ajit; Xiao, Hui; Wang, Yinsheng; Maloney, David J; Zhuang, Zhihao

2014-01-01

Protein ubiquitination and deubiquitination are central to the control of a large number of cellular pathways and signaling networks in eukaryotes. Although the essential roles of ubiquitination have been established in the eukaryotic DNA damage response, the deubiquitination process remains poorly defined. Chemical probes that perturb the activity of deubiquitinases (DUBs) are needed to characterize the cellular function of deubiquitination. Here we report ML323 (2), a highly potent inhibitor of the USP1-UAF1 deubiquitinase complex with excellent selectivity against human DUBs, deSUMOylase, deneddylase and unrelated proteases. Using ML323, we interrogated deubiquitination in the cellular response to UV- and cisplatin-induced DNA damage and revealed new insights into the requirement of deubiquitination in the DNA translesion synthesis and Fanconi anemia pathways. Moreover, ML323 potentiates cisplatin cytotoxicity in non-small cell lung cancer and osteosarcoma cells. Our findings point to USP1-UAF1 as a key regulator of the DNA damage response and a target for overcoming resistance to the platinum-based anticancer drugs. PMID:24531842

Metallic ions released from stainless steel, nickel-free, and titanium orthodontic alloys: toxicity and DNA damage.

PubMed

Ortiz, Antonio José; Fernández, Esther; Vicente, Ascensión; Calvo, José L; Ortiz, Clara

2011-09-01

The aims of this study were to determine the amounts of metallic ions that stainless steel, nickel-free, and titanium alloys release to a culture medium, and to evaluate the cellular viability and DNA damage of cultivated human fibroblasts with those mediums. The metals were extracted from 10 samples (each consisting of 4 buccal tubes and 20 brackets) of the 3 orthodontic alloys that were submerged for 30 days in minimum essential medium. Next, the determination of metals was performed by using inductively coupled plasma mass spectrometry, cellular viability was assessed by using the tetrazolium reduction assay (MTT assay) (3-[4,5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide), and DNA damage was determined with the Comet assay. The metals measured in all the samples were Ti(47), Cr(52), Mn(55), Co(59), Ni(60), Mo(92), Fe(56), Cu(63), Zn(66), As(75), Se(78), Cd(111), and Pb(208). The cellular viability of the cultured fibroblasts incubated for 7 days with minimum essential medium, with the stainless steel alloy submerged, was close to 0%. Moreover, high concentrations of titanium, chromium, manganese, cobalt, nickel, molybdenum, iron, copper, and zinc were detected. The nickel-free alloy released lower amounts of ions to the medium. The greatest damage in the cellular DNA, measured as the olive moment, was also produced by the stainless steel alloy followed by the nickel-free alloy. Conversely, the titanium alloy had an increased cellular viability and did not damage the cellular DNA, as compared with the control values. The titanium brackets and tubes are the most biocompatible of the 3 alloys studied. Copyright © 2011 American Association of Orthodontists. Published by Mosby, Inc. All rights reserved.

Cidofovir is active against human papillomavirus positive and negative head and neck and cervical tumor cells by causing DNA damage as one of its working mechanisms.

PubMed

Mertens, Barbara; Nogueira, Tatiane; Stranska, Ruzena; Naesens, Lieve; Andrei, Graciela; Snoeck, Robert

2016-07-26

Human papillomavirus (HPV) causes cervical cancer and a large fraction of head and neck squamous cell carcinomas (HNSCC). Cidofovir (CDV) proved efficacious in the treatment of several HPV-induced benign and malignant hyper proliferations. To provide a better insight into how CDV selectively eradicates transformed cells, HPV+ and HPV- cervical carcinoma and HNSCC cell lines were compared to normal cells for antiproliferative effects, CDV metabolism, drug incorporation into cellular DNA, and DNA damage. Incorporation of CDV into cellular DNA was higher in tumor cells than in normal cells and correlated with CDV antiproliferative effects, which were independent of HPV status. Increase in phospho-ATM levels was detected following CDV exposure and higher levels of γ-H2AX (a quantitative marker of double-strand breaks) were measured in tumor cells compared to normal cells. A correlation between DNA damage and CDV incorporation into DNA was found but not between DNA damage and CDV antiproliferative effects. These data indicate that CDV antiproliferative effects result from incorporation of the drug into DNA causing DNA damage. However, the anti-tumor effects of CDV cannot be exclusively ascribed to DNA damage. Furthermore, CDV can be considered a promising broad spectrum anti-cancer agent, not restricted to HPV+ lesions.

The Role of Non-Targeted Effects as Mediators in the Biological Effects of Proton Irradiation

NASA Technical Reports Server (NTRS)

Cucinotta, Francis A.; Dicello, John F.

2006-01-01

In recent years, the hypothesis that non-DNA targets are primary initiators and mediators of the biological effects of ionizing radiation, such as proton beams and heavy ions, has gained much interest. These phenomena have been denoted as non-targeted or bystander effects to distinguish them from the more traditionally studied model that focuses on direct damage to DNA causing chromosomal rearrangements and mutations as causative of most biological endpoints such as cell killing, tissue damage, and cancer. We review cellular and extra-cellular structures and signal transduction pathways that have been implemented in these recent studies. Non-targeted effects of interest include oxidative damage to the cytoplasm and mitochondria, disruption of the extra-cellular matrix, and modification of cytokine signaling including TGF-beta, and gap junction communication. We present an introduction to these targets and pathways, and contrast there role with DNA damage pathways.

What’s the Damage? The Impact of Pathogens on Pathways that Maintain Host Genome Integrity

PubMed Central

Weitzman, Matthew D.; Weitzman, Jonathan B.

2014-01-01

Maintaining genome integrity and transmission of intact genomes is critical for cellular, organismal, and species survival. Cells can detect damaged DNA, activate checkpoints, and either enable DNA repair or trigger apoptosis to eliminate the damaged cell. Aberrations in these mechanisms lead to somatic mutations and genetic instability, which are hallmarks of cancer. Considering the long history of host-microbe coevolution, an impact of microbial infection on host genome integrity is not unexpected, and emerging links between microbial infections and oncogenesis further reinforce this idea. In this review, we compare strategies employed by viruses, bacteria, and parasites to alter, subvert, or otherwise manipulate host DNA damage and repair pathways. We highlight how microbes contribute to tumorigenesis by directly inducing DNA damage, inactivating checkpoint controls, or manipulating repair processes. We also discuss indirect effects resulting from inflammatory responses, changes in cellular metabolism, nuclear architecture, and epigenome integrity, and the associated evolutionary tradeoffs. PMID:24629335

A damaged DNA binding protein 2 mutation disrupting interaction with proliferating-cell nuclear antigen affects DNA repair and confers proliferation advantage.

PubMed

Perucca, Paola; Mocchi, Roberto; Guardamagna, Isabella; Bassi, Elisabetta; Sommatis, Sabrina; Nardo, Tiziana; Prosperi, Ennio; Stivala, Lucia Anna; Cazzalini, Ornella

2018-06-01

In mammalian cells, Nucleotide Excision Repair (NER) plays a role in removing DNA damage induced by UV radiation. In Global Genome-NER subpathway, DDB2 protein forms a complex with DDB1 (UV-DDB), recognizing photolesions. During DNA repair, DDB2 interacts directly with PCNA through a conserved region in N-terminal tail and this interaction is important for DDB2 degradation. In this work, we sought to investigate the role of DDB2-PCNA association in DNA repair and cell proliferation after UV-induced DNA damage. To this end, stable clones expressing DDB2 Wt and DDB2 PCNA- were used. We have found that cells expressing a mutant DDB2 show inefficient photolesions removal, and a concomitant lack of binding to damaged DNA in vitro. Unexpected cellular behaviour after DNA damage, such as UV-resistance, increased cell growth and motility were found in DDB2 PCNA- stable cell clones, in which the most significant defects in cell cycle checkpoint were observed, suggesting a role in the new cellular phenotype. Based on these findings, we propose that DDB2-PCNA interaction may contribute to a correct DNA damage response for maintaining genome integrity. Copyright © 2018 Elsevier B.V. All rights reserved.

Toxicity evaluation of e-juice and its soluble aerosols generated by electronic cigarettes using recombinant bioluminescent bacteria responsive to specific cellular damages.

PubMed

Bharadwaj, Shiv; Mitchell, Robert J; Qureshi, Anjum; Niazi, Javed H

2017-04-15

Electronic-cigarettes (e-cigarette) are widely used as an alternative to traditional cigarettes but their safety is not well established. Herein, we demonstrate and validate an analytical method to discriminate the deleterious effects of e-cigarette refills (e-juice) and soluble e-juice aerosol (SEA) by employing stress-specific bioluminescent recombinant bacterial cells (RBCs) as whole-cell biosensors. These RBCs carry luxCDABE-operon tightly controlled by promoters that specifically induced to DNA damage (recA), superoxide radicals (sodA), heavy metals (copA) and membrane damage (oprF). The responses of the RBCs following exposure to various concentrations of e-juice/SEA was recorded in real-time that showed dose-dependent stress specific-responses against both the e-juice and vaporized e-juice aerosols produced by the e-cigarette. We also established that high doses of e-juice (4-folds diluted) lead to cell death by repressing the cellular machinery responsible for repairing DNA-damage, superoxide toxicity, ion homeostasis and membrane damage. SEA also caused the cellular damages but the cells showed enhanced bioluminescence expression without significant growth inhibition, indicating that the cells activated their global defense system to repair these damages. DNA fragmentation assay also revealed the disintegration of total cellular DNA at sub-toxic doses of e-juice. Despite their state of matter, the e-juice and its aerosols induce cytotoxicity and alter normal cellular functions, respectively that raises concerns on use of e-cigarettes as alternative to traditional cigarette. The ability of RBCs in detecting both harmful effects and toxicity mechanisms provided a fundamental understanding of biological response to e-juice and aerosols. Copyright © 2016 Elsevier B.V. All rights reserved.

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.

PubMed

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, P<0.001). We show that if melanin binds directly to DNA, it acts as a direct photosensitizer of mtDNA damage during UVA irradiation (P<0.001), providing evidence for the dual roles of melanin. Copyright © 2011 Elsevier Inc. All rights reserved.

Effects of Spaceflight on Molecular and Cellular Responses to Bleomycin-induced DNA Damages in Confluent Human Fibroblasts

NASA Astrophysics Data System (ADS)

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

2016-07-01

Spaceflights expose human beings to various risk factors. Among them are microgravity related physiological stresses in immune, cytoskeletal, and cardiovascular systems, and space radiation related elevation of cancer risk. Cosmic radiation consists of energetic protons and other heavier charged particles that induce DNA damages. Effective DNA damage response and repair mechanism is important to maintain genomic integrity and reduce cancer risk. There were studies on effects of spaceflight and microgravity on DNA damage response in cell and animal models, but the published results were mostly conflicting and inconsistent. To investigate effects of spaceflight on molecular and cellular responses to DNA damages, bleomycin, an anti-cancer drug and radiomimetic reagent, was used to induce DNA damages in confluent human fibroblasts flown to the International Space Station (ISS) and on ground. After exposure to 1.0 mg/ml bleomycin for 3 hours, cells were fixed for immunofluorescence assays and for RNA preparation. Extents of DNA damages were quantified by focus pattern and focus number counting of phosphorylated histone protein H2AX (γg-H2AX). The cells on the ISS showed modestly increased average focus counts per nucleus while the distribution of patterns was similar to that on the ground. PCR array analysis showed that expressions of several genes, including CDKN1A and PCNA, were significantly changed in response to DNA damages induced by bleomycin in both flight and ground control cells. However, there were no significant differences in the overall expression profiles of DNA damage response genes between the flight and ground samples. Analysis of cellular proliferation status with Ki-67 staining showed a slightly higher proliferating population in cells on the ISS than those on ground. Our results suggested that the difference in γg-H2AX focus counts between flight and ground was due to the higher percentage of proliferating cells in space, but spaceflight did not significantly affect initial transcriptional responses to bleomycin treatment in the selected genes in the DNA damage signaling pathways.

Increased oxidative phosphorylation in response to acute and chronic DNA damage

PubMed Central

Brace, Lear E; Vose, Sarah C; Stanya, Kristopher; Gathungu, Rose M; Marur, Vasant R; Longchamp, Alban; Treviño-Villarreal, Humberto; Mejia, Pedro; Vargas, Dorathy; Inouye, Karen; Bronson, Roderick T; Lee, Chih-Hao; Neilan, Edward; Kristal, Bruce S; Mitchell, James R

2016-01-01

Accumulation of DNA damage is intricately linked to aging, aging-related diseases and progeroid syndromes such as Cockayne syndrome (CS). Free radicals from endogenous oxidative energy metabolism can damage DNA, however the potential of acute or chronic DNA damage to modulate cellular and/or organismal energy metabolism remains largely unexplored. We modeled chronic endogenous genotoxic stress using a DNA repair-deficient Csa−/−|Xpa−/− mouse model of CS. Exogenous genotoxic stress was modeled in mice in vivo and primary cells in vitro treated with different genotoxins giving rise to diverse spectrums of lesions, including ultraviolet radiation, intrastrand crosslinking agents and ionizing radiation. Both chronic endogenous and acute exogenous genotoxic stress increased mitochondrial fatty acid oxidation (FAO) on the organismal level, manifested by increased oxygen consumption, reduced respiratory exchange ratio, progressive adipose loss and increased FAO in tissues ex vivo. In multiple primary cell types, the metabolic response to different genotoxins manifested as a cell-autonomous increase in oxidative phosphorylation (OXPHOS) subsequent to a transient decline in steady-state NAD+ and ATP levels, and required the DNA damage sensor PARP-1 and energy-sensing kinase AMPK. We conclude that increased FAO/OXPHOS is a general, beneficial, adaptive response to DNA damage on cellular and organismal levels, illustrating a fundamental link between genotoxic stress and energy metabolism driven by the energetic cost of DNA damage. Our study points to therapeutic opportunities to mitigate detrimental effects of DNA damage on primary cells in the context of radio/chemotherapy or progeroid syndromes. PMID:28721274

Nanoparticles can cause DNA damage across a cellular barrier

NASA Astrophysics Data System (ADS)

Bhabra, Gevdeep; Sood, Aman; Fisher, Brenton; Cartwright, Laura; Saunders, Margaret; Evans, William Howard; Surprenant, Annmarie; Lopez-Castejon, Gloria; Mann, Stephen; Davis, Sean A.; Hails, Lauren A.; Ingham, Eileen; Verkade, Paul; Lane, Jon; Heesom, Kate; Newson, Roger; Case, Charles Patrick

2009-12-01

The increasing use of nanoparticles in medicine has raised concerns over their ability to gain access to privileged sites in the body. Here, we show that cobalt-chromium nanoparticles (29.5 +/- 6.3 nm in diameter) can damage human fibroblast cells across an intact cellular barrier without having to cross the barrier. The damage is mediated by a novel mechanism involving transmission of purine nucleotides (such as ATP) and intercellular signalling within the barrier through connexin gap junctions or hemichannels and pannexin channels. The outcome, which includes DNA damage without significant cell death, is different from that observed in cells subjected to direct exposure to nanoparticles. Our results suggest the importance of indirect effects when evaluating the safety of nanoparticles. The potential damage to tissues located behind cellular barriers needs to be considered when using nanoparticles for targeting diseased states.

Approaches for characterizing threshold dose-response relationships for DNA-damage pathways involved in carcinogenicity in vivo and micronuclei formation in vitro.

PubMed

Clewell, Rebecca A; Andersen, Melvin E

2016-05-01

Assessing the shape of dose-response curves for DNA-damage in cellular systems and for the consequences of DNA damage in intact animals remains a controversial topic. This overview looks at aspects of the pharmacokinetics (PK) and pharmacodynamics (PD) of cellular DNA-damage/repair and their role in defining the shape of dose-response curves using an in vivo example with formaldehyde and in vitro examples for micronuclei (MN) formation with several test compounds. Formaldehyde is both strongly mutagenic and an endogenous metabolite in cells. With increasing inhaled concentrations, there were transitions in gene changes, from activation of selective stress pathway genes at low concentrations, to activation of pathways for cell-cycle control, p53-DNA damage, and stem cell niche pathways at higher exposures. These gene expression changes were more consistent with dose-dependent transitions in the PD responses to formaldehyde in epithelial cells in the intact rat rather than the low-dose linear extrapolation methods currently used for carcinogens. However, more complete PD explanations of non-linear dose response for creation of fixed damage in cells require detailed examination of cellular responses in vitro using measures of DNA damage and repair that are not easily accessible in the intact animal. In the second section of the article, we illustrate an approach from our laboratory that develops fit-for-purpose, in vitro assays and evaluates the PD of DNA damage and repair through studies using prototypical DNA-damaging agents. Examination of a broad range of responses in these cells showed that transcriptional upregulation of cell cycle control and DNA repair pathways only occurred at doses higher than those causing overt damage fixed damage-measured as MN formation. Lower levels of damage appear to be handled by post-translational repair process using pre-existing proteins. In depth evaluation of the PD properties of one such post-translational process (formation of DNA repair centers; DRCs) has indicated that the formation of DRCs and their ability to complete repair before replication are consistent with threshold behaviours for mutagenesis and, by extension, with chemical carcinogenesis. © The Author 2016. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Close encounters for the first time: Helicase interactions with DNA damage.

PubMed

Khan, Irfan; Sommers, Joshua A; Brosh, Robert M

2015-09-01

DNA helicases are molecular motors that harness the energy of nucleoside triphosphate hydrolysis to unwinding structured DNA molecules that must be resolved during cellular replication, DNA repair, recombination, and transcription. In vivo, DNA helicases are expected to encounter a wide spectrum of covalent DNA modifications to the sugar phosphate backbone or the nitrogenous bases; these modifications can be induced by endogenous biochemical processes or exposure to environmental agents. The frequency of lesion abundance can vary depending on the lesion type. Certain adducts such as oxidative base modifications can be quite numerous, and their effects can be helix-distorting or subtle perturbations to DNA structure. Helicase encounters with specific DNA lesions and more novel forms of DNA damage will be discussed. We will also review the battery of assays that have been used to characterize helicase-catalyzed unwinding of damaged DNA substrates. Characterization of the effects of specific DNA adducts on unwinding by various DNA repair and replication helicases has proven to be insightful for understanding mechanistic and biological aspects of helicase function in cellular DNA metabolism. Published by Elsevier B.V.

Repair of Oxidative DNA Damage in Saccharomyces cerevisiae.

PubMed

Chalissery, Jisha; Jalal, Deena; Al-Natour, Zeina; Hassan, Ahmed H

2017-03-01

Malfunction of enzymes that detoxify reactive oxygen species leads to oxidative attack on biomolecules including DNA and consequently activates various DNA repair pathways. The nature of DNA damage and the cell cycle stage at which DNA damage occurs determine the appropriate repair pathway to rectify the damage. Oxidized DNA bases are primarily repaired by base excision repair and nucleotide incision repair. Nucleotide excision repair acts on lesions that distort DNA helix, mismatch repair on mispaired bases, and homologous recombination and non-homologous end joining on double stranded breaks. Post-replication repair that overcomes replication blocks caused by DNA damage also plays a crucial role in protecting the cell from the deleterious effects of oxidative DNA damage. Mitochondrial DNA is also prone to oxidative damage and is efficiently repaired by the cellular DNA repair machinery. In this review, we discuss the DNA repair pathways in relation to the nature of oxidative DNA damage in Saccharomyces cerevisiae. Copyright © 2017 Elsevier B.V. All rights reserved.

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

NASA Technical Reports Server (NTRS)

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 bleomycin treatment.

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

NASA Technical Reports Server (NTRS)

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

2015-01-01

Living organisms are constantly exposed to space radiation that consists of energetic protons and other heavier charged particles. Whether spaceflight factors, microgravity in particular, affects on the cellular response to DNA damage induced by exposures to radiation or other toxic chemicals will have an impact on the radiation risks for the astronauts, as well as on the mutation rate in microorganisms, is still an open question. Although the 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 the effects of spaceflight on the cellular response 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 induces DNA damages including the double strand breaks (DSB) similar to the ionizing radiation. Damage in the DNA was measured by the phosphorylation of a histone protein H2AX (-H2AX), which showed slightly more foci in the cells on ISS than in the ground control. The expression of genes involved in the 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 Ti-67 signals. Our results suggested that the difference in -H2AX between flight and ground was due to the faster growth rate of the cells in space, but spaceflight did not affect the response of the DNA damage response genes to bleomycin treatment.

Oxidative DNA damage during sleep periods among nightshift workers.

PubMed

Bhatti, Parveen; Mirick, Dana K; Randolph, Timothy W; Gong, Jicheng; Buchanan, Diana Taibi; Zhang, Junfeng Jim; Davis, Scott

2016-08-01

Oxidative DNA damage may be increased among nightshift workers because of suppression of melatonin, a cellular antioxidant, and/or inflammation related to sleep disruption. However, oxidative DNA damage has received limited attention in previous studies of nightshift work. From two previous cross-sectional studies, urine samples collected during a night sleep period for 217 dayshift workers and during day and night sleep (on their first day off) periods for 223 nightshift workers were assayed for 8-hydroxydeoxyguanosine (8-OH-dG), a marker of oxidative DNA damage, using high-performance liquid chromatography with electrochemical detection. Urinary measures of 6-sulfatoxymelatonin (aMT6s), a marker of circulating melatonin levels, and actigraphy-based sleep quality data were also available. Nightshift workers during their day sleep periods excreted 83% (p=0.2) and 77% (p=0.03) of the 8-OH-dG that dayshift workers and they themselves, respectively, excreted during their night sleep periods. Among nightshift workers, higher aMT6s levels were associated with higher urinary 8-OH-dG levels, and an inverse U-shaped trend was observed between 8-OH-dG levels and sleep efficiency and sleep duration. Reduced excretion of 8-OH-dG among nightshift workers during day sleep may reflect reduced functioning of DNA repair machinery, which could potentially lead to increased cellular levels of oxidative DNA damage. Melatonin disruption among nightshift workers may be responsible for the observed effect, as melatonin is known to enhance repair of oxidative DNA damage. Quality of sleep may similarly impact DNA repair. Cellular levels of DNA damage will need to be evaluated in future studies to help interpret these findings. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/

Monitoring regulation of DNA repair activities of cultured cells in-gel using the comet assay

PubMed Central

Nickson, Catherine M.; Parsons, Jason L.

2014-01-01

Base excision repair (BER) is the predominant cellular mechanism by which human cells repair DNA base damage, sites of base loss, and DNA single strand breaks of various complexity, that are generated in their thousands in every human cell per day as a consequence of cellular metabolism and exogenous agents, including ionizing radiation. Over the last three decades the comet assay has been employed in scientific research to examine the cellular response to these types of DNA damage in cultured cells, therefore revealing the efficiency and capacity of BER. We have recently pioneered new research demonstrating an important role for post-translational modifications (particularly ubiquitylation) in the regulation of cellular levels of BER proteins, and that subtle changes (∼20–50%) in protein levels following siRNA knockdown of E3 ubiquitin ligases or deubiquitylation enzymes can manifest in significant changes in DNA repair capacity monitored using the comet assay. For example, we have shown that the E3 ubiquitin ligase Mule, the tumor suppressor protein ARF, and the deubiquitylation enzyme USP47 modulate DNA repair by controlling cellular levels of DNA polymerase β, and also that polynucleotide kinase phosphatase levels are controlled by ATM-dependant phosphorylation and Cul4A–DDB1–STRAP-dependent ubiquitylation. In these studies we employed a modification of the comet assay whereby cultured cells, following DNA damage treatment, are embedded in agarose and allowed to repair in-gel prior to lysis and electrophoresis. Whilst this method does have its limitations, it avoids the extensive cell culture-based processing associated with the traditional approach using attached cells and also allows for the examination of much more precise DNA repair kinetics. In this review we will describe, using this modified comet assay, our accumulating evidence that ubiquitylation-dependant regulation of BER proteins has important consequences for overall cellular DNA repair capacity. PMID:25076968

Dimer monomer transition and dimer re-formation play important role for ATM cellular function during DNA repair

DOE Office of Scientific and Technical Information (OSTI.GOV)

Du, Fengxia; Zhang, Minjie; University of Chinese Academy of Sciences, Beijing 100049

2014-10-03

Highlights: • ATM phosphorylates the opposite strand of the dimer in response to DNA damage. • The PETPVFRLT box of ATM plays a key role in its dimer dissociation in DNA repair. • The dephosphorylation of ATM is critical for dimer re-formation after DNA repair. - Abstract: The ATM protein kinase, is a serine/threonine protein kinase that is recruited and activated by DNA double-strand breaks, mediates responses to ionizing radiation in mammalian cells. Here we show that ATM is held inactive in unirradiated cells as a dimer and phosphorylates the opposite strand of the dimer in response to DNA damage.more » Cellular irradiation induces rapid intermolecular autophosphorylation of serine 1981 that causes dimer dissociation and initiates cellular ATM kinase activity. ATM cannot phosphorylate the substrates when it could not undergo dimer monomer transition. After DNA repair, the active monomer will undergo dephosphorylation to form dimer again and dephosphorylation is critical for dimer re-formation. Our work reveals novel function of ATM dimer monomer transition and explains why ATM dimer monomer transition plays such important role for ATM cellular activity during DNA repair.« less

DNA Damage Response, Redox Status and Hematopoiesis

PubMed Central

Weiss, Cary N.; Ito, Keisuke

2013-01-01

The ability of hematopoietic stem cells (HSCs) to self-renew and differentiate into progenitors is essential for homeostasis of the hematopoietic system. The longevity of HSCs makes them vulnerable to accumulating DNA damage, which may be leukemogenic or result in senescence and cell death. Additionally, the ability of HSCs to self-renew and differentiate allows DNA damage to spread throughout the hematologic system, leaving the organism vulnerable to disease. In this review we discuss cell fate decisions made in the face of DNA damage and other cellular stresses, and the role of reactive oxygen species in the long-term maintenance of HSCs and their DNA damage response. PMID:24041596

Salivary α-amylase, serum albumin, and myoglobin protect against DNA-damaging activities of ingested dietary agents in vitro

PubMed Central

Hossain, M. Zulfiquer; Patel, Kalpesh; Kern, Scott E.

2014-01-01

Potent DNA-damaging activities were seen in vitro from dietary chemicals found in coffee, tea, and liquid smoke. A survey of tea varieties confirmed genotoxic activity to be widespread. Constituent pyrogallol-like polyphenols (PLPs) such as epigallocatechin-3-gallate (EGCG), pyrogallol, and gallic acid were proposed as a major source of DNA-damaging activities, inducing DNA double-strand breaks in the p53R assay, a well characterized assay sensitive to DNA strand breaks, and comet assay. Paradoxically, their consumption does not lead to the kind of widespread cellular toxicity and acute disease that might be expected from genotoxic exposure. Existing physiological mechanisms could limit DNA damage from dietary injurants. Serum albumin and salivary α-amylase are known to bind EGCG. Salivary α-amylase, serum albumin, and myoglobin, but not salivary proline-rich proteins, reduced damage from tea, coffee, and PLPs, but did not inhibit damage from the chemotherapeutics etoposide and camptothecin. This represents a novel function for saliva in addition to its known functions including protection against tannins. Cell populations administered repeated pyrogallol exposures had abatement of measured DNA damage by two weeks, indicating an innate cellular adaptation. We suggest that layers of physiological protections may exist toward natural dietary products to which animals have had high-level exposure over evolution. PMID:24842839

Salivary α-amylase, serum albumin, and myoglobin protect against DNA-damaging activities of ingested dietary agents in vitro.

PubMed

Hossain, M Zulfiquer; Patel, Kalpesh; Kern, Scott E

2014-08-01

Potent DNA-damaging activities were seen in vitro from dietary chemicals found in coffee, tea, and liquid smoke. A survey of tea varieties confirmed genotoxic activity to be widespread. Constituent pyrogallol-like polyphenols (PLPs) such as epigallocatechin-3-gallate (EGCG), pyrogallol, and gallic acid were proposed as a major source of DNA-damaging activities, inducing DNA double-strand breaks in the p53R assay, a well characterized assay sensitive to DNA strand breaks, and comet assay. Paradoxically, their consumption does not lead to the kind of widespread cellular toxicity and acute disease that might be expected from genotoxic exposure. Existing physiological mechanisms could limit DNA damage from dietary injurants. Serum albumin and salivary α-amylase are known to bind EGCG. Salivary α-amylase, serum albumin, and myoglobin, but not salivary proline-rich proteins, reduced damage from tea, coffee, and PLPs, but did not inhibit damage from the chemotherapeutics etoposide and camptothecin. This represents a novel function for saliva in addition to its known functions including protection against tannins. Cell populations administered repeated pyrogallol exposures had abatement of measured DNA damage by two weeks, indicating an innate cellular adaptation. We suggest that layers of physiological protections may exist toward natural dietary products to which animals have had high-level exposure over evolution. Copyright © 2014 Elsevier Ltd. All rights reserved.

Interplay between Ubiquitin, SUMO, and Poly(ADP-Ribose) in the Cellular Response to Genotoxic Stress

PubMed Central

Pellegrino, Stefania; Altmeyer, Matthias

2016-01-01

Cells employ a complex network of molecular pathways to cope with endogenous and exogenous genotoxic stress. This multilayered response ensures that genomic lesions are efficiently detected and faithfully repaired in order to safeguard genome integrity. The molecular choreography at sites of DNA damage relies heavily on post-translational modifications (PTMs). Protein modifications with ubiquitin and the small ubiquitin-like modifier SUMO have recently emerged as important regulatory means to coordinate DNA damage signaling and repair. Both ubiquitylation and SUMOylation can lead to extensive chain-like protein modifications, a feature that is shared with yet another DNA damage-induced PTM, the modification of proteins with poly(ADP-ribose) (PAR). Chains of ubiquitin, SUMO, and PAR all contribute to the multi-protein assemblies found at sites of DNA damage and regulate their spatio-temporal dynamics. Here, we review recent advancements in our understanding of how ubiquitin, SUMO, and PAR coordinate the DNA damage response and highlight emerging examples of an intricate interplay between these chain-like modifications during the cellular response to genotoxic stress. PMID:27148359

Mechanisms of DNA Damage Response to Targeted Irradiation in Organotypic 3D Skin Cultures

PubMed Central

Acheva, Anna; Ghita, Mihaela; Patel, Gaurang; Prise, Kevin M.; Schettino, Giuseppe

2014-01-01

DNA damage (caused by direct cellular exposure and bystander signaling) and the complex pathways involved in its repair are critical events underpinning cellular and tissue response following radiation exposures. There are limited data addressing the dynamics of DNA damage induction and repair in the skin particularly in areas not directly exposed. Here we investigate the mechanisms regulating DNA damage, repair, intracellular signalling and their impact on premature differentiation and development of inflammatory-like response in the irradiated and surrounding areas of a 3D organotypic skin model. Following localized low-LET irradiation (225 kVp X-rays), low levels of 53BP1 foci were observed in the 3D model (3.8±0.28 foci/Gy/cell) with foci persisting and increasing in size up to 48 h post irradiation. In contrast, in cell monolayers 14.2±0.6 foci/Gy/cell and biphasic repair kinetics with repair completed before 24 h was observed. These differences are linked to differences in cellular status with variable level of p21 driving apoptotic signalling in 2D and accelerated differentiation in both the directly irradiated and bystander areas of the 3D model. The signalling pathways utilized by irradiated keratinocytes to induce DNA damage in non-exposed areas of the skin involved the NF-κB transcription factor and its downstream target COX-2. PMID:24505255

Effects of methyl and inorganic mercury exposure on genome homeostasis and mitochondrial function in Caenorhabditis elegans.

PubMed

Wyatt, Lauren H; Luz, Anthony L; Cao, Xiou; Maurer, Laura L; Blawas, Ashley M; Aballay, Alejandro; Pan, William K Y; Meyer, Joel N

2017-04-01

Mercury toxicity mechanisms have the potential to induce DNA damage and disrupt cellular processes, like mitochondrial function. Proper mitochondrial function is important for cellular bioenergetics and immune signaling and function. Reported impacts of mercury on the nuclear genome (nDNA) are conflicting and inconclusive, and mitochondrial DNA (mtDNA) impacts are relatively unknown. In this study, we assessed genotoxic (mtDNA and nDNA), metabolic, and innate immune impacts of inorganic and organic mercury exposure in Caenorhabditis elegans. Genotoxic outcomes measured included DNA damage, DNA damage repair (nucleotide excision repair, NER; base excision repair, BER), and genomic copy number following MeHg and HgCl 2 exposure alone and in combination with known DNA damage-inducing agents ultraviolet C radiation (UVC) and hydrogen peroxide (H 2 O 2 ), which cause bulky DNA lesions and oxidative DNA damage, respectively. Following exposure to both MeHg and HgCl 2 , low-level DNA damage (∼0.25 lesions/10kb mtDNA and nDNA) was observed. Unexpectedly, a higher MeHg concentration reduced damage in both genomes compared to controls. However, this observation was likely the result of developmental delay. In co-exposure treatments, both mercury compounds increased initial DNA damage (mtDNA and nDNA) in combination with H 2 O 2 exposure, but had no impact in combination with UVC exposure. Mercury exposure both increased and decreased DNA damage removal via BER. DNA repair after H 2 O 2 exposure in mercury-exposed nematodes resulted in damage levels lower than measured in controls. Impacts to NER were not detected. mtDNA copy number was significantly decreased in the MeHg-UVC and MeHg-H 2 O 2 co-exposure treatments. Mercury exposure had metabolic impacts (steady-state ATP levels) that differed between the compounds; HgCl 2 exposure decreased these levels, while MeHg slightly increased levels or had no impact. Both mercury species reduced mRNA levels for immune signaling-related genes, but had mild or no effects on survival on pathogenic bacteria. Overall, mercury exposure disrupted mitochondrial endpoints in a mercury-compound dependent fashion. Copyright © 2017 Elsevier B.V. All rights reserved.

Effects of methyl and inorganic mercury exposure on genome homeostasis and mitochondrial function in Caenorhabditis elegans

PubMed Central

Wyatt, Lauren H.; Luz, Anthony L.; Cao, Xiou; Maurer, Laura L.; Blawas, Ashley M.; Aballay, Alejandro; Pan, William K.; Meyer, Joel N.

2017-01-01

Mercury toxicity mechanisms have the potential to induce DNA damage and disrupt cellular processes, like mitochondrial function. Proper mitochondrial function is important for cellular bioenergetics and immune signaling and function. Impacts of mercury on the nuclear genome (nDNA) are conflicting and inconclusive, and mitochondrial DNA (mtDNA) impacts are relatively unknown. In this study, we assessed genotoxic (mtDNA and nDNA), metabolic, and innate immune impacts of inorganic and organic mercury exposure in Caenorhabditis elegans. Genotoxic outcomes measured included DNA damage, DNA damage repair (nucleotide excision repair, NER; base excision repair, BER), and genomic copy number following MeHg and HgCl2 exposure alone and in combination with known DNA damage-inducing agents ultraviolet C radiation (UVC) and hydrogen peroxide (H2O2), which cause bulky DNA lesions and oxidative DNA damage, respectively. Following exposure to both MeHg and HgCl2, low-level DNA damage (~0.25 lesions/10 kb mtDNA and nDNA) was observed. Unexpectedly, a higher MeHg concentration reduced damage in both genomes compared to controls. However, this observation was likely the result of developmental delay. In co-exposure treatments, both mercury compounds increased initial DNA damage (mtDNA and nDNA) in combination with H2O2 exposure, but had no impact in combination with UVC exposure. Mercury exposure both increased and decreased DNA damage removal via BER. DNA repair after H2O2 exposure in mercury-exposed nematodes resulted in damage levels lower than measured in controls. Impacts to NER were not detected. mtDNA copy number was significantly decreased in the MeHg-UVC and MeHg-H2O2 co-exposure treatments. Mercury exposure had metabolic impacts (steady-state ATP levels) that differed between the compounds; HgCl2 exposure decreased these levels, while MeHg slightly increased levels or had no impact. Both mercury species reduced mRNA levels for immune signaling-related genes, but had mild or no effects on survival on pathogenic bacteria. Overall, mercury exposure disrupted mitochondrial endpoints in a mercury-compound dependent fashion. PMID:28242054

SMC1-Mediated Intra-S-Phase Arrest Facilitates Bocavirus DNA Replication

PubMed Central

Luo, Yong; Deng, Xuefeng; Cheng, Fang; Li, Yi

2013-01-01

Activation of a host DNA damage response (DDR) is essential for DNA replication of minute virus of canines (MVC), a member of the genus Bocavirus of the Parvoviridae family; however, the mechanism by which DDR contributes to viral DNA replication is unknown. In the current study, we demonstrate that MVC infection triggers the intra-S-phase arrest to slow down host cellular DNA replication and to recruit cellular DNA replication factors for viral DNA replication. The intra-S-phase arrest is regulated by ATM (ataxia telangiectasia-mutated kinase) signaling in a p53-independent manner. Moreover, we demonstrate that SMC1 (structural maintenance of chromosomes 1) is the key regulator of the intra-S-phase arrest induced during infection. Either knockdown of SMC1 or complementation with a dominant negative SMC1 mutant blocks both the intra-S-phase arrest and viral DNA replication. Finally, we show that the intra-S-phase arrest induced during MVC infection was caused neither by damaged host cellular DNA nor by viral proteins but by replicating viral genomes physically associated with the DNA damage sensor, the Mre11-Rad50-Nbs1 (MRN) complex. In conclusion, the feedback loop between MVC DNA replication and the intra-S-phase arrest is mediated by ATM-SMC1 signaling and plays a critical role in MVC DNA replication. Thus, our findings unravel the mechanism underlying DDR signaling-facilitated MVC DNA replication and demonstrate a novel strategy of DNA virus-host interaction. PMID:23365434

Involvement of DNA polymerase beta in repairing oxidative damages induced by antitumor drug adriamycin

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liu Shukun; Wu Mei; Zhang Zunzhen, E-mail: zhangzunzhen@163.co

2010-08-01

Adriamycin (ADM) is a widely used antineoplastic drug. However, the increasing cellular resistance has become a serious limitation to ADM clinical application. The most important mechanism related to ADM-induced cell death is oxidative DNA damage mediated by reactive oxygen species (ROS). Base excision repair (BER) is a major pathway in the repair of DNA single strand break (SSB) and oxidized base. In this study, we firstly applied the murine embryo fibroblasts wild-type (pol {beta} +/+) and homozygous pol {beta} null cell (pol {beta} -/-) as a model to investigate ADM DNA-damaging effects and the molecular basis underlying these effects. Here,more » cellular sensitivity to ADM was examined using colorimetric assay and colony forming assay. ADM-induced cellular ROS level and the alteration of superoxide dismutase (SOD) activity were measured by commercial kits. Further, DNA strand break, chromosomal damage and gene mutation were assessed by comet assay, micronucleus test and hprt gene mutation assay, respectively. The results showed that pol {beta} -/- cells were more sensitive to ADM compared with pol {beta} +/+ cells and more severe SSB and chromosomal damage as well as higher hprt gene mutation frequency were observed in pol {beta} -/- cells. ROS level in pol {beta} -/- cells increased along with decreased activity of SOD. These results demonstrated that pol {beta} deficiency could enable ROS accumulation with SOD activity decrease, further elevate oxidative DNA damage, and subsequently result in SSB, chromosome cleavage as well as gene mutation, which may be partly responsible for the cytotoxicity of ADM and the hypersensitivity of pol {beta} -/- cells to ADM. These findings suggested that pol {beta} is vital for repairing oxidative damage induced by ADM.« less

Transforming growth factor-beta1 mediates cellular response to DNA damage in situ

NASA Technical Reports Server (NTRS)

Ewan, Kenneth B.; Henshall-Powell, Rhonda L.; Ravani, Shraddha A.; Pajares, Maria Jose; Arteaga, Carlos; Warters, Ray; Akhurst, Rosemary J.; Barcellos-Hoff, Mary Helen

2002-01-01

Transforming growth factor (TGF)-beta1 is rapidly activated after ionizing radiation, but its specific role in cellular responses to DNA damage is not known. Here we use Tgfbeta1 knockout mice to show that radiation-induced apoptotic response is TGF-beta1 dependent in the mammary epithelium, and that both apoptosis and inhibition of proliferation in response to DNA damage decrease as a function of TGF-beta1 gene dose in embryonic epithelial tissues. Because apoptosis in these tissues has been shown previously to be p53 dependent, we then examined p53 protein activation. TGF-beta1 depletion, by either gene knockout or by using TGF-beta neutralizing antibodies, resulted in decreased p53 Ser-18 phosphorylation in irradiated mammary gland. These data indicate that TGF-beta1 is essential for rapid p53-mediated cellular responses that mediate cell fate decisions in situ.

Comparing the effects of mitochondrial targeted and localized antioxidants with cellular antioxidants in human skin cells exposed to UVA and hydrogen peroxide.

PubMed

Oyewole, Anne O; Wilmot, Marie-Claire; Fowler, Mark; Birch-Machin, Mark A

2014-01-01

Skin cancer and aging are linked to increased cellular reactive oxygen species (ROS), particularly following exposure to ultraviolet A (UVA) in sunlight. As mitochondria are the main source of cellular ROS, this study compared the protective effects of mitochondria-targeted and -localized antioxidants (MitoQ and tiron, respectively) with cellular antioxidants against oxidative stress-induced [UVA and hydrogen peroxide (H2O2)] mitochondrial DNA (mtDNA) damage in human dermal fibroblasts. With the use of a long quantitative PCR assay, tiron (EC50 10 mM) was found to confer complete (100%) protection (P<0.001) against both UVA- and H2O2-induced mtDNA damage, whereas MitoQ (EC50 750 nM) provided less protection (17 and 32%, respectively; P<0.05). This particular protective effect of tiron was greater than a range of cellular antioxidants investigated. The nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathway provides cellular protection against oxidative stress. An ELISA assay for the Nrf2 target gene heme oxygenase-1 (HO-1) and studies using Nrf2 small interfering RNA both indicated that tiron's mode of action was Nrf2 independent. The comet assay showed that tiron's protective effect against H2O2-induced nuclear DNA damage was greater than the cellular antioxidants and MitoQ (P<0.001). This study provides a platform to investigate molecules with similar structure to tiron as potent and clinically relevant antioxidants.

ATM Is Required for the Prolactin-Induced HSP90-Mediated Increase in Cellular Viability and Clonogenic Growth After DNA Damage.

PubMed

Karayazi Atici, Ödül; Urbanska, Anna; Gopinathan, Sesha Gopal; Boutillon, Florence; Goffin, Vincent; Shemanko, Carrie S

2018-02-01

Prolactin (PRL) acts as a survival factor for breast cancer cells, but the PRL signaling pathway and the mechanism are unknown. Previously, we identified the master chaperone, heat shock protein 90 (HSP90) α, as a prolactin-Janus kinase 2 (JAK2)-signal transducer and activator of transcription 5 (STAT5) target gene involved in survival, and here we investigated the role of HSP90 in the mechanism of PRL-induced viability in response to DNA damage. The ataxia-telangiectasia mutated kinase (ATM) protein plays a critical role in the cellular response to double-strand DNA damage. We observed that PRL increased viability of breast cancer cells treated with doxorubicin or etoposide. The increase in cellular resistance is specific to the PRL receptor, because the PRL receptor antagonist, Δ1-9-G129R-hPRL, prevented the increase in viability. Two different HSP90 inhibitors, 17-allylamino-17-demethoxygeldanamycin and BIIB021, reduced the PRL-mediated increase in cell viability of doxorubicin-treated cells and led to a decrease in JAK2, ATM, and phosphorylated ATM protein levels. Inhibitors of JAK2 (G6) and ATM (KU55933) abolished the PRL-mediated increase in cell viability of DNA-damaged cells, supporting the involvement of each, as well as the crosstalk of ATM with the PRL pathway in the context of DNA damage. Drug synergism was detected between the ATM inhibitor (KU55933) and doxorubicin and between the HSP90 inhibitor (BIIB021) and doxorubicin. Short interfering RNA directed against ATM prevented the PRL-mediated increase in cell survival in two-dimensional cell culture, three-dimensional collagen gel cultures, and clonogenic cell survival, after doxorubicin treatment. Our results indicate that ATM contributes to the PRL-JAK2-STAT5-HSP90 pathway in mediating cellular resistance to DNA-damaging agents. Copyright © 2018 Endocrine Society.

Functions of Fun30 Chromatin Remodeler in Regulating Cellular Resistance to Genotoxic Stress

PubMed Central

Bi, Xin; Yu, Qun; Siler, Jasmine; Li, Chong; Khan, Ali

2015-01-01

The Saccharomyces cerevisiae Fun30 chromatin remodeler has recently been shown to facilitate long-range resection of DNA double strand break (DSB) ends, which proceeds homologous recombination (HR). This is believed to underlie the role of Fun30 in promoting cellular resistance to DSB inducing agent camptothecin. We show here that Fun30 also contributes to cellular resistance to genotoxins methyl methanesulfonate (MMS) and hydroxyurea (HU) that can stall the progression of DNA replication. We present evidence implicating DNA end resection in Fun30-dependent MMS-resistance. On the other hand, we show that Fun30 deletion suppresses the MMS- and HU-sensitivity of cells lacking the Rad5/Mms2/Ubc13-dependent error-free DNA damage tolerance mechanism. This suppression is not the result of a reduction in DNA end resection, and is dependent on the key HR component Rad51. We further show that Fun30 negatively regulates the recovery of rad5Δ mutant from MMS induced G2/M arrest. Therefore, Fun30 has two functions in DNA damage repair: one is the promotion of cellular resistance to genotoxic stress by aiding in DNA end resection, and the other is the negative regulation of a Rad51-dependent, DNA end resection-independent mechanism for countering replicative stress. The latter becomes manifest when Rad5 dependent DNA damage tolerance is impaired. In addition, we find that the putative ubiquitin-binding CUE domain of Fun30 serves to restrict the ability of Fun30 to hinder MMS- and HU-tolerance in the absence of Rad5. PMID:25806814

The FANC pathway is activated by adenovirus infection and promotes viral replication-dependent recombination

PubMed Central

Cherubini, Gioia; Naim, Valeria; Caruso, Paola; Burla, Romina; Bogliolo, Massimo; Cundari, Enrico; Benihoud, Karim; Saggio, Isabella; Rosselli, Filippo

2011-01-01

Deciphering the crosstalk between a host cell and a virus during infection is important not only to better define viral biology but also to improve our understanding of cellular processes. We identified the FANC pathway as a helper of viral replication and recombination by searching for cellular targets that are modified by adenovirus (Ad) infection and are involved in its outcome. This pathway, which is involved in the DNA damage response and checkpoint control, is altered in Fanconi anaemia, a rare cancer predisposition syndrome. We show here that Ad5 infection activates the FANC pathway independent of the classical DNA damage response. Infection with a non-replicating Ad shows that the presence of viral DNA is not sufficient to induce the monoubiquitination of FANCD2 but still activates the DNA damage response coordinated by phospho-NBS1 and phospho-CHK1. E1A expression alone fails to induce FANCD2 monoubiquitination, indicating that a productive viral infection and/or replication is required for FANC pathway activation. Our data indicate that Ad5 infection induces FANCD2 activation to promote its own replication. Specifically, we show that FANCD2 is involved in the recombination process that accompanies viral DNA replication. This study provides evidence of a DNA damage-independent function of the FANC pathway and identifies a cellular system involved in Ad5 recombination. PMID:21421559

Differential sensitivities of cellular XPA and PARP-1 to arsenite inhibition and zinc rescue.

PubMed

Ding, Xiaofeng; Zhou, Xixi; Cooper, Karen L; Huestis, Juliana; Hudson, Laurie G; Liu, Ke Jian

2017-09-15

Arsenite directly binds to the zinc finger domains of the DNA repair protein poly (ADP ribose) polymerase (PARP)-1, and inhibits PARP-1 activity in the base excision repair (BER) pathway. PARP inhibition by arsenite enhances ultraviolet radiation (UVR)-induced DNA damage in keratinocytes, and the increase in DNA damage is reduced by zinc supplementation. However, little is known about the effects of arsenite and zinc on the zinc finger nucleotide excision repair (NER) protein xeroderma pigmentosum group A (XPA). In this study, we investigated the difference in response to arsenite exposure between XPA and PARP-1, and the differential effectiveness of zinc supplementation in restoring protein DNA binding and DNA damage repair. Arsenite targeted both XPA and PARP-1 in human keratinocytes, resulting in zinc loss from each protein and a pronounced decrease in XPA and PARP-1 binding to chromatin as demonstrated by Chip-on-Western assays. Zinc effectively restored DNA binding of PARP-1 and XPA to chromatin when zinc concentrations were equal to those of arsenite. In contrast, zinc was more effective in rescuing arsenite-augmented direct UVR-induced DNA damage than oxidative DNA damage. Taken together, our findings indicate that arsenite interferes with PARP-1 and XPA binding to chromatin, and that zinc supplementation fully restores DNA binding activity to both proteins in the cellular context. Interestingly, rescue of arsenite-inhibited DNA damage repair by supplemental zinc was more sensitive for DNA damage repaired by the XPA-associated NER pathway than for the PARP-1-dependent BER pathway. This study expands our understanding of arsenite's role in DNA repair inhibition and co-carcinogenesis. Copyright © 2017 Elsevier Inc. All rights reserved.

HSV-I and the cellular DNA damage response.

PubMed

Smith, Samantha; Weller, Sandra K

2015-04-01

Peter Wildy first observed genetic recombination between strains of HSV in 1955. At the time, knowledge of DNA repair mechanisms was limited, and it has only been in the last decade that particular DNA damage response (DDR) pathways have been examined in the context of viral infections. One of the first reports addressing the interaction between a cellular DDR protein and HSV-1 was the observation by Lees-Miller et al . that DNA-dependent protein kinase catalytic subunit levels were depleted in an ICP0-dependent manner during Herpes simplex virus 1 infection. Since then, there have been numerous reports describing the interactions between HSV infection and cellular DDR pathways. Due to space limitations, this review will focus predominantly on the most recent observations regarding how HSV navigates a potentially hostile environment to replicate its genome.

Electronic cigarette aerosols suppress cellular antioxidant defenses and induce significant oxidative DNA damage

PubMed Central

Ganapathy, Vengatesh; Manyanga, Jimmy; Brame, Lacy; McGuire, Dehra; Sadhasivam, Balaji; Floyd, Evan; Rubenstein, David A.; Ramachandran, Ilangovan; Wagener, Theodore

2017-01-01

Background Electronic cigarette (EC) aerosols contain unique compounds in addition to toxicants and carcinogens traditionally found in tobacco smoke. Studies are warranted to understand the public health risks of ECs. Objective The aim of this study was to determine the genotoxicity and the mechanisms induced by EC aerosol extracts on human oral and lung epithelial cells. Methods Cells were exposed to EC aerosol or mainstream smoke extracts and DNA damage was measured using the primer anchored DNA damage detection assay (q-PADDA) and 8-oxo-dG ELISA assay. Cell viability, reactive oxygen species (ROS) and total antioxidant capacity (TAC) were measured using standard methods. mRNA and protein expression were evaluated by RT-PCR and western blot, respectively. Results EC aerosol extracts induced DNA damage in a dose-dependent manner, but independently of nicotine concentration. Overall, EC aerosol extracts induced significantly less DNA damage than mainstream smoke extracts, as measured by q-PADDA. However, the levels of oxidative DNA damage, as indicated by the presence of 8-oxo-dG, a highly mutagenic DNA lesion, were similar or slightly higher after exposure to EC aerosol compared to mainstream smoke extracts. Mechanistically, while exposure to EC extracts significantly increased ROS, it decreased TAC as well as the expression of 8-oxoguanine DNA glycosylase (OGG1), an enzyme essential for the removal of oxidative DNA damage. Conclusions Exposure to EC aerosol extracts suppressed the cellular antioxidant defenses and led to significant DNA damage. These findings emphasize the urgent need to investigate the potential long-term cancer risk of exposure to EC aerosol for vapers and the general public. PMID:28542301

WE-DE-202-01: Connecting Nanoscale Physics to Initial DNA Damage Through Track Structure Simulations

DOE Office of Scientific and Technical Information (OSTI.GOV)

Schuemann, J.

Radiation therapy for the treatment of cancer has been established as a highly precise and effective way to eradicate a localized region of diseased tissue. To achieve further significant gains in the therapeutic ratio, we need to move towards biologically optimized treatment planning. To achieve this goal, we need to understand how the radiation-type dependent patterns of induced energy depositions within the cell (physics) connect via molecular, cellular and tissue reactions to treatment outcome such as tumor control and undesirable effects on normal tissue. Several computational biology approaches have been developed connecting physics to biology. Monte Carlo simulations are themore » most accurate method to calculate physical dose distributions at the nanometer scale, however simulations at the DNA scale are slow and repair processes are generally not simulated. Alternative models that rely on the random formation of individual DNA lesions within one or two turns of the DNA have been shown to reproduce the clusters of DNA lesions, including single strand breaks (SSBs), double strand breaks (DSBs) without the need for detailed track structure simulations. Efficient computational simulations of initial DNA damage induction facilitate computational modeling of DNA repair and other molecular and cellular processes. Mechanistic, multiscale models provide a useful conceptual framework to test biological hypotheses and help connect fundamental information about track structure and dosimetry at the sub-cellular level to dose-response effects on larger scales. In this symposium we will learn about the current state of the art of computational approaches estimating radiation damage at the cellular and sub-cellular scale. How can understanding the physics interactions at the DNA level be used to predict biological outcome? We will discuss if and how such calculations are relevant to advance our understanding of radiation damage and its repair, or, if the underlying biological processes are too complex for a mechanistic approach. Can computer simulations be used to guide future biological research? We will debate the feasibility of explaining biology from a physicists’ perspective. Learning Objectives: Understand the potential applications and limitations of computational methods for dose-response modeling at the molecular, cellular and tissue levels Learn about mechanism of action underlying the induction, repair and biological processing of damage to DNA and other constituents Understand how effects and processes at one biological scale impact on biological processes and outcomes on other scales J. Schuemann, NCI/NIH grantsS. McMahon, Funding: European Commission FP7 (grant EC FP7 MC-IOF-623630)« less

Cellular redistribution of Rad51 in response to DNA damage: novel role for Rad51C.

PubMed

Gildemeister, Otto S; Sage, Jay M; Knight, Kendall L

2009-11-13

Exposure of cells to DNA-damaging agents results in a rapid increase in the formation of subnuclear complexes containing Rad51. To date, it has not been determined to what extent DNA damage-induced cytoplasmic to nuclear transport of Rad51 may contribute to this process. We have analyzed subcellular fractions of HeLa and HCT116 cells and found a significant increase in nuclear Rad51 levels following exposure to a modest dose of ionizing radiation (2 grays). We also observed a DNA damage-induced increase in nuclear Rad51 in the Brca2-defective cell line Capan-1. To address a possible Brca2-independent mechanism for Rad51 nuclear transport, we analyzed subcellular fractions for two other Rad51-interacting proteins, Rad51C and Xrcc3. Rad51C has a functional nuclear localization signal, and although we found that the subcellular distribution of Xrcc3 was not significantly affected by DNA damage, there was a damage-induced increase in nuclear Rad51C. Furthermore, RNA interference-mediated depletion of Rad51C in HeLa and Capan-1 cells resulted in lower steady-state levels of nuclear Rad51 as well as a diminished DNA damage-induced increase. Our results provide important insight into the cellular regulation of Rad51 nuclear entry and a role for Rad51C in this process.

A Novel ATM/TP53/p21-Mediated Checkpoint Only Activated by Chronic γ-Irradiation

PubMed Central

Sasatani, Megumi; Iizuka, Daisuke; Masuda, Yuji; Inaba, Toshiya; Suzuki, Keiji; Ootsuyama, Akira; Umata, Toshiyuki; Kamiya, Kenji; Suzuki, Fumio

2014-01-01

Different levels or types of DNA damage activate distinct signaling pathways that elicit various cellular responses, including cell-cycle arrest, DNA repair, senescence, and apoptosis. Whereas a range of DNA-damage responses have been characterized, mechanisms underlying subsequent cell-fate decision remain elusive. Here we exposed cultured cells and mice to different doses and dose rates of γ-irradiation, which revealed cell-type-specific sensitivities to chronic, but not acute, γ-irradiation. Among tested cell types, human fibroblasts were associated with the highest levels of growth inhibition in response to chronic γ-irradiation. In this context, fibroblasts exhibited a reversible G1 cell-cycle arrest or an irreversible senescence-like growth arrest, depending on the irradiation dose rate or the rate of DNA damage. Remarkably, when the same dose of γ-irradiation was delivered chronically or acutely, chronic delivery induced considerably more cellular senescence. A similar effect was observed with primary cells isolated from irradiated mice. We demonstrate a critical role for the ataxia telangiectasia mutated (ATM)/tumor protein p53 (TP53)/p21 pathway in regulating DNA-damage-associated cell fate. Indeed, blocking the ATM/TP53/p21 pathway deregulated DNA damage responses, leading to micronucleus formation in chronically irradiated cells. Together these results provide insights into the mechanisms governing cell-fate determination in response to different rates of DNA damage. PMID:25093836

Spatiotemporal dynamics of DNA repair proteins following laser microbeam induced DNA damage – When is a DSB not a DSB?☆

PubMed Central

Reynolds, Pamela; Botchway, Stanley W.; Parker, Anthony W.; O’Neill, Peter

2013-01-01

The formation of DNA lesions poses a constant threat to cellular stability. Repair of endogenously and exogenously produced lesions has therefore been extensively studied, although the spatiotemporal dynamics of the repair processes has yet to be fully understood. One of the most recent advances to study the kinetics of DNA repair has been the development of laser microbeams to induce and visualize recruitment and loss of repair proteins to base damage in live mammalian cells. However, a number of studies have produced contradictory results that are likely caused by the different laser systems used reflecting in part the wavelength dependence of the damage induced. Additionally, the repair kinetics of laser microbeam induced DNA lesions have generally lacked consideration of the structural and chemical complexity of the DNA damage sites, which are known to greatly influence their reparability. In this review, we highlight the key considerations when embarking on laser microbeam experiments and interpreting the real time data from laser microbeam irradiations. We compare the repair kinetics from live cell imaging with biochemical and direct quantitative cellular measurements for DNA repair. PMID:23688615

Combined loss of three DNA damage response pathways renders C. elegans intolerant to light.

PubMed

van Bostelen, Ivo; Tijsterman, Marcel

2017-06-01

Infliction of DNA damage initiates a complex cellular reaction - the DNA damage response - that involves both signaling and DNA repair networks with many redundancies and parallel pathways. Here, we reveal the three strategies that the simple multicellular eukaryote, C. elegans, uses to deal with DNA damage induced by light. Separately inactivating repair or replicative bypass of photo-lesions results in cellular hypersensitivity towards UV-light, but impeding repair of replication associated DNA breaks does not. Yet, we observe an unprecedented synergistic relationship when these pathways are inactivated in combination. C. elegans mutants that lack nucleotide excision repair (NER), translesion synthesis (TLS) and alternative end joining (altEJ) grow undisturbed in the dark, but become sterile when grown in light. Even exposure to very low levels of normal daylight impedes animal growth. We show that NER and TLS operate to suppress the formation of lethal DNA breaks that require polymerase theta-mediated end joining (TMEJ) for their repair. Our data testifies to the enormous genotoxicity of light and to the demand of multiple layers of protection against an environmental threat that is so common. Copyright © 2017 Elsevier B.V. All rights reserved.

Interferon antagonist NSs of La Crosse virus triggers a DNA damage response-like degradation of transcribing RNA polymerase II.

PubMed

Verbruggen, Paul; Ruf, Marius; Blakqori, Gjon; Överby, Anna K; Heidemann, Martin; Eick, Dirk; Weber, Friedemann

2011-02-04

La Crosse encephalitis virus (LACV) is a mosquito-borne member of the negative-strand RNA virus family Bunyaviridae. We have previously shown that the virulence factor NSs of LACV is an efficient inhibitor of the antiviral type I interferon system. A recombinant virus unable to express NSs (rLACVdelNSs) strongly induced interferon transcription, whereas the corresponding wt virus (rLACV) suppressed it. Here, we show that interferon induction by rLACVdelNSs mainly occurs through the signaling pathway leading from the pattern recognition receptor RIG-I to the transcription factor IRF-3. NSs expressed by rLACV, however, acts downstream of IRF-3 by specifically blocking RNA polymerase II-dependent transcription. Further investigations revealed that NSs induces proteasomal degradation of the mammalian RNA polymerase II subunit RPB1. NSs thereby selectively targets RPB1 molecules of elongating RNA polymerase II complexes, the so-called IIo form. This phenotype has similarities to the cellular DNA damage response, and NSs was indeed found to transactivate the DNA damage response gene pak6. Moreover, NSs expressed by rLACV boosted serine 139 phosphorylation of histone H2A.X, one of the earliest cellular reactions to damaged DNA. However, other DNA damage response markers such as up-regulation and serine 15 phosphorylation of p53 or serine 1524 phosphorylation of BRCA1 were not triggered by LACV infection. Collectively, our data indicate that the strong suppression of interferon induction by LACV NSs is based on a shutdown of RNA polymerase II transcription and that NSs achieves this by exploiting parts of the cellular DNA damage response pathway to degrade IIo-borne RPB1 subunits.

Damage to cellular DNA from particulate radiations, the efficacy of its processing and the radiosensitivity of mammalian cells. Emphasis on DNA double strand breaks and chromatin breaks

NASA Technical Reports Server (NTRS)

Lett, J. T.

1992-01-01

For several years, it has been evident that cellular radiation biology is in a necessary period of consolidation and transition (Lett 1987, 1990; Lett et al. 1986, 1987). Both changes are moving apace, and have been stimulated by studies with heavy charged particles. From the standpoint of radiation chemistry, there is now a consensus of opinion that the DNA hydration shell must be distinguished from bulk water in the cell nucleus and treated as an integral part of DNA (chromatin) (Lett 1987). Concomitantly, sentiment is strengthening for the abandonment of the classical notions of "direct" and "indirect" action (Fielden and O'Neill 1991; O'Neill 1991; O'Neill et al. 1991; Schulte-Frohlinde and Bothe 1991 and references therein). A layer of water molecules outside, or in the outer edge of, the DNA (chromatin) hydration shell influences cellular radiosensitivity in ways not fully understood. Charge and energy transfer processes facilitated by, or involving, DNA hydration must be considered in rigorous theories of radiation action on cells. The induction and processing of double stand breaks (DSBs) in DNA (chromatin) seem to be the predominant determinants of the radiotoxicity of normally radioresistant mammalian cells, the survival curves of which reflect the patterns of damage induced and the damage present after processing ceases, and can be modelled in formal terms by the use of reaction (enzyme) kinetics. Incongruities such as sublethal damage are neither scientifically sound nor relevant to cellular radiation biology (Calkins 1991; Lett 1990; Lett et al. 1987a). Increases in linear energy transfer (LET infinity) up to 100-200 keV micron-1 cause increases in the extents of neighboring chemical and physical damage in DNA denoted by the general term DSB. Those changes are accompanied by decreasing abilities of cells normally radioresistant to sparsely ionizing radiations to process DSBs in DNA and chromatin and to recover from radiation exposure, so they make significant contributions to the relative biological effectiveness (RBE) of a given radiation.(ABSTRACT TRUNCATED AT 400 WORDS).

Ubiquitylation-dependent regulation of NEIL1 by Mule and TRIM26 is required for the cellular DNA damage response.

PubMed

Edmonds, Matthew J; Carter, Rachel J; Nickson, Catherine M; Williams, Sarah C; Parsons, Jason L

2017-01-25

Endonuclease VIII-like protein 1 (NEIL1) is a DNA glycosylase involved in initiating the base excision repair pathway, the major cellular mechanism for repairing DNA base damage. Here, we have purified the major E3 ubiquitin ligases from human cells responsible for regulation of NEIL1 by ubiquitylation. Interestingly, we have identified two enzymes that catalyse NEIL1 polyubiquitylation, Mcl-1 ubiquitin ligase E3 (Mule) and tripartite motif 26 (TRIM26). We demonstrate that these enzymes are capable of polyubiquitylating NEIL1 in vitro, and that both catalyse ubiquitylation of NEIL1 within the same C-terminal lysine residues. An siRNA-mediated knockdown of Mule or TRIM26 leads to stabilisation of NEIL1, demonstrating that these enzymes are important in regulating cellular NEIL1 steady state protein levels. Similarly, a mutant NEIL1 protein lacking residues for ubiquitylation is more stable than the wild type protein in vivo We also demonstrate that cellular NEIL1 protein is induced in response to ionising radiation (IR), although this occurs specifically in a Mule-dependent manner. Finally we show that stabilisation of NEIL1, particularly following TRIM26 siRNA, contributes to cellular resistance to IR. This highlights the importance of Mule and TRIM26 in maintaining steady state levels of NEIL1, but also those required for the cellular DNA damage response. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Pairing of heterochromatin in response to cellular stress.

PubMed

Abdel-Halim, H I; Mullenders, L H F; Boei, J J W A

2006-07-01

We previously reported that exposure of human cells to DNA-damaging agents (X-rays and mitomycin C (MMC)) induces pairing of the homologous paracentromeric heterochromatin of chromosome 9 (9q12-13). Here, we show that UV irradiation and also heat shock treatment of human cells lead to similar effects. Since the various agents induce very different types and frequencies of damage to cellular constituents, the data suggest a general stress response as the underlying mechanism. Moreover, local UV irradiation experiments revealed that pairing of heterochromatin is an event that can be triggered without induction of DNA damage in the heterochromatic sequences. The repair deficient xeroderma pigmentosum cells (group F) previously shown to fail pairing after MMC displayed elevated pairing after heat shock treatment but not after UV exposure. Taken together, the present results indicate that pairing of heterochromatin following exposure to DNA-damaging agents is initiated by a general stress response and that the sensing of stress or the maintenance of the paired status of the heterochromatin might be dependent on DNA repair.

Persistent response of Fanconi anemia haematopoietic stem and progenitor cells to oxidative stress.

PubMed

Li, Yibo; Amarachintha, Surya; Wilson, Andrew F; Li, Xue; Du, Wei

2017-06-18

Oxidative stress is considered as an important pathogenic factor in many human diseases including Fanconi anemia (FA), an inherited bone marrow failure syndrome with extremely high risk of leukemic transformation. Members of the FA protein family are involved in DNA damage and other cellular stress responses. Loss of FA proteins renders cells hypersensitive to oxidative stress and cancer transformation. However, how FA cells respond to oxidative DNA damage remains unclear. By using an in vivo stress-response mouse strain expressing the Gadd45β-luciferase transgene, we show here that haematopoietic stem and progenitor cells (HSPCs) from mice deficient for the FA gene Fanca or Fancc persistently responded to oxidative stress. Mechanistically, we demonstrated that accumulation of unrepaired DNA damage, particularly in oxidative damage-sensitive genes, was responsible for the long-lasting response in FA HSPCs. Furthermore, genetic correction of Fanca deficiency almost completely abolished the persistent oxidative stress-induced G 2 /M arrest and DNA damage response in vivo. Our study suggests that FA pathway is an integral part of a versatile cellular mechanism by which HSPCs respond to oxidative stress.

Persistent response of Fanconi anemia haematopoietic stem and progenitor cells to oxidative stress

PubMed Central

Wilson, Andrew F.; Li, Xue

2017-01-01

ABSTRACT Oxidative stress is considered as an important pathogenic factor in many human diseases including Fanconi anemia (FA), an inherited bone marrow failure syndrome with extremely high risk of leukemic transformation. Members of the FA protein family are involved in DNA damage and other cellular stress responses. Loss of FA proteins renders cells hypersensitive to oxidative stress and cancer transformation. However, how FA cells respond to oxidative DNA damage remains unclear. By using an in vivo stress-response mouse strain expressing the Gadd45β-luciferase transgene, we show here that haematopoietic stem and progenitor cells (HSPCs) from mice deficient for the FA gene Fanca or Fancc persistently responded to oxidative stress. Mechanistically, we demonstrated that accumulation of unrepaired DNA damage, particularly in oxidative damage-sensitive genes, was responsible for the long-lasting response in FA HSPCs. Furthermore, genetic correction of Fanca deficiency almost completely abolished the persistent oxidative stress-induced G2/M arrest and DNA damage response in vivo. Our study suggests that FA pathway is an integral part of a versatile cellular mechanism by which HSPCs respond to oxidative stress. PMID:28475398

DNA Damage Observed in Unaffected Individuals with Family History of T2DM

NASA Astrophysics Data System (ADS)

Ramesh, Nikhila; Abilash, V. G.

2017-11-01

Diabetes has been documented to cause high levels of DNA fragmentation in some cases. As diabetes is inheritable and influenced by both genetic and environmental factors, an investigation into the genomic stability of individuals who are strongly at risk of inheriting diabetes was conducted by inducing oxidative stress, as DNA damage in unaffected individuals could be a sign of onset of the disease or the presence of genetic alterations that reduce cellular defences against reactive oxygen species. In this study, alkaline comet assay was performed on isolated human leukocytes to determine whether individuals with a family history of Type 2 Diabetes Mellitus (T2DM) are more prone to DNA damage under oxidative stress. Visual scoring of comets showed that these individuals have higher degree of DNA damage compared to a control individual with no family history of Type 2 Diabetes Mellitus. Further studies with large sample could determine the presence of disabled cellular defences against oxidative stress in unaffected individuals and intervention with antioxidants could prevent or manage Type 2 Diabetes Mellitus and its complications.

Differential responses of juvenile and adult South African abalone (Haliotis midae Linnaeus) to low and high oxygen levels.

PubMed

Vosloo, Andre; Laas, Anél; Vosloo, Dalene

2013-01-01

Marine invertebrates have evolved multiple responses to naturally variable environmental oxygen, all aimed at either maintaining cellular oxygen homeostasis or limiting cellular damage during or after hypoxic or hyperoxic events. We assessed organismal (rates of oxygen consumption and ammonia excretion) and cellular (heat shock protein expression, anti-oxidant enzymes) responses of juvenile and adult abalone exposed to low (~83% of saturation), intermediate (~95% of saturation) and high (~115% of saturation) oxygen levels for one month. Using the Comet assay, we measured DNA damage to determine whether the observed trends in the protective responses were sufficient to prevent oxidative damage to cells. Juveniles were unaffected by moderately hypoxic and hyperoxic conditions. Elevated basal rates of superoxide dismutase, glutathione peroxidase and catalase were sufficient to prevent DNA fragmentation and protein damage. Adults, with their lower basal rate of anti-oxidant enzymes, had increased DNA damage under hypoxic and hyperoxic conditions, indicating that the antioxidant enzymes were unable to prevent oxidative damage under hypoxic and hyperoxic conditions. The apparent insensitivity of juvenile abalone to decreased and increased oxygen might be related to their life history and development in algal and diatom biofilms where they are exposed to extreme diurnal fluctuations in dissolved oxygen levels. Copyright © 2012 Elsevier Inc. All rights reserved.

Radiation-Induced Loss of Salivary Gland Function Is Driven by Cellular Senescence and Prevented by IL6 Modulation.

PubMed

Marmary, Yitzhak; Adar, Revital; Gaska, Svetlana; Wygoda, Annette; Maly, Alexander; Cohen, Jonathan; Eliashar, Ron; Mizrachi, Lina; Orfaig-Geva, Carmit; Baum, Bruce J; Rose-John, Stefan; Galun, Eithan; Axelrod, Jonathan H

2016-03-01

Head and neck cancer patients treated by radiation commonly suffer from a devastating side effect known as dry-mouth syndrome, which results from the irreversible loss of salivary gland function via mechanisms that are not completely understood. In this study, we used a mouse model of radiation-induced salivary hypofunction to investigate the outcomes of DNA damage in the head and neck region. We demonstrate that the loss of salivary function was closely accompanied by cellular senescence, as evidenced by a persistent DNA damage response (γH2AX and 53BP1) and the expression of senescence-associated markers (SA-βgal, p19ARF, and DcR2) and secretory phenotype (SASP) factors (PAI-1 and IL6). Notably, profound apoptosis or necrosis was not observed in irradiated regions. Signs of cellular senescence were also apparent in irradiated salivary glands surgically resected from human patients who underwent radiotherapy. Importantly, using IL6 knockout mice, we found that sustained expression of IL6 in the salivary gland long after initiation of radiation-induced DNA damage was required for both senescence and hypofunction. Additionally, we demonstrate that IL6 pretreatment prevented both senescence and salivary gland hypofunction via a mechanism involving enhanced DNA damage repair. Collectively, these results indicate that cellular senescence is a fundamental mechanism driving radiation-induced damage in the salivary gland and suggest that IL6 pretreatment may represent a promising therapeutic strategy to preserve salivary gland function in head and neck cancer patients undergoing radiotherapy. ©2016 American Association for Cancer Research.

Repair of DNA Damage Induced by the Cytidine Analog Zebularine Requires ATR and ATM in Arabidopsis[OPEN

PubMed Central

Liu, Chun-Hsin; Finke, Andreas; Díaz, Mariana; Rozhon, Wilfried; Poppenberger, Brigitte; Baubec, Tuncay; Pecinka, Ales

2015-01-01

DNA damage repair is an essential cellular mechanism that maintains genome stability. Here, we show that the nonmethylable cytidine analog zebularine induces a DNA damage response in Arabidopsis thaliana, independent of changes in DNA methylation. In contrast to genotoxic agents that induce damage in a cell cycle stage-independent manner, zebularine induces damage specifically during strand synthesis in DNA replication. The signaling of this damage is mediated by additive activity of ATAXIA TELANGIECTASIA MUTATED AND RAD3-RELATED and ATAXIA TELANGIECTASIA MUTATED kinases, which cause postreplicative cell cycle arrest and increased endoreplication. The repair requires a functional STRUCTURAL MAINTENANCE OF CHROMOSOMES5 (SMC5)-SMC6 complex and is accomplished predominantly by synthesis-dependent strand-annealing homologous recombination. Here, we provide insight into the response mechanism for coping with the genotoxic effects of zebularine and identify several components of the zebularine-induced DNA damage repair pathway. PMID:26023162

Mechanisms of DNA damage, repair and mutagenesis

PubMed Central

Chatterjee, Nimrat; Walker, Graham C.

2017-01-01

Living organisms are continuously exposed to a myriad of DNA damaging agents that can impact health and modulate disease-states. However, robust DNA repair and damage-bypass mechanisms faithfully protect the DNA by either removing or tolerating the damage to ensure an overall survival. Deviations in this fine-tuning are known to destabilize cellular metabolic homeostasis, as exemplified in diverse cancers where disruption or deregulation of DNA repair pathways results in genome instability. Because routinely used biological, physical and chemical agents impact human health, testing their genotoxicity and regulating their use have become important. In this introductory review, we will delineate mechanisms of DNA damage and the counteracting repair/tolerance pathways to provide insights into the molecular basis of genotoxicity in cells that lays the foundation for subsequent articles in this issue. PMID:28485537

Deoxyribonucleoprotein structure and radiation injury - Cellular radiosensitivity is determined by LET-infinity-dependent DNA damage in hydrated deoxyribonucleoproteins and the extent of its repair

NASA Technical Reports Server (NTRS)

Lett, J. T.; Peters, E. L.

1992-01-01

Until recently, OH radicals formed in bulk nuclear water were believed to be the major causes of DNA damage that results in cell death, especially for sparsely ionizing radiations. That hypothesis has now been challenged, if not refuted. Lethal genomic DNA damage is determined mainly by energy deposition in deoxyribonucleoproteins, and their hydration shells, and charge (energy) transfer processes within those structures.

Attenuated DNA damage repair by trichostatin A through BRCA1 suppression.

PubMed

Zhang, Yin; Carr, Theresa; Dimtchev, Alexandre; Zaer, Naghmeh; Dritschilo, Anatoly; Jung, Mira

2007-07-01

Recent studies have demonstrated that some histone deacetylase (HDAC) inhibitors enhance cellular radiation sensitivity. However, the underlying mechanism for such a radiosensitizing effect remains unexplored. Here we show evidence that treatment with the HDAC inhibitor trichostatin A (TSA) impairs radiation-induced repair of DNA damage. The effect of TSA on the kinetics of DNA damage repair was measured by performing the comet assay and gamma-H2AX focus analysis in radioresistant human squamous carcinoma cells (SQ-20B). TSA exposure increased the amount of radiation-induced DNA damage and slowed the repair kinetics. Gene expression profiling also revealed that a majority of the genes that control cell cycle, DNA replication and damage repair processes were down-regulated after TSA exposure, including BRCA1. The involvement of BRCA1 was further demonstrated by expressing ectopic wild-type BRCA1 in a BRCA1 null cell line (HCC-1937). TSA treatment enhanced radiation sensitivity of HCC-1937/wtBRCA1 clonal cells, which restored cellular radiosensitivity (D(0) = 1.63 Gy), to the control level (D(0) = 1.03 Gy). However, TSA had no effect on the level of radiosensitivity of BRCA1 null cells. Our data demonstrate for the first time that TSA treatment modulates the radiation-induced DNA damage repair process, in part by suppressing BRCA1 gene expression, suggesting that BRCA1 is one of molecular targets of TSA.

Transcriptional upregulation of p19INK4d upon diverse genotoxic stress is critical for optimal DNA damage response.

PubMed

Ceruti, Julieta M; Scassa, María E; Marazita, Mariela C; Carcagno, Abel C; Sirkin, Pablo F; Cánepa, Eduardo T

2009-06-01

p19INK4d promotes survival of several cell lines after UV irradiation due to enhanced DNA repair, independently of CDK4 inhibition. To further understand the action of p19INK4d in the cellular response to DNA damage, we aimed to elucidate whether this novel regulator plays a role only in mechanisms triggered by UV or participates in diverse mechanisms initiated by different genotoxics. We found that p19INK4d is induced in cells injured with cisplatin or beta-amyloid peptide as robustly as with UV. The mentioned genotoxics transcriptionally activate p19INK4d expression as demonstrated by run-on assay without influencing its mRNA stability and with partial requirement of protein synthesis. It is not currently known whether DNA damage-inducible genes are turned on by the DNA damage itself or by the consequences of that damage. Experiments carried out in cells transfected with distinct damaged DNA structures revealed that the damage itself is not responsible for the observed up-regulation. It is also not known whether the increased expression of DNA-damage-inducible genes is related to immediate protective responses such as DNA repair or to more delayed responses such as cell cycle arrest or apoptosis. We found that ectopic expression of p19INK4d improves DNA repair ability and protects neuroblastoma cells from apoptosis caused by cisplatin or beta-amyloid peptide. Using clonal cell lines where p19INK4d levels can be modified at will, we show that p19INK4d expression correlates with increased survival and clonogenicity. The results presented here, prompted us to suggest that p19INK4d displays an important role in an early stage of cellular DNA damage response.

Oxidative DNA Base Damage in MCF-10A Breast Epithelial Cells at Clinically Achievable Concentrations of Doxorubicin

PubMed Central

Gajewski, Ewa; Gaur, Shikha; Akman, Steven A.; Matsumoto, Linda; van Balgooy, Josephus N.A.; Doroshow, James H.

2009-01-01

The cellular metabolism of doxorubicin generates reactive oxygen species with significant potential to damage DNA. Such DNA damage can result in mutations if not adequately repaired by cellular DNA repair pathways. Secondary malignancies have been reported in patients who have received doxorubicin-containing chemotherapeutic regimens; however, the underlying molecular mechanism(s) to explain the development of these tumors remains under active investigation. We have previously demonstrated the presence of DNA bases modified by oxidation in the peripheral blood mononuclear cells of patients with breast cancer following treatment with doxorubicin. In those studies, doxorubicin was administered by continuous infusion over 96 hours to minimize the risk of cardiac toxicity. To evaluate potential mechanisms underlying doxorubicin-induced DNA base oxidation in non-malignant tissues, MCF-10A breast epithelial cells were cultured for 96 hours with the same doxorubicin concentration achieved in vivo (0.1 μM). During doxorubicin exposure, MCF-10A cells underwent growth arrest and apoptosis, developed elevated levels of reactive oxygen species, and demonstrated a time-dependent and significant increase in the levels of 11 oxidized DNA bases, as determined by gas chromatography/mass spectroscopy. Diminished expression of DNA repair enzymes was also observed over the same time course. Thus, clinically achievable concentrations of doxorubicin induce a level of oxidative stress in MCF-10A cells that is capable of oxidizing DNA bases and significantly altering cellular proliferation. PMID:17445777

Mechanisms of free radical-induced damage to DNA.

PubMed

Dizdaroglu, Miral; Jaruga, Pawel

2012-04-01

Endogenous and exogenous sources cause free radical-induced DNA damage in living organisms by a variety of mechanisms. The highly reactive hydroxyl radical reacts with the heterocyclic DNA bases and the sugar moiety near or at diffusion-controlled rates. Hydrated electron and H atom also add to the heterocyclic bases. These reactions lead to adduct radicals, further reactions of which yield numerous products. These include DNA base and sugar products, single- and double-strand breaks, 8,5'-cyclopurine-2'-deoxynucleosides, tandem lesions, clustered sites and DNA-protein cross-links. Reaction conditions and the presence or absence of oxygen profoundly affect the types and yields of the products. There is mounting evidence for an important role of free radical-induced DNA damage in the etiology of numerous diseases including cancer. Further understanding of mechanisms of free radical-induced DNA damage, and cellular repair and biological consequences of DNA damage products will be of outmost importance for disease prevention and treatment.

Control of G1 arrest after DNA damage.

PubMed Central

Kastan, M B; Kuerbitz, S J

1993-01-01

The temporal relationship between DNA damage and DNA replication may be critical in determining whether the genetic changes necessary for cellular transformation occur after DNA damage. Recent characterization of the mechanisms responsible for alterations in cell-cycle progression after DNA damage in our laboratory have implicated the p53 (tumor suppressor) protein in the G1 arrest that occurs after certain types of DNA damage. In particular, we found that levels of p53 protein increased rapidly and transiently after nonlethal doses of gamma irradiation (XRT) in hematopoietic cells with wild-type, but not mutant, p53 genes. These changes in p53 protein levels were temporally linked to a transient G1 arrest in these cells. Hematopoietic cells with mutant or absent p53 genes did not exhibit this G1 arrest, through they continued to demonstrate a G2 arrest. We recently extended these observations of a tight correlation between the status of the endogenous p53 genes and this G1 arrest after XRT and this cell-cycle alteration after XRT was then established by transfecting cells lacking endogenous p53 genes with a wild-type gene and observing acquisition of the G1 arrest and by transfecting cells processing endogenous wild-type p53 genes with a mutant p53 gene and observing loss of the G1 arrest after XRT. These observations and their significance for our understanding of the mechanisms of DNA damage-induced cellular transformation are discussed. PMID:8013425

WE-DE-202-02: Are Track Structure Simulations Truly Needed for Radiobiology at the Cellular and Tissue Levels?

DOE Office of Scientific and Technical Information (OSTI.GOV)

Stewart, R.

Radiation therapy for the treatment of cancer has been established as a highly precise and effective way to eradicate a localized region of diseased tissue. To achieve further significant gains in the therapeutic ratio, we need to move towards biologically optimized treatment planning. To achieve this goal, we need to understand how the radiation-type dependent patterns of induced energy depositions within the cell (physics) connect via molecular, cellular and tissue reactions to treatment outcome such as tumor control and undesirable effects on normal tissue. Several computational biology approaches have been developed connecting physics to biology. Monte Carlo simulations are themore » most accurate method to calculate physical dose distributions at the nanometer scale, however simulations at the DNA scale are slow and repair processes are generally not simulated. Alternative models that rely on the random formation of individual DNA lesions within one or two turns of the DNA have been shown to reproduce the clusters of DNA lesions, including single strand breaks (SSBs), double strand breaks (DSBs) without the need for detailed track structure simulations. Efficient computational simulations of initial DNA damage induction facilitate computational modeling of DNA repair and other molecular and cellular processes. Mechanistic, multiscale models provide a useful conceptual framework to test biological hypotheses and help connect fundamental information about track structure and dosimetry at the sub-cellular level to dose-response effects on larger scales. In this symposium we will learn about the current state of the art of computational approaches estimating radiation damage at the cellular and sub-cellular scale. How can understanding the physics interactions at the DNA level be used to predict biological outcome? We will discuss if and how such calculations are relevant to advance our understanding of radiation damage and its repair, or, if the underlying biological processes are too complex for a mechanistic approach. Can computer simulations be used to guide future biological research? We will debate the feasibility of explaining biology from a physicists’ perspective. Learning Objectives: Understand the potential applications and limitations of computational methods for dose-response modeling at the molecular, cellular and tissue levels Learn about mechanism of action underlying the induction, repair and biological processing of damage to DNA and other constituents Understand how effects and processes at one biological scale impact on biological processes and outcomes on other scales J. Schuemann, NCI/NIH grantsS. McMahon, Funding: European Commission FP7 (grant EC FP7 MC-IOF-623630)« less

Recognition and repair of chemically heterogeneous structures at DNA ends

PubMed Central

Andres, Sara N.; Schellenberg, Matthew J.; Wallace, Bret D.; Tumbale, Percy; Williams, R. Scott

2014-01-01

Exposure to environmental toxicants and stressors, radiation, pharmaceutical drugs, inflammation, cellular respiration, and routine DNA metabolism all lead to the production of cytotoxic DNA strand breaks. Akin to splintered wood, DNA breaks are not “clean”. Rather, DNA breaks typically lack DNA 5'-phosphate and 3'-hydroxyl moieties required for DNA synthesis and DNA ligation. Failure to resolve damage at DNA ends can lead to abnormal DNA replication and repair, and is associated with genomic instability, mutagenesis, neurological disease, ageing and carcinogenesis. An array of chemically heterogeneous DNA termini arises from spontaneously generated DNA single-strand and double-strand breaks (SSBs and DSBs), and also from normal and/or inappropriate DNA metabolism by DNA polymerases, DNA ligases and topoisomerases. As a front line of defense to these genotoxic insults, eukaryotic cells have accrued an arsenal of enzymatic first responders that bind and protect damaged DNA termini, and enzymatically tailor DNA ends for DNA repair synthesis and ligation. These nucleic acid transactions employ direct damage reversal enzymes including Aprataxin (APTX), Polynucleotide kinase phosphatase (PNK), the tyrosyl DNA phosphodiesterases (TDP1 and TDP2), the Ku70/80 complex and DNA polymerase β (POLβ). Nucleolytic processing enzymes such as the MRE11/RAD50/NBS1/CtIP complex, Flap endonuclease (FEN1) and the apurinic endonucleases (APE1 and APE2) also act in the chemical "cleansing" of DNA breaks to prevent genomic instability and disease, and promote progression of DNA- and RNA-DNA damage response (DDR and RDDR) pathways. Here, we provide an overview of cellular first responders dedicated to the detection and repair of abnormal DNA termini. PMID:25111769

Nuclear translocation of p19INK4d in response to oxidative DNA damage promotes chromatin relaxation.

PubMed

Sonzogni, Silvina V; Ogara, María F; Castillo, Daniela S; Sirkin, Pablo F; Radicella, J Pablo; Cánepa, Eduardo T

2015-01-01

DNA is continuously exposed to damaging agents that can lead to changes in the genetic information with adverse consequences. Nonetheless, eukaryotic cells have mechanisms such as the DNA damage response (DDR) to prevent genomic instability. The DNA of eukaryotic cells is packaged into nucleosomes, which fold the genome into highly condensed chromatin, but relatively little is known about the role of chromatin accessibility in DNA repair. p19INK4d, a cyclin-dependent kinase inhibitor, plays an important role in cell cycle regulation and cellular DDR. Extensive data indicate that p19INK4d is a critical factor in the maintenance of genomic integrity and cell survival. p19INK4d is upregulated by various genotoxics, improving the repair efficiency for a variety of DNA lesions. The evidence of p19INK4d translocation into the nucleus and its low sequence specificity in its interaction with DNA prompted us to hypothesize that p19INK4d plays a role at an early stage of cellular DDR. In the present study, we demonstrate that upon oxidative DNA damage, p19INK4d strongly binds to and relaxes chromatin. Furthermore, in vitro accessibility assays show that DNA is more accessible to a restriction enzyme when a chromatinized plasmid is incubated in the presence of a protein extract with high levels of p19INK4d. Nuclear protein extracts from cells overexpressing p19INK4d are better able to repair a chromatinized and damaged plasmid. These observations support the notion that p19INK4d would act as a chromatin accessibility factor that allows the access of the repair machinery to the DNA damage site.

Optimised detection of mitochondrial DNA strand breaks.

PubMed

Hanna, Rebecca; Crowther, Jonathan M; Bulsara, Pallav A; Wang, Xuying; Moore, David J; Birch-Machin, Mark A

2018-05-04

Intrinsic and extrinsic factors that induce cellular oxidative stress damage tissue integrity and promote ageing, resulting in accumulative strand breaks to the mitochondrial DNA (mtDNA) genome. Limited repair mechanisms and close proximity to superoxide generation make mtDNA a prominent biomarker of oxidative damage. Using human DNA we describe an optimised long-range qPCR methodology that sensitively detects mtDNA strand breaks relative to a suite of short mitochondrial and nuclear DNA housekeeping amplicons, which control for any variation in mtDNA copy number. An application is demonstrated by detecting 16-36-fold mtDNA damage in human skin cells induced by hydrogen peroxide and solar simulated radiation. Copyright © 2018 Elsevier B.V. and Mitochondria Research Society. All rights reserved.

Sensitivity of spiral ganglion neurons to damage caused by mobile phone electromagnetic radiation will increase in lipopolysaccharide-induced inflammation in vitro model.

PubMed

Zuo, Wen-Qi; Hu, Yu-Juan; Yang, Yang; Zhao, Xue-Yan; Zhang, Yuan-Yuan; Kong, Wen; Kong, Wei-Jia

2015-05-29

With the increasing popularity of mobile phones, the potential hazards of radiofrequency electromagnetic radiation (RF-EMR) on the auditory system remain unclear. Apart from RF-EMR, humans are also exposed to various physical and chemical factors. We established a lipopolysaccharide (LPS)-induced inflammation in vitro model to investigate whether the possible sensitivity of spiral ganglion neurons to damage caused by mobile phone electromagnetic radiation (at specific absorption rates: 2, 4 W/kg) will increase. Spiral ganglion neurons (SGN) were obtained from neonatal (1- to 3-day-old) Sprague Dawley® (SD) rats. After the SGN were treated with different concentrations (0, 20, 40, 50, 100, 200, and 400 μg/ml) of LPS, the Cell Counting Kit-8 (CCK-8) and alkaline comet assay were used to quantify cellular activity and DNA damage, respectively. The SGN were treated with the moderate LPS concentrations before RF-EMR exposure. After 24 h intermittent exposure at an absorption rate of 2 and 4 W/kg, DNA damage was examined by alkaline comet assay, ultrastructure changes were detected by transmission electron microscopy, and expression of the autophagy markers LC3-II and Beclin1 were examined by immunofluorescence and confocal laser scanning microscopy. Reactive oxygen species (ROS) production was quantified by the dichlorofluorescin-diacetate assay. LPS (100 μg/ml) induced DNA damage and suppressed cellular activity (P < 0.05). LPS (40 μg/ml) did not exhibit cellular activity changes or DNA damage (P > 0.05); therefore, 40 μg/ml was used to pretreat the concentration before exposure to RF-EMR. RF-EMR could not directly induce DNA damage. However, the 4 W/kg combined with LPS (40 μg/ml) group showed mitochondria vacuoles, karyopyknosis, presence of lysosomes and autophagosome, and increasing expression of LC3-II and Beclin1. The ROS values significantly increased in the 4 W/kg exposure, 4 W/kg combined with LPS (40 μg/ml) exposure, and H2O2 groups (P < 0.05, 0.01). Short-term exposure to radiofrequency electromagnetic radiation could not directly induce DNA damage in normal spiral ganglion neurons, but it could cause the changes of cellular ultrastructure at special SAR 4.0 W/kg when cells are in fragile or micro-damaged condition. It seems that the sensitivity of SGN to damage caused by mobile phone electromagnetic radiation will increase in a lipopolysaccharide-induced inflammation in vitro model.

Interplay of space radiation and microgravity in DNA damage and DNA damage response.

PubMed

Moreno-Villanueva, María; Wong, Michael; Lu, Tao; Zhang, Ye; Wu, Honglu

2017-01-01

In space, multiple unique environmental factors, particularly microgravity and space radiation, pose constant threat to the DNA integrity of living organisms. Specifically, space radiation can cause damage to DNA directly, through the interaction of charged particles with the DNA molecules themselves, or indirectly through the production of free radicals. Although organisms have evolved strategies on Earth to confront such damage, space environmental conditions, especially microgravity, can impact DNA repair resulting in accumulation of severe DNA lesions. Ultimately these lesions, namely double strand breaks, chromosome aberrations, micronucleus formation, or mutations, can increase the risk for adverse health effects, such as cancer. How spaceflight factors affect DNA damage and the DNA damage response has been investigated since the early days of the human space program. Over the years, these experiments have been conducted either in space or using ground-based analogs. This review summarizes the evidence for DNA damage induction by space radiation and/or microgravity as well as spaceflight-related impacts on the DNA damage response. The review also discusses the conflicting results from studies aimed at addressing the question of potential synergies between microgravity and radiation with regard to DNA damage and cellular repair processes. We conclude that further experiments need to be performed in the true space environment in order to address this critical question.

Expression Profile of DNA Damage Signaling Genes in Proton Exposed Mouse Brain

NASA Astrophysics Data System (ADS)

Ramesh, Govindarajan; Wu, Honglu

Exposure of living systems to radiation results in a wide assortment of lesions, the most signif-icant of is damage to genomic DNA which induce several cellular functions such as cell cycle arrest, repair, apoptosis etc. The radiation induced DNA damage investigation is one of the im-portant area in biology, but still the information available regarding the effects of proton is very limited. In this report, we investigated the differential gene expression pattern of DNA damage signaling genes particularly, damaged DNA binding, repair, cell cycle arrest, checkpoints and apoptosis using quantitative real-time RT-PCR array in proton exposed mouse brain tissues. The expression profiles showed significant changes in DNA damage related genes in 2Gy proton exposed mouse brain tissues as compared with control brain tissues. Furthermore, we also show that significantly increased levels of apoptotic related genes, caspase-3 and 8 activities in these cells, suggesting that in addition to differential expression of DNA damage genes, the alteration of apoptosis related genes may also contribute to the radiation induced DNA damage followed by programmed cell death. In summary, our findings suggest that proton exposed brain tissue undergo severe DNA damage which in turn destabilize the chromatin stability.

THE ATM-SMC1 PATHWAY IS ESSENTIAL FOR ACTIVATION OF THE CHROMIUM[VI]-INDUCED S-PHASE CHECKPOINT

EPA Science Inventory

Hexavalent chromium (Cr[VI] is a common industrial waste product, an environmental pollutant, and a recognized human carcinogen. Following cellular uptake, Cr[VI] can cause DNA damage, however, the mechanisims by which mammalian cells respond to Cr-induced DNA damage remain to b...

PARP-1 dependent recruitment of the amyotrophic lateral sclerosis-associated protein FUS/TLS to sites of oxidative DNA damage

PubMed Central

Rulten, Stuart L.; Rotheray, Amy; Green, Ryan L.; Grundy, Gabrielle J.; Moore, Duncan A. Q.; Gómez-Herreros, Fernando; Hafezparast, Majid; Caldecott, Keith W

2014-01-01

Amyotrophic lateral sclerosis (ALS) is associated with progressive degeneration of motor neurons. Several of the genes associated with this disease encode proteins involved in RNA processing, including fused-in-sarcoma/translocated-in-sarcoma (FUS/TLS). FUS is a member of the heterogeneous nuclear ribonucleoprotein (hnRNP) family of proteins that bind thousands of pre-mRNAs and can regulate their splicing. Here, we have examined the possibility that FUS is also a component of the cellular response to DNA damage. We show that both GFP-tagged and endogenous FUS re-localize to sites of oxidative DNA damage induced by UVA laser, and that FUS recruitment is greatly reduced or ablated by an inhibitor of poly (ADP-ribose) polymerase activity. Consistent with this, we show that recombinant FUS binds directly to poly (ADP-ribose) in vitro, and that both GFP-tagged and endogenous FUS fail to accumulate at sites of UVA laser induced damage in cells lacking poly (ADP-ribose) polymerase-1. Finally, we show that GFP-FUSR521G, harbouring a mutation that is associated with ALS, exhibits reduced ability to accumulate at sites of UVA laser-induced DNA damage. Together, these data suggest that FUS is a component of the cellular response to DNA damage, and that defects in this response may contribute to ALS. PMID:24049082

Cytologic Effects of Air Force Chemicals

DTIC Science & Technology

1978-09-01

pCi/ml, 60 Ci/mmole), hydroxyurea (10- 2M) to suppress replicative DNA synthesis, and with or without 4-nitroquinoline-l-oxide (4NQO, a DNA-damaging...organ cultures. The tissues were minced in cold, buffered saline and then incubated with 3 H-thymidine, hydroxyurea and 4NQO to damage cellular DNA...incorporation under these conditions is taken as an indication of DNA repair activity, and incorporation of 3H-thymidine in the absence of hydroxyurea and

DNA Damage Signalling and Repair Inhibitors: The Long-Sought-After Achilles’ Heel of Cancer

PubMed Central

Velic, Denis; Couturier, Anthony M.; Ferreira, Maria Tedim; Rodrigue, Amélie; Poirier, Guy G.; Fleury, Fabrice; Masson, Jean-Yves

2015-01-01

For decades, radiotherapy and chemotherapy were the two only approaches exploiting DNA repair processes to fight against cancer. Nowadays, cancer therapeutics can be a major challenge when it comes to seeking personalized targeted medicine that is both effective and selective to the malignancy. Over the last decade, the discovery of new targeted therapies against DNA damage signalling and repair has offered the possibility of therapeutic improvements in oncology. In this review, we summarize the current knowledge of DNA damage signalling and repair inhibitors, their molecular and cellular effects, and future therapeutic use. PMID:26610585

Unrepaired clustered DNA lesions induce chromosome breakage in human cells

PubMed Central

Asaithamby, Aroumougame; Hu, Burong; Chen, David J.

2011-01-01

Clustered DNA damage induced by ionizing radiation is refractory to repair and may trigger carcinogenic events for reasons that are not well understood. Here, we used an in situ method to directly monitor induction and repair of clustered DNA lesions in individual cells. We showed, consistent with biophysical modeling, that the kinetics of loss of clustered DNA lesions was substantially compromised in human fibroblasts. The unique spatial distribution of different types of DNA lesions within the clustered damages, but not the physical location of these damages within the subnuclear domains, determined the cellular ability to repair the damage. We then examined checkpoint arrest mechanisms and yield of gross chromosomal aberrations. Induction of nonrepairable clustered damage affected only G2 accumulation but not the early G2/M checkpoint. Further, cells that were released from the G2/M checkpoint with unrepaired clustered damage manifested a spectrum of chromosome aberrations in mitosis. Difficulties associated with clustered DNA damage repair and checkpoint release before the completion of clustered DNA damage repair appear to promote genome instability that may lead to carcinogenesis. PMID:21527720

Chromosome territories reposition during DNA damage-repair response

PubMed Central

2013-01-01

Background Local higher-order chromatin structure, dynamics and composition of the DNA are known to determine double-strand break frequencies and the efficiency of repair. However, how DNA damage response affects the spatial organization of chromosome territories is still unexplored. Results Our report investigates the effect of DNA damage on the spatial organization of chromosome territories within interphase nuclei of human cells. We show that DNA damage induces a large-scale spatial repositioning of chromosome territories that are relatively gene dense. This response is dose dependent, and involves territories moving from the nuclear interior to the periphery and vice versa. Furthermore, we have found that chromosome territory repositioning is contingent upon double-strand break recognition and damage sensing. Importantly, our results suggest that this is a reversible process where, following repair, chromosome territories re-occupy positions similar to those in undamaged control cells. Conclusions Thus, our report for the first time highlights DNA damage-dependent spatial reorganization of whole chromosomes, which might be an integral aspect of cellular damage response. PMID:24330859

RNF8- and Ube2S-Dependent Ubiquitin Lysine 11-Linkage Modification in Response to DNA Damage.

PubMed

Paul, Atanu; Wang, Bin

2017-05-18

Ubiquitin modification of proteins plays pivotal roles in the cellular response to DNA damage. Given the complexity of ubiquitin conjugation due to the formation of poly-conjugates of different linkages, functional roles of linkage-specific ubiquitin modification at DNA damage sites are largely unclear. We identify that Lys11-linkage ubiquitin modification occurs at DNA damage sites in an ATM-dependent manner, and ubiquitin-modifying enzymes, including Ube2S E2-conjugating enzyme and RNF8 E3 ligase, are responsible for the assembly of Lys11-linkage conjugates on damaged chromatin, including histone H2A/H2AX. We show that RNF8- and Ube2S-dependent Lys11-linkage ubiquitin conjugation plays an important role in regulating DNA damage-induced transcriptional silencing, distinct from the role of Lys63-linkage ubiquitin in the recruitment of DNA damage repair proteins 53BP1 and BRCA1. Thus, our study highlights the importance of linkage-specific ubiquitination at DNA damage sites, and it reveals that Lys11-linkage ubiquitin modification plays a crucial role in the DNA damage response. Copyright © 2017 Elsevier Inc. All rights reserved.

Polyhydroxylated fatty alcohols derived from avocado suppress inflammatory response and provide non-sunscreen protection against UV-induced damage in skin cells.

PubMed

Rosenblat, Gennady; Meretski, Shai; Segal, Joseph; Tarshis, Mark; Schroeder, Avi; Zanin-Zhorov, Alexandra; Lion, Gilead; Ingber, Arieh; Hochberg, Malka

2011-05-01

Exposing skin to ultraviolet (UV) radiation contributes to photoaging and to the development of skin cancer by DNA lesions and triggering inflammatory and other harmful cellular cascades. The present study tested the ability of unique lipid molecules, polyhydroxylated fatty alcohols (PFA), extracted from avocado, to reduce UVB-induced damage and inflammation in skin. Introducing PFA to keratinocytes prior to their exposure to UVB exerted a protective effect, increasing cell viability, decreasing the secretion of IL-6 and PGE(2), and enhancing DNA repair. In human skin explants, treating with PFA reduced significantly UV-induced cellular damage. These results support the idea that PFA can play an important role as a photo-protective agent in UV-induced skin damage.

Pre-exposure to 50 Hz magnetic fields modifies menadione-induced genotoxic effects in human SH-SY5Y neuroblastoma cells.

PubMed

Luukkonen, Jukka; Liimatainen, Anu; Höytö, Anne; Juutilainen, Jukka; Naarala, Jonne

2011-03-23

Extremely low frequency (ELF) magnetic fields (MF) are generated by power lines and various electric appliances. They have been classified as possibly carcinogenic by the International Agency for Research on Cancer, but a mechanistic explanation for carcinogenic effects is lacking. A previous study in our laboratory showed that pre-exposure to ELF MF altered cancer-relevant cellular responses (cell cycle arrest, apoptosis) to menadione-induced DNA damage, but it did not include endpoints measuring actual genetic damage. In the present study, we examined whether pre-exposure to ELF MF affects chemically induced DNA damage level, DNA repair rate, or micronucleus frequency in human SH-SY5Y neuroblastoma cells. Exposure to 50 Hz MF was conducted at 100 µT for 24 hours, followed by chemical exposure for 3 hours. The chemicals used for inducing DNA damage and subsequent micronucleus formation were menadione and methyl methanesulphonate (MMS). Pre-treatment with MF enhanced menadione-induced DNA damage, DNA repair rate, and micronucleus formation in human SH-SY5Y neuroblastoma cells. Although the results with MMS indicated similar effects, the differences were not statistically significant. No effects were observed after MF exposure alone. The results confirm our previous findings showing that pre-exposure to MFs as low as 100 µT alters cellular responses to menadione, and show that increased genotoxicity results from such interaction. The present findings also indicate that complementary data at several chronological points may be critical for understanding the MF effects on DNA damage, repair, and post-repair integrity of the genome.

Cytotoxicity and DNA cleavage with core-shell nanocomposites functionalized by a KH domain DNA binding peptide

NASA Astrophysics Data System (ADS)

Bazak, Remon; Ressl, Jan; Raha, Sumita; Doty, Caroline; Liu, William; Wanzer, Beau; Salam, Seddik Abdel; Elwany, Samy; Paunesku, Tatjana; Woloschak, Gayle E.

2013-11-01

A nanoconjugate was composed of metal oxide nanoparticles decorated with peptides and fluorescent dye and tested for DNA cleavage following UV light activation. The peptide design was based on a DNA binding domain, the so called KH domain of the hnRNPK protein. This ``KH peptide'' enabled cellular uptake of nanoconjugates and their entry into cell nuclei. The control nanoconjugate carried no peptide; it consisted only of the metal oxide nanoparticle prepared as Fe3O4@TiO2 nanocomposite and the fluorescent dye alizarin red S. These components of either construct are responsible for nanoconjugate activation by UV light and the resultant production of reactive oxygen species (ROS). Production of ROS at different subcellular locations causes damage to different components of cells: only nanoconjugates inside cell nuclei can be expected to cause DNA cleavage. Degradation of cellular DNA with KH peptide decorated nanoconjugates exceeded the DNA damage obtained from control, no-peptide nanoconjugate counterparts. Moreover, caspase activation and cell death were more extensive in the same cells.A nanoconjugate was composed of metal oxide nanoparticles decorated with peptides and fluorescent dye and tested for DNA cleavage following UV light activation. The peptide design was based on a DNA binding domain, the so called KH domain of the hnRNPK protein. This ``KH peptide'' enabled cellular uptake of nanoconjugates and their entry into cell nuclei. The control nanoconjugate carried no peptide; it consisted only of the metal oxide nanoparticle prepared as Fe3O4@TiO2 nanocomposite and the fluorescent dye alizarin red S. These components of either construct are responsible for nanoconjugate activation by UV light and the resultant production of reactive oxygen species (ROS). Production of ROS at different subcellular locations causes damage to different components of cells: only nanoconjugates inside cell nuclei can be expected to cause DNA cleavage. Degradation of cellular DNA with KH peptide decorated nanoconjugates exceeded the DNA damage obtained from control, no-peptide nanoconjugate counterparts. Moreover, caspase activation and cell death were more extensive in the same cells. Electronic supplementary information (ESI) available: http://janus.northwestern.edu/wololab/auxiliary/supplementary_data_2013.docx. See DOI: 10.1039/c3nr02203j

Put a RING on it: regulation and inhibition of RNF8 and RNF168 RING finger E3 ligases at DNA damage sites

PubMed Central

Bartocci, Cristina; Denchi, Eros Lazzerini

2013-01-01

RING (Really Interesting New Gene) domain-containing E3 ubiquitin ligases comprise a large family of enzymes that in combination with an E2 ubiquitin-conjugating enzyme, modify target proteins by attaching ubiquitin moieties. A number of RING E3s play an essential role in the cellular response to DNA damage highlighting a crucial contribution for ubiquitin-mediated signaling to the genome surveillance pathway. Among the RING E3s, RNF8 and RNF168 play a critical role in the response to double stranded breaks, one of the most deleterious types of DNA damage. These proteins act as positive regulators of the signaling cascade that initiates at DNA lesions. Inactivation of these enzymes is sufficient to severely impair the ability of cells to respond to DNA damage. Given their central role in the pathway, several layers of regulation act at this nodal signaling point. Here we will summarize current knowledge on the roles of RNF8 and RNF168 in maintaining genome integrity with particular emphasis on recent insights into the multiple layers of regulation that act on these enzymes to fine-tune the cellular response to DNA lesions. PMID:23847653

HIPK2 restricts SIRT1 activity upon severe DNA damage by a phosphorylation-controlled mechanism

PubMed Central

Conrad, E; Polonio-Vallon, T; Meister, M; Matt, S; Bitomsky, N; Herbel, C; Liebl, M; Greiner, V; Kriznik, B; Schumacher, S; Krieghoff-Henning, E; Hofmann, T G

2016-01-01

Upon severe DNA damage a cellular signalling network initiates a cell death response through activating tumour suppressor p53 in association with promyelocytic leukaemia (PML) nuclear bodies. The deacetylase Sirtuin 1 (SIRT1) suppresses cell death after DNA damage by antagonizing p53 acetylation. To facilitate efficient p53 acetylation, SIRT1 function needs to be restricted. How SIRT1 activity is regulated under these conditions remains largely unclear. Here we provide evidence that SIRT1 activity is limited upon severe DNA damage through phosphorylation by the DNA damage-responsive kinase HIPK2. We found that DNA damage provokes interaction of SIRT1 and HIPK2, which phosphorylates SIRT1 at Serine 682 upon lethal damage. Furthermore, upon DNA damage SIRT1 and HIPK2 colocalize at PML nuclear bodies, and PML depletion abrogates DNA damage-induced SIRT1 Ser682 phosphorylation. We show that Ser682 phosphorylation inhibits SIRT1 activity and impacts on p53 acetylation, apoptotic p53 target gene expression and cell death. Mechanistically, we found that DNA damage-induced SIRT1 Ser682 phosphorylation provokes disruption of the complex between SIRT1 and its activator AROS. Our findings indicate that phosphorylation-dependent restriction of SIRT1 activity by HIPK2 shapes the p53 response. PMID:26113041

Aeroallergens Induce Reactive Oxygen Species Production and DNA Damage and Dampen Antioxidant Responses in Bronchial Epithelial Cells.

PubMed

Chan, Tze Khee; Tan, W S Daniel; Peh, Hong Yong; Wong, W S Fred

2017-07-01

Exposure to environmental allergens is a major risk factor for asthma development. Allergens possess proteolytic activity that is capable of disrupting the airway epithelium. Although there is increasing evidence pointing to asthma as an epithelial disease, the underlying mechanism that drives asthma has not been fully elucidated. In this study, we investigated the direct DNA damage potential of aeroallergens on human bronchial epithelial cells and elucidated the mechanisms mediating the damage. Human bronchial epithelial cells, BEAS-2B, directly exposed to house dust mites (HDM) resulted in enhanced DNA damage, as measured by the CometChip and the staining of DNA double-strand break marker, γH2AX. HDM stimulated cellular reactive oxygen species production, increased mitochondrial oxidative stress, and promoted nitrosative stress. Notably, expression of nuclear factor erythroid 2-related factor 2-dependent antioxidant genes was reduced immediately after HDM exposure, suggesting that HDM altered antioxidant responses. HDM exposure also reduced cell proliferation and induced cell death. Importantly, HDM-induced DNA damage can be prevented by the antioxidants glutathione and catalase, suggesting that HDM-induced reactive oxygen and nitrogen species can be neutralized by antioxidants. Mechanistic studies revealed that HDM-induced cellular injury is NADPH oxidase (NOX)-dependent, and apocynin, a NOX inhibitor, protected cells from double-strand breaks induced by HDM. Our results show that direct exposure of bronchial epithelial cells to HDM leads to the production of reactive oxygen and nitrogen species that damage DNA and induce cytotoxicity. Antioxidants and NOX inhibitors can prevent HDM-induced DNA damage, revealing a novel role for antioxidants and NOX inhibitors in mitigating allergic airway disease. Copyright © 2017 by The American Association of Immunologists, Inc.

Comprehensive Profiling of Radiosensitive Human Cell Lines with DNA Damage Response Assays Identifies the Neutral Comet Assay as a Potential Surrogate for Clonogenic Survival

PubMed Central

Nahas, Shareef A.; Davies, Robert; Fike, Francesca; Nakamura, Kotoka; Du, Liutao; Kayali, Refik; Martin, Nathan T.; Concannon, Patrick; Gatti, Richard A.

2015-01-01

In an effort to explore the possible causes of human radiosensitivity and identify more rapid assays for cellular radiosensitivity, we interrogated a set of assays that evaluate cellular functions involved in recognition and repair of DNA double-strand breaks: (1) neutral comet assay, (2) radiation-induced γ-H2AX focus formation, (3) the temporal kinetics of structural maintenance of chromosomes 1 phosphorylation, (4) intra-S-phase checkpoint integrity, and (5) mitochondrial respiration. We characterized a unique panel of 19 “radiosensitive” human lymphoblastoid cell lines from individuals with undiagnosed diseases suggestive of a DNA repair disorder. Radiosensitivity was defined by reduced cellular survival using a clonogenic survival assay. Each assay identified cell lines with defects in DNA damage response functions. The highest concordance rate observed, 89% (17/19), was between an abnormal neutral comet assay and reduced survival by the colony survival assay. Our data also suggested that the neutral comet assay would be a more rapid surrogate for analyzing DNA repair/processing disorders. PMID:21962002

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

PubMed Central

Persson, Henrik; Købler, Carsten; Mølhave, Kristian; Samuelson, Lars; Tegenfeldt, Jonas O; Oredsson, Stina; Prinz, Christelle N

2013-01-01

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 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 damage. These results are important guidelines to the design and interpretation of experiments involving nanowire-based transfection and electrical characterization of living cells. PMID:23813871

Linking abnormal mitosis to the acquisition of DNA damage

PubMed Central

Pellman, David

2012-01-01

Cellular defects that impair the fidelity of mitosis promote chromosome missegregation and aneuploidy. Increasing evidence reveals that errors in mitosis can also promote the direct and indirect acquisition of DNA damage and chromosome breaks. Consequently, deregulated cell division can devastate the integrity of the normal genome and unleash a variety of oncogenic stimuli that may promote transformation. Recent work has shed light on the mechanisms that link abnormal mitosis with the development of DNA damage, how cells respond to such affronts, and the potential impact on tumorigenesis. PMID:23229895

WE-DE-202-00: Connecting Radiation Physics with Computational Biology

DOE Office of Scientific and Technical Information (OSTI.GOV)

NONE

Radiation therapy for the treatment of cancer has been established as a highly precise and effective way to eradicate a localized region of diseased tissue. To achieve further significant gains in the therapeutic ratio, we need to move towards biologically optimized treatment planning. To achieve this goal, we need to understand how the radiation-type dependent patterns of induced energy depositions within the cell (physics) connect via molecular, cellular and tissue reactions to treatment outcome such as tumor control and undesirable effects on normal tissue. Several computational biology approaches have been developed connecting physics to biology. Monte Carlo simulations are themore » most accurate method to calculate physical dose distributions at the nanometer scale, however simulations at the DNA scale are slow and repair processes are generally not simulated. Alternative models that rely on the random formation of individual DNA lesions within one or two turns of the DNA have been shown to reproduce the clusters of DNA lesions, including single strand breaks (SSBs), double strand breaks (DSBs) without the need for detailed track structure simulations. Efficient computational simulations of initial DNA damage induction facilitate computational modeling of DNA repair and other molecular and cellular processes. Mechanistic, multiscale models provide a useful conceptual framework to test biological hypotheses and help connect fundamental information about track structure and dosimetry at the sub-cellular level to dose-response effects on larger scales. In this symposium we will learn about the current state of the art of computational approaches estimating radiation damage at the cellular and sub-cellular scale. How can understanding the physics interactions at the DNA level be used to predict biological outcome? We will discuss if and how such calculations are relevant to advance our understanding of radiation damage and its repair, or, if the underlying biological processes are too complex for a mechanistic approach. Can computer simulations be used to guide future biological research? We will debate the feasibility of explaining biology from a physicists’ perspective. Learning Objectives: Understand the potential applications and limitations of computational methods for dose-response modeling at the molecular, cellular and tissue levels Learn about mechanism of action underlying the induction, repair and biological processing of damage to DNA and other constituents Understand how effects and processes at one biological scale impact on biological processes and outcomes on other scales J. Schuemann, NCI/NIH grantsS. McMahon, Funding: European Commission FP7 (grant EC FP7 MC-IOF-623630)« less

DOE Office of Scientific and Technical Information (OSTI.GOV)

McMahon, S.

Radiation therapy for the treatment of cancer has been established as a highly precise and effective way to eradicate a localized region of diseased tissue. To achieve further significant gains in the therapeutic ratio, we need to move towards biologically optimized treatment planning. To achieve this goal, we need to understand how the radiation-type dependent patterns of induced energy depositions within the cell (physics) connect via molecular, cellular and tissue reactions to treatment outcome such as tumor control and undesirable effects on normal tissue. Several computational biology approaches have been developed connecting physics to biology. Monte Carlo simulations are themore » most accurate method to calculate physical dose distributions at the nanometer scale, however simulations at the DNA scale are slow and repair processes are generally not simulated. Alternative models that rely on the random formation of individual DNA lesions within one or two turns of the DNA have been shown to reproduce the clusters of DNA lesions, including single strand breaks (SSBs), double strand breaks (DSBs) without the need for detailed track structure simulations. Efficient computational simulations of initial DNA damage induction facilitate computational modeling of DNA repair and other molecular and cellular processes. Mechanistic, multiscale models provide a useful conceptual framework to test biological hypotheses and help connect fundamental information about track structure and dosimetry at the sub-cellular level to dose-response effects on larger scales. In this symposium we will learn about the current state of the art of computational approaches estimating radiation damage at the cellular and sub-cellular scale. How can understanding the physics interactions at the DNA level be used to predict biological outcome? We will discuss if and how such calculations are relevant to advance our understanding of radiation damage and its repair, or, if the underlying biological processes are too complex for a mechanistic approach. Can computer simulations be used to guide future biological research? We will debate the feasibility of explaining biology from a physicists’ perspective. Learning Objectives: Understand the potential applications and limitations of computational methods for dose-response modeling at the molecular, cellular and tissue levels Learn about mechanism of action underlying the induction, repair and biological processing of damage to DNA and other constituents Understand how effects and processes at one biological scale impact on biological processes and outcomes on other scales J. Schuemann, NCI/NIH grantsS. McMahon, Funding: European Commission FP7 (grant EC FP7 MC-IOF-623630)« less

DNA-PKcs, ATM, and ATR Interplay Maintains Genome Integrity during Neurogenesis.

PubMed

Enriquez-Rios, Vanessa; Dumitrache, Lavinia C; Downing, Susanna M; Li, Yang; Brown, Eric J; Russell, Helen R; McKinnon, Peter J

2017-01-25

The DNA damage response (DDR) orchestrates a network of cellular processes that integrates cell-cycle control and DNA repair or apoptosis, which serves to maintain genome stability. DNA-PKcs (the catalytic subunit of the DNA-dependent kinase, encoded by PRKDC), ATM (ataxia telangiectasia, mutated), and ATR (ATM and Rad3-related) are related PI3K-like protein kinases and central regulators of the DDR. Defects in these kinases have been linked to neurodegenerative or neurodevelopmental syndromes. In all cases, the key neuroprotective function of these kinases is uncertain. It also remains unclear how interactions between the three DNA damage-responsive kinases coordinate genome stability, particularly in a physiological context. Here, we used a genetic approach to identify the neural function of DNA-PKcs and the interplay between ATM and ATR during neurogenesis. We found that DNA-PKcs loss in the mouse sensitized neuronal progenitors to apoptosis after ionizing radiation because of excessive DNA damage. DNA-PKcs was also required to prevent endogenous DNA damage accumulation throughout the adult brain. In contrast, ATR coordinated the DDR during neurogenesis to direct apoptosis in cycling neural progenitors, whereas ATM regulated apoptosis in both proliferative and noncycling cells. We also found that ATR controls a DNA damage-induced G 2 /M checkpoint in cortical progenitors, independent of ATM and DNA-PKcs. These nonoverlapping roles were further confirmed via sustained murine embryonic or cortical development after all three kinases were simultaneously inactivated. Thus, our results illustrate how DNA-PKcs, ATM, and ATR have unique and essential roles during the DDR, collectively ensuring comprehensive genome maintenance in the nervous system. The DNA damage response (DDR) is essential for prevention of a broad spectrum of different human neurologic diseases. However, a detailed understanding of the DDR at a physiological level is lacking. In contrast to many in vitro cellular studies, here we demonstrate independent biological roles for the DDR kinases DNA-PKcs, ATM, and ATR during neurogenesis. We show that DNA-PKcs is central to DNA repair in nonproliferating cells, and restricts DNA damage accumulation, whereas ATR controls damage-induced G 2 checkpoint control and apoptosis in proliferating cells. Conversely, ATM is critical for controlling apoptosis in immature noncycling neural cells after DNA damage. These data demonstrate functionally distinct, but cooperative, roles for each kinase in preserving genome stability in the nervous system. Copyright © 2017 the authors 0270-6474/17/370893-13$15.00/0.

Dissecting DNA damage response pathways by analyzing protein localization and abundance changes during DNA replication stress

PubMed Central

Tkach, Johnny M.; Yimit, Askar; Lee, Anna Y.; Riffle, Michael; Costanzo, Michael; Jaschob, Daniel; Hendry, Jason A.; Ou, Jiongwen; Moffat, Jason; Boone, Charles; Davis, Trisha N.; Nislow, Corey; Brown, Grant W.

2012-01-01

Re-localization of proteins is a hallmark of the DNA damage response. We use high-throughput microscopic screening of the yeast GFP fusion collection to develop a systems-level view of protein re-organization following drug-induced DNA replication stress. Changes in protein localization and abundance reveal drug-specific patterns of functional enrichments. Classification of proteins by sub-cellular destination allows the identification of pathways that respond to replication stress. We analyzed pairwise combinations of GFP fusions and gene deletion mutants to define and order two novel DNA damage responses. In the first, Cmr1 forms subnuclear foci that are regulated by the histone deacetylase Hos2 and are distinct from the typical Rad52 repair foci. In a second example, we find that the checkpoint kinases Mec1/Tel1 and the translation regulator Asc1 regulate P-body formation. This method identifies response pathways that were not detected in genetic and protein interaction screens, and can be readily applied to any form of chemical or genetic stress to reveal cellular response pathways. PMID:22842922

Oligodendroglia are particularly vulnerable to oxidative damage after neurotrauma in vivo.

PubMed

Giacci, Marcus K; Bartlett, Carole A; Smith, Nicole M; Iyer, K Swaminathan; Toomey, Lillian M; Jiang, Haibo; Guagliardo, Paul; Kilburn, Matt R; Fitzgerald, Melinda

2018-06-18

Loss of function following injury to the central nervous system is worsened by secondary degeneration of neurons and glia surrounding the injury and initiated by oxidative damage. However, it is not yet known which cellular populations and structures are most vulnerable to oxidative damage in vivo Using Nanoscale secondary ion mass spectrometry (NanoSIMS), oxidative damage was semi-quantified within cellular subpopulations and structures of optic nerve vulnerable to secondary degeneration, following a partial transection of the optic nerve in adult female PVG rats. Simultaneous assessment of cellular subpopulations and structures revealed oligodendroglia as the most vulnerable to DNA oxidation following injury. 5-ethynyl-2'-deoxyuridine (EdU) was used to label cells that proliferated in the first 3 days after injury. Injury led to increases in DNA, protein and lipid damage in OPCs and mature oligodendrocytes at 3 days, regardless of proliferative state, associated with a decline in the numbers of OPCs at 7 days. O4+ pre-oligodendrocytes also exhibited increased lipid peroxidation. Interestingly, EdU+ mature oligodendrocytes derived after injury demonstrated increased early susceptibility to DNA damage and lipid peroxidation. However, EdU- mature oligodendrocytes with high 8OHdG immunoreactivity were more likely to be caspase3+. By day 28, newly derived mature oligodendrocytes had significantly reduced MYRF mRNA indicating that the myelination potential of these cells may be reduced. The proportion of caspase3+ oligodendrocytes remained higher in EdU- cells. Innovative use of NanoSIMS together with traditional immunohistochemistry and in situ hybridisation have enabled the first demonstration of subpopulation specific oligodendroglial vulnerability to oxidative damage, due to secondary degeneration in vivo. SIGNIFICANCE STATEMENT Injury to the central nervous system is characterised by oxidative damage in areas adjacent to the injury. However, the cellular subpopulations and structures most vulnerable to this damage remain to be elucidated. Here we use powerful NanoSIMS techniques to show increased oxidative damage in oligodendroglia and axons and to demonstrate that cells early in the oligodendroglial lineage are the most vulnerable to DNA oxidation. Further immunohistochemical and in situ hybridisation investigation reveals that mature oligodendrocytes derived after injury are more vulnerable to oxidative damage than their counterparts existing at the time of injury and have reduced MYRF mRNA, yet pre-existing oligodendrocytes are more likely to die. Copyright © 2018 the authors.

Biochemical changes to fibroblast cells subjected to ionizing radiation.

PubMed

Jones, Pamala; Benghuzzi, Hamed; Tucci, Michelle; Richards, Latoya; Harrison, George; Patel, Ramesh

2008-01-01

High energy X-rays are capable of interacting with biological membranes to cause both functional and structural modifications. The goal of the present study was to investigate the effects human fibroblast cells exposed multiple times to 10 Gy over time. Following exposures of 2, 3, or 4 times to 10 Gy/10min the cells were evaluated for cell number changes, membrane damage, and intracellular glutathione content after 24, 48 and 72 hours. Twenty-four hours following exposure the cell numbers were reduced and increased levels of cellular membrane damage was evident. This trend was observed for the duration of the study. Interestingly, there was not an exposure dependent increase in cell damage or cell loss with time. Intracellular antioxidant systems were activated as indicated by anincrease in total cellular glutathione content. Additional studies are needed to determine if the cellular reduction is caused by a direct effect of the X-rays targeting the DNA or an indirect effect of the X-ray targeting the cellular membrane, which then generates radicals that target cell cycle checkpoints or DNA damage. In conclusion, fibroblast cells can be used to determine early and late events of cellular function following exposure to harmful levels of radiation exposure and results of exposure can be seen within twenty four hours.

DNA damage and repair in plants under ultraviolet and ionizing radiations.

PubMed

Gill, Sarvajeet S; Anjum, Naser A; Gill, Ritu; Jha, Manoranjan; Tuteja, Narendra

2015-01-01

Being sessile, plants are continuously exposed to DNA-damaging agents present in the environment such as ultraviolet (UV) and ionizing radiations (IR). Sunlight acts as an energy source for photosynthetic plants; hence, avoidance of UV radiations (namely, UV-A, 315-400 nm; UV-B, 280-315 nm; and UV-C, <280 nm) is unpreventable. DNA in particular strongly absorbs UV-B; therefore, it is the most important target for UV-B induced damage. On the other hand, IR causes water radiolysis, which generates highly reactive hydroxyl radicals (OH(•)) and causes radiogenic damage to important cellular components. However, to maintain genomic integrity under UV/IR exposure, plants make use of several DNA repair mechanisms. In the light of recent breakthrough, the current minireview (a) introduces UV/IR and overviews UV/IR-mediated DNA damage products and (b) critically discusses the biochemistry and genetics of major pathways responsible for the repair of UV/IR-accrued DNA damage. The outcome of the discussion may be helpful in devising future research in the current context.

ATM directs DNA damage responses and proteostasis via genetically separable pathways

PubMed Central

Lee, Ji-Hoon; Mand, Michael R.; Kao, Chung-Hsuan; Zhou, Yi; Ryu, Seung W.; Richards, Alicia L.; Coon, Joshua J.; Paull, Tanya T.

2018-01-01

The protein kinase ATM is a master regulator of the DNA damage response but also responds directly to oxidative stress. Loss of ATM causes Ataxia telangiectasia, a neurodegenerative disorder with pleiotropic symptoms that include cerebellar dysfunction, cancer, diabetes, and premature aging. Here, we genetically separated DNA damage activation of ATM from oxidative activation using separation-of-function mutations. We found that deficiency in ATM activation by Mre11-Rad50-Nbs1 and DNA double-strand breaks resulted in loss of cell viability, checkpoint activation, and DNA end resection in response to DNA damage. In contrast, loss of oxidative activation of ATM had minimal effects on DNA damage-related outcomes but blocked ATM-mediated initiation of checkpoint responses after oxidative stress and resulted in deficiencies in mitochondrial function and autophagy. In addition, expression of ATM lacking oxidative activation generates widespread protein aggregation. These results indicate a direct relationship between the mechanism of ATM activation and its effects on cellular metabolism and DNA damage responses in human cells and implicates ATM in the control of protein homeostasis. PMID:29317520

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

PubMed

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.

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

PubMed Central

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

Creating Age Asymmetry: Consequences of Inheriting Damaged Goods in Mammalian Cells.

PubMed

Moore, Darcie L; Jessberger, Sebastian

2017-01-01

Accumulating evidence suggests that mammalian cells asymmetrically segregate cellular components ranging from genomic DNA to organelles and damaged proteins during cell division. Asymmetric inheritance upon mammalian cell division may be specifically important to ensure cellular fitness and propagate cellular potency to individual progeny, for example in the context of somatic stem cell division. We review here recent advances in the field and discuss potential effects and underlying mechanisms that mediate asymmetric segregation of cellular components during mammalian cell division. Copyright © 2016 Elsevier Ltd. All rights reserved.

Efficient DNA Repair: A Cell’s Fountain of Youth? | Center for Cancer Research

Cancer.gov

Given the central importance of the genome to a cell’s function, it is not surprising that there are a number of proteins devoted to sensing and repairing DNA damage. But what happens when these repair proteins do not work properly? Cancer is one possible outcome, and a growing body of evidence also indicates that the cellular response to DNA damage plays a key role in the

TRAIP promotes DNA damage response during genome replication and is mutated in primordial dwarfism.

PubMed

Harley, Margaret E; Murina, Olga; Leitch, Andrea; Higgs, Martin R; Bicknell, Louise S; Yigit, Gökhan; Blackford, Andrew N; Zlatanou, Anastasia; Mackenzie, Karen J; Reddy, Kaalak; Halachev, Mihail; McGlasson, Sarah; Reijns, Martin A M; Fluteau, Adeline; Martin, Carol-Anne; Sabbioneda, Simone; Elcioglu, Nursel H; Altmüller, Janine; Thiele, Holger; Greenhalgh, Lynn; Chessa, Luciana; Maghnie, Mohamad; Salim, Mahmoud; Bober, Michael B; Nürnberg, Peter; Jackson, Stephen P; Hurles, Matthew E; Wollnik, Bernd; Stewart, Grant S; Jackson, Andrew P

2016-01-01

DNA lesions encountered by replicative polymerases threaten genome stability and cell cycle progression. Here we report the identification of mutations in TRAIP, encoding an E3 RING ubiquitin ligase, in patients with microcephalic primordial dwarfism. We establish that TRAIP relocalizes to sites of DNA damage, where it is required for optimal phosphorylation of H2AX and RPA2 during S-phase in response to ultraviolet (UV) irradiation, as well as fork progression through UV-induced DNA lesions. TRAIP is necessary for efficient cell cycle progression and mutations in TRAIP therefore limit cellular proliferation, providing a potential mechanism for microcephaly and dwarfism phenotypes. Human genetics thus identifies TRAIP as a component of the DNA damage response to replication-blocking DNA lesions.

Aldehydes with high and low toxicities inactivate cells by damaging distinct cellular targets.

PubMed

Xie, Ming-Zhang; Shoulkamy, Mahmoud I; Salem, Amir M H; Oba, Shunya; Goda, Mizuki; Nakano, Toshiaki; Ide, Hiroshi

2016-04-01

Aldehydes are genotoxic and cytotoxic molecules and have received considerable attention for their associations with the pathogenesis of various human diseases. In addition, exposure to anthropogenic aldehydes increases human health risks. The general mechanism of aldehyde toxicity involves adduct formation with biomolecules such as DNA and proteins. Although the genotoxic effects of aldehydes such as mutations and chromosomal aberrations are directly related to DNA damage, the role of DNA damage in the cytotoxic effects of aldehydes is poorly understood because concurrent protein damage by aldehydes has similar effects. In this study, we have analysed how saturated and α,β-unsaturated aldehydes exert cytotoxic effects through DNA and protein damage. Interestingly, DNA repair is essential for alleviating the cytotoxic effect of weakly toxic aldehydes such as saturated aldehydes but not highly toxic aldehydes such as long α,β-unsaturated aldehydes. Thus, highly toxic aldehydes inactivate cells exclusively by protein damage. Our data suggest that DNA interstrand crosslinks, but not DNA-protein crosslinks and DNA double-strand breaks, are the critical cytotoxic DNA damage induced by aldehydes. Further, we show that the depletion of intracellular glutathione and the oxidation of thioredoxin 1 partially account for the DNA damage-independent cytotoxicity of aldehydes. On the basis of these findings, we have proposed a mechanistic model of aldehyde cytotoxicity mediated by DNA and protein damage. Copyright © 2016 Elsevier B.V. All rights reserved.

Overexpression of the base excision repair NTHL1 glycosylase causes genomic instability and early cellular hallmarks of cancer

PubMed Central

Limpose, Kristin L; Trego, Kelly S; Li, Zhentian; Leung, Sara W; Sarker, Altaf H; Shah, Jason A; Ramalingam, Suresh S; Werner, Erica M; Dynan, William S; Cooper, Priscilla K; Corbett, Anita H; Doetsch, Paul W

2018-01-01

Abstract Base excision repair (BER), which is initiated by DNA N-glycosylase proteins, is the frontline for repairing potentially mutagenic DNA base damage. The NTHL1 glycosylase, which excises DNA base damage caused by reactive oxygen species, is thought to be a tumor suppressor. However, in addition to NTHL1 loss-of-function mutations, our analysis of cancer genomic datasets reveals that NTHL1 frequently undergoes amplification or upregulation in some cancers. Whether NTHL1 overexpression could contribute to cancer phenotypes has not yet been explored. To address the functional consequences of NTHL1 overexpression, we employed transient overexpression. Both NTHL1 and a catalytically-dead NTHL1 (CATmut) induce DNA damage and genomic instability in non-transformed human bronchial epithelial cells (HBEC) when overexpressed. Strikingly, overexpression of either NTHL1 or CATmut causes replication stress signaling and a decrease in homologous recombination (HR). HBEC cells that overexpress NTHL1 or CATmut acquire the ability to grow in soft agar and exhibit loss of contact inhibition, suggesting that a mechanism independent of NTHL1 catalytic activity contributes to acquisition of cancer-related cellular phenotypes. We provide evidence that NTHL1 interacts with the multifunctional DNA repair protein XPG suggesting that interference with HR is a possible mechanism that contributes to acquisition of early cellular hallmarks of cancer. PMID:29522130

Involvement of oxidatively damaged DNA and repair in cancer development and aging

PubMed Central

Tudek, Barbara; Winczura, Alicja; Janik, Justyna; Siomek, Agnieszka; Foksinski, Marek; Oliński, Ryszard

2010-01-01

DNA damage and DNA repair may mediate several cellular processes, like replication and transcription, mutagenesis and apoptosis and thus may be important factors in the development and pathology of an organism, including cancer. DNA is constantly damaged by reactive oxygen species (ROS) and reactive nitrogen species (RNS) directly and also by products of lipid peroxidation (LPO), which form exocyclic adducts to DNA bases. A wide variety of oxidatively-generated DNA lesions are present in living cells. 8-oxoguanine (8-oxoGua) is one of the best known DNA lesions due to its mutagenic properties. Among LPO-derived DNA base modifications the most intensively studied are ethenoadenine and ethenocytosine, highly miscoding DNA lesions considered as markers of oxidative stress and promutagenic DNA damage. Although at present it is impossible to directly answer the question concerning involvement of oxidatively damaged DNA in cancer etiology, it is likely that oxidatively modified DNA bases may serve as a source of mutations that initiate carcinogenesis and are involved in aging (i.e. they may be causal factors responsible for these processes). To counteract the deleterious effect of oxidatively damaged DNA, all organisms have developed several DNA repair mechanisms. The efficiency of oxidatively damaged DNA repair was frequently found to be decreased in cancer patients. The present work reviews the basis for the biological significance of DNA damage, particularly effects of 8-oxoGua and ethenoadduct occurrence in DNA in the aspect of cancer development, drawing attention to the multiplicity of proteins with repair activities. PMID:20589166

DNA Damage as a Driver for Growth Delay: Chromosome Instability Syndromes with Intrauterine Growth Retardation

PubMed Central

Hernández-Gómez, Mariana

2017-01-01

DNA is constantly exposed to endogenous and exogenous mutagenic stimuli that are capable of producing diverse lesions. In order to protect the integrity of the genetic material, a wide array of DNA repair systems that can target each specific lesion has evolved. Despite the availability of several repair pathways, a common general program known as the DNA damage response (DDR) is stimulated to promote lesion detection, signaling, and repair in order to maintain genetic integrity. The genes that participate in these pathways are subject to mutation; a loss in their function would result in impaired DNA repair and genomic instability. When the DDR is constitutionally altered, every cell of the organism, starting from development, will show DNA damage and subsequent genomic instability. The cellular response to this is either uncontrolled proliferation and cell cycle deregulation that ensues overgrowth, or apoptosis and senescence that result in tissue hypoplasia. These diverging growth abnormalities can clinically translate as cancer or growth retardation; both features can be found in chromosome instability syndromes (CIS). The analysis of the clinical, cellular, and molecular phenotypes of CIS with intrauterine growth retardation allows inferring that replication alteration is their unifying feature. PMID:29238724

Mechanisms of ultraviolet disinfection and chlorination of Escherichia coli: Culturability, membrane permeability, metabolism, and genetic damage.

PubMed

Xu, Limei; Zhang, Chongmiao; Xu, Pengcheng; Wang, Xiaochang C

2018-03-01

Traditional culture methods may underestimate the tolerance of microorganisms to disinfectants because of the existence of viable but nonculturable or sublethally injured cells after disinfection. The selection of a strict method is crucial for the evaluation of disinfection performance. The actions of 2 typical disinfectants - ultraviolet (UV) and chlorine - on the fecal indicator Escherichia coli were investigated by the detection of culturability, membrane permeability, metabolic activity, deoxyribonucleic acid (DNA), and messenger ribonucleic acid (mRNA). During UV disinfection, the irreversible damages in the cell membrane and cellular adenosine triphosphate (ATP) were negligible at low UV doses (<80mJ/cm 2 ). However, membrane permeability was damaged at low doses of chlorine (<5mg/L), leading to leakage of cellular ATP. Our study showed that a slight lesion in DNA was detected even at high doses of UV (400mJ/cm 2 ) and chlorine (>5mg/L) treatments. The decay of mRNA was more rapid than that of DNA. The degradation level of mRNA depended on the choice of target genes. After exposure to 50mJ/cm 2 UV dose or 5mg/L chlorine for 30min, the DNA damage repair function (RecA mRNA) was inhibited. The mRNA involved in the DNA damage repair function can be a potential indicator of bacterial viability. Copyright © 2017. Published by Elsevier B.V.

Prediction of cellular radiosensitivity from DNA damage induced by gamma-rays and carbon ion irradiation in canine tumor cells.

PubMed

Wada, Seiichi; Van Khoa, Tran; Kobayashi, Yasuhiko; Funayama, Tomoo; Ogihara, Kikumi; Ueno, Shunji; Ito, Nobuhiko

2005-11-01

Diseases of companion animals are shifting from infectious diseases to neoplasms (cancer), and since radiation therapy is one of the effective choices available for cancer treatment, the application of radiotherapy in veterinary medicine is likely to increase. However tumor tissues have different radiosensitivities, and therefore it is important to determine the intrinsic radiosensitivity of tumors in individual patients in advance of radiotherapy. We have studied the relationship between the surviving cell fraction measured by a clonogenic assay and DNA double strand breaks detected by a comet assay under neutral conditions in three canine tumor cell lines, after gamma-ray and carbon ion irradiation. In all the cell lines, cell death assessed by the clonogenic assay was much higher following irradiation with carbon ions than with gamma-rays. The initial and residual (4 hr) DNA damage due to gamma-ray and carbon ion irradiation were higher in a radiosensitive cell line than in a radioresistant cell line. The surviving cell fraction at 2 Gy (SF2) showed a tendency for correlation with both the initial and residual DNA damage. In particular, the residual damage per Gy was significantly correlated with SF2, regardless of the type of radiation. This indicates that cellular radiosensitivity can be predicted by detection of radiation-induced residual DNA damage.

Systematic random sampling of the comet assay.

PubMed

McArt, Darragh G; Wasson, Gillian R; McKerr, George; Saetzler, Kurt; Reed, Matt; Howard, C Vyvyan

2009-07-01

The comet assay is a technique used to quantify DNA damage and repair at a cellular level. In the assay, cells are embedded in agarose and the cellular content is stripped away leaving only the DNA trapped in an agarose cavity which can then be electrophoresed. The damaged DNA can enter the agarose and migrate while the undamaged DNA cannot and is retained. DNA damage is measured as the proportion of the migratory 'tail' DNA compared to the total DNA in the cell. The fundamental basis of these arbitrary values is obtained in the comet acquisition phase using fluorescence microscopy with a stoichiometric stain in tandem with image analysis software. Current methods deployed in such an acquisition are expected to be both objectively and randomly obtained. In this paper we examine the 'randomness' of the acquisition phase and suggest an alternative method that offers both objective and unbiased comet selection. In order to achieve this, we have adopted a survey sampling approach widely used in stereology, which offers a method of systematic random sampling (SRS). This is desirable as it offers an impartial and reproducible method of comet analysis that can be used both manually or automated. By making use of an unbiased sampling frame and using microscope verniers, we are able to increase the precision of estimates of DNA damage. Results obtained from a multiple-user pooled variation experiment showed that the SRS technique attained a lower variability than that of the traditional approach. The analysis of a single user with repetition experiment showed greater individual variances while not being detrimental to overall averages. This would suggest that the SRS method offers a better reflection of DNA damage for a given slide and also offers better user reproducibility.

Regulatory roles of tankyrase 1 at telomeres and in DNA repair: suppression of T-SCE and stabilization of DNA-PKcs

PubMed Central

Dregalla, Ryan C.; Zhou, Junqing; Idate, Rupa R.; Battaglia, Christine L.R.; Liber, Howard L.; Bailey, Susan M.

2010-01-01

Intrigued by the dynamics of the seemingly contradictory yet integrated cellular responses to the requisites of preserving telomere integrity while also efficiently repairing damaged DNA, we investigated roles of the telomere associated poly(adenosine diphosphate [ADP]-ribose) polymerase (PARP) tankyrase 1 in both telomere function and the DNA damage response following exposure to ionizing radiation. Tankyrase 1 siRNA knockdown in human cells significantly elevated recombination specifically within telomeres, a phenotype with the potential of accelerating cellular senescence. Additionally, depletion of tankyrase 1 resulted in concomitant and rapid reduction of the nonhomologous end-joining protein DNA-PKcs, while Ku86 and ATM protein levels remained unchanged; DNA-PKcs mRNA levels were also unaffected. We found that the requirement of tankyrase 1 for DNA-PKcs protein stability reflects the necessity of its PARP enzymatic activity. We also demonstrated that depletion of tankyrase 1 resulted in proteasome-mediated DNA-PKcs degradation, explaining the associated defective damage response observed; i.e., increased sensitivity to ionizing radiation-induced cell killing, mutagenesis, chromosome aberration and telomere fusion. We provide the first evidence for regulation of DNA-PKcs by tankyrase 1 PARP activity and taken together, identify roles of tankyrase 1 with implications not only for DNA repair and telomere biology, but also for cancer and aging. PMID:21037379

Ablation of XP-V gene causes adipose tissue senescence and metabolic abnormalities

PubMed Central

Chen, Yih-Wen; Harris, Robert A.; Hatahet, Zafer; Chou, Kai-ming

2015-01-01

Obesity and the metabolic syndrome have evolved to be major health issues throughout the world. Whether loss of genome integrity contributes to this epidemic is an open question. DNA polymerase η (pol η), encoded by the xeroderma pigmentosum (XP-V) gene, plays an essential role in preventing cutaneous cancer caused by UV radiation-induced DNA damage. Herein, we demonstrate that pol η deficiency in mice (pol η−/−) causes obesity with visceral fat accumulation, hepatic steatosis, hyperleptinemia, hyperinsulinemia, and glucose intolerance. In comparison to WT mice, adipose tissue from pol η−/− mice exhibits increased DNA damage and a greater DNA damage response, indicated by up-regulation and/or phosphorylation of ataxia telangiectasia mutated (ATM), phosphorylated H2AX (γH2AX), and poly[ADP-ribose] polymerase 1 (PARP-1). Concomitantly, increased cellular senescence in the adipose tissue from pol η−/− mice was observed and measured by up-regulation of senescence markers, including p53, p16Ink4a, p21, senescence-associated (SA) β-gal activity, and SA secretion of proinflammatory cytokines interleukin 6 (IL-6) and tumor necrosis factor α (TNF-α) as early as 4 wk of age. Treatment of pol η−/− mice with a p53 inhibitor, pifithrin-α, reduced adipocyte senescence and attenuated the metabolic abnormalities. Furthermore, elevation of adipocyte DNA damage with a high-fat diet or sodium arsenite exacerbated adipocyte senescence and metabolic abnormalities in pol η−/− mice. In contrast, reduction of adipose DNA damage with N-acetylcysteine or metformin ameliorated cellular senescence and metabolic abnormalities. These studies indicate that elevated DNA damage is a root cause of adipocyte senescence, which plays a determining role in the development of obesity and insulin resistance. PMID:26240351

Pre-Exposure to 50 Hz Magnetic Fields Modifies Menadione-Induced Genotoxic Effects in Human SH-SY5Y Neuroblastoma Cells

PubMed Central

Luukkonen, Jukka; Liimatainen, Anu; Höytö, Anne; Juutilainen, Jukka; Naarala, Jonne

2011-01-01

Background Extremely low frequency (ELF) magnetic fields (MF) are generated by power lines and various electric appliances. They have been classified as possibly carcinogenic by the International Agency for Research on Cancer, but a mechanistic explanation for carcinogenic effects is lacking. A previous study in our laboratory showed that pre-exposure to ELF MF altered cancer-relevant cellular responses (cell cycle arrest, apoptosis) to menadione-induced DNA damage, but it did not include endpoints measuring actual genetic damage. In the present study, we examined whether pre-exposure to ELF MF affects chemically induced DNA damage level, DNA repair rate, or micronucleus frequency in human SH-SY5Y neuroblastoma cells. Methodology/Principal Findings Exposure to 50 Hz MF was conducted at 100 µT for 24 hours, followed by chemical exposure for 3 hours. The chemicals used for inducing DNA damage and subsequent micronucleus formation were menadione and methyl methanesulphonate (MMS). Pre-treatment with MF enhanced menadione-induced DNA damage, DNA repair rate, and micronucleus formation in human SH-SY5Y neuroblastoma cells. Although the results with MMS indicated similar effects, the differences were not statistically significant. No effects were observed after MF exposure alone. Conclusions The results confirm our previous findings showing that pre-exposure to MFs as low as 100 µT alters cellular responses to menadione, and show that increased genotoxicity results from such interaction. The present findings also indicate that complementary data at several chronological points may be critical for understanding the MF effects on DNA damage, repair, and post-repair integrity of the genome. PMID:21448285

DNA damage induction and/or repair as mammalian cell biomarker for the prediction of cellular radiation response

NASA Astrophysics Data System (ADS)

Baumstark-Khan, C.

DNA damage and its repair processes are key factors in cancer induction and also in the treatment of malignancies. Cancer prevention during extended space missions becomes a topic of great importance for space radiobiology. The knowledge of individual responsiveness would allow the protection strategy to be tailored optimally in each case. Radiobiological analysis of cultured cells derived from tissue explants from individuals has shown that measurement of the surviving fraction after 2 Gy (SF2) may be used to predict the individual responsiveness. However, clonogenic assays are timeconsuming, thus alternative assays for the determination of radiore-sponse are being sought. For that reason CHO cell strains having different repair capacities were used for examining whether DNA strand break repair is a suitable experimental design to allow predictive statements. Cellular survival (CFA assay) and DNA strand breaks (total DNA strand breaks: FADU technique; DSBs: non-denaturing elution) were determined in parallel immediately after irradiation as well as after a 24 hour recovery period according to dose. There were no correlations between the dose-response curves of the initial level of DNA strand breaks and parameters that describe clonogenic survival curves (SF2). A good correlation exists between intrinsic cellular radioresistance and the extent of residual DNA strand breaks.

Dynamical analysis of cellular ageing by modeling of gene regulatory network based attractor landscape.

PubMed

Chong, Ket Hing; Zhang, Xiaomeng; Zheng, Jie

2018-01-01

Ageing is a natural phenomenon that is inherently complex and remains a mystery. Conceptual model of cellular ageing landscape was proposed for computational studies of ageing. However, there is a lack of quantitative model of cellular ageing landscape. This study aims to investigate the mechanism of cellular ageing in a theoretical model using the framework of Waddington's epigenetic landscape. We construct an ageing gene regulatory network (GRN) consisting of the core cell cycle regulatory genes (including p53). A model parameter (activation rate) is used as a measure of the accumulation of DNA damage. Using the bifurcation diagrams to estimate the parameter values that lead to multi-stability, we obtained a conceptual model for capturing three distinct stable steady states (or attractors) corresponding to homeostasis, cell cycle arrest, and senescence or apoptosis. In addition, we applied a Monte Carlo computational method to quantify the potential landscape, which displays: I) one homeostasis attractor for low accumulation of DNA damage; II) two attractors for cell cycle arrest and senescence (or apoptosis) in response to high accumulation of DNA damage. Using the Waddington's epigenetic landscape framework, the process of ageing can be characterized by state transitions from landscape I to II. By in silico perturbations, we identified the potential landscape of a perturbed network (inactivation of p53), and thereby demonstrated the emergence of a cancer attractor. The simulated dynamics of the perturbed network displays a landscape with four basins of attraction: homeostasis, cell cycle arrest, senescence (or apoptosis) and cancer. Our analysis also showed that for the same perturbed network with low DNA damage, the landscape displays only the homeostasis attractor. The mechanistic model offers theoretical insights that can facilitate discovery of potential strategies for network medicine of ageing-related diseases such as cancer.

Regulation of ATM-Dependent DNA Damage Responses in Breast Cancer by the RhoGEF Net1

DTIC Science & Technology

2014-04-01

1998) Science 279: 509-514. 12. Harper JW, et al., (2007) The DNA Damage Response: Ten years after. Mol. Cell 28; 739-745. 13. Hill R, et al., (2010...RhoGTPases: Biochemistry and Biology. Annu. Rev. Cell Dev. Biol. 21:247-269. 17. Khanna KK, et al., (2001) ATM, a central controller of cellular

Oxidative DNA damage background estimated by a system model of base excision repair

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sokhansanj, B A; Wilson, III, D M

Human DNA can be damaged by natural metabolism through free radical production. It has been suggested that the equilibrium between innate damage and cellular DNA repair results in an oxidative DNA damage background that potentially contributes to disease and aging. Efforts to quantitatively characterize the human oxidative DNA damage background level based on measuring 8-oxoguanine lesions as a biomarker have led to estimates varying over 3-4 orders of magnitude, depending on the method of measurement. We applied a previously developed and validated quantitative pathway model of human DNA base excision repair, integrating experimentally determined endogenous damage rates and model parametersmore » from multiple sources. Our estimates of at most 100 8-oxoguanine lesions per cell are consistent with the low end of data from biochemical and cell biology experiments, a result robust to model limitations and parameter variation. Our results show the power of quantitative system modeling to interpret composite experimental data and make biologically and physiologically relevant predictions for complex human DNA repair pathway mechanisms and capacity.« less

Kinetic theory approach to modeling of cellular repair mechanisms under genome stress.

PubMed

Qi, Jinpeng; Ding, Yongsheng; Zhu, Ying; Wu, Yizhi

2011-01-01

Under acute perturbations from outer environment, a normal cell can trigger cellular self-defense mechanism in response to genome stress. To investigate the kinetics of cellular self-repair process at single cell level further, a model of DNA damage generating and repair is proposed under acute Ion Radiation (IR) by using mathematical framework of kinetic theory of active particles (KTAP). Firstly, we focus on illustrating the profile of Cellular Repair System (CRS) instituted by two sub-populations, each of which is made up of the active particles with different discrete states. Then, we implement the mathematical framework of cellular self-repair mechanism, and illustrate the dynamic processes of Double Strand Breaks (DSBs) and Repair Protein (RP) generating, DSB-protein complexes (DSBCs) synthesizing, and toxins accumulating. Finally, we roughly analyze the capability of cellular self-repair mechanism, cellular activity of transferring DNA damage, and genome stability, especially the different fates of a certain cell before and after the time thresholds of IR perturbations that a cell can tolerate maximally under different IR perturbation circumstances.

Chemical determination of free radical-induced damage to DNA.

PubMed

Dizdaroglu, M

1991-01-01

Free radical-induced damage to DNA in vivo can result in deleterious biological consequences such as the initiation and promotion of cancer. Chemical characterization and quantitation of such DNA damage is essential for an understanding of its biological consequences and cellular repair. Methodologies incorporating the technique of gas chromatography/mass spectrometry (GC/MS) have been developed in recent years for measurement of free radical-induced DNA damage. The use of GC/MS with selected-ion monitoring (SIM) facilitates unequivocal identification and quantitation of a large number of products of all four DNA bases produced in DNA by reactions with hydroxyl radical, hydrated electron, and H atom. Hydroxyl radical-induced DNA-protein cross-links in mammalian chromatin, and products of the sugar moiety in DNA are also unequivocally identified and quantitated. The sensitivity and selectivity of the GC/MS-SIM technique enables the measurement of DNA base products even in isolated mammalian chromatin without the necessity of first isolating DNA, and despite the presence of histones. Recent results reviewed in this article demonstrate the usefulness of the GC/MS technique for chemical determination of free radical-induced DNA damage in DNA as well as in mammalian chromatin under a vast variety of conditions of free radical production.

Linking JNK Activity to the DNA Damage Response

PubMed Central

Picco, Vincent

2013-01-01

The activity of c-Jun N-terminal kinase (JNK) was initially described as ultraviolet- and oncogene-induced kinase activity on c-Jun. Shortly after this initial discovery, JNK activation was reported for a wider variety of DNA-damaging agents, including γ-irradiation and chemotherapeutic compounds. As the DNA damage response mechanisms were progressively uncovered, the mechanisms governing the activation of JNK upon genotoxic stresses became better understood. In particular, a recent set of papers links the physical breakage in DNA, the activation of the transcription factor NF-κB, the secretion of TNF-α, and an autocrine activation of the JNK pathway. In this review, we will focus on the pathway that is initiated by a physical break in the DNA helix, leading to JNK activation and the resultant cellular consequences. The implications of these findings will be discussed in the context of cancer therapy with DNA-damaging agents. PMID:24349633

Understanding radiation damage on sub-cellular scale using RADAMOL simulation tool

NASA Astrophysics Data System (ADS)

Štěpán, Václav; Davídková, Marie

2016-11-01

We present an overview of the biophysical model RADAMOL developed as a Monte Carlo simulation tool for physical, physico-chemical and chemical stages of ionizing radiation action. Direct and indirect radiation damage by 10 keV electrons, and protons and alpha particles with energies from 1 MeV up to 30 MeV to a free DNA oligomer or DNA in the complex with lac repressor protein is analyzed. The role of radiation type and energy, oxygen concentration and DNA interaction with proteins on yields and distributions of primary biomolecular damage is demonstrated and discussed.

Comparative analysis of different laser systems to study cellular responses to DNA damage in mammalian cells

PubMed Central

Kong, Xiangduo; Mohanty, Samarendra K.; Stephens, Jared; Heale, Jason T.; Gomez-Godinez, Veronica; Shi, Linda Z.; Kim, Jong-Soo; Yokomori, Kyoko; Berns, Michael W.

2009-01-01

Proper recognition and repair of DNA damage is critical for the cell to protect its genomic integrity. Laser microirradiation ranging in wavelength from ultraviolet A (UVA) to near-infrared (NIR) can be used to induce damage in a defined region in the cell nucleus, representing an innovative technology to effectively analyze the in vivo DNA double-strand break (DSB) damage recognition process in mammalian cells. However, the damage-inducing characteristics of the different laser systems have not been fully investigated. Here we compare the nanosecond nitrogen 337 nm UVA laser with and without bromodeoxyuridine (BrdU), the nanosecond and picosecond 532 nm green second-harmonic Nd:YAG, and the femtosecond NIR 800 nm Ti:sapphire laser with regard to the type(s) of damage and corresponding cellular responses. Crosslinking damage (without significant nucleotide excision repair factor recruitment) and single-strand breaks (with corresponding repair factor recruitment) were common among all three wavelengths. Interestingly, UVA without BrdU uniquely produced base damage and aberrant DSB responses. Furthermore, the total energy required for the threshold H2AX phosphorylation induction was found to vary between the individual laser systems. The results indicate the involvement of different damage mechanisms dictated by wavelength and pulse duration. The advantages and disadvantages of each system are discussed. PMID:19357094

Efficient DNA Repair: A Cell’s Fountain of Youth? | Center for Cancer Research

Cancer.gov

Given the central importance of the genome to a cell’s function, it is not surprising that there are a number of proteins devoted to sensing and repairing DNA damage. But what happens when these repair proteins do not work properly? Cancer is one possible outcome, and a growing body of evidence also indicates that the cellular response to DNA damage plays a key role in the aging process. This concept is supported by the fact that many premature aging syndromes are caused by mutations in DNA repair proteins.

Glutamine deficiency induces DNA alkylation damage and sensitizes cancer cells to alkylating agents through inhibition of ALKBH enzymes.

PubMed

Tran, Thai Q; Ishak Gabra, Mari B; Lowman, Xazmin H; Yang, Ying; Reid, Michael A; Pan, Min; O'Connor, Timothy R; Kong, Mei

2017-11-01

Driven by oncogenic signaling, glutamine addiction exhibited by cancer cells often leads to severe glutamine depletion in solid tumors. Despite this nutritional environment that tumor cells often experience, the effect of glutamine deficiency on cellular responses to DNA damage and chemotherapeutic treatment remains unclear. Here, we show that glutamine deficiency, through the reduction of alpha-ketoglutarate, inhibits the AlkB homolog (ALKBH) enzymes activity and induces DNA alkylation damage. As a result, glutamine deprivation or glutaminase inhibitor treatment triggers DNA damage accumulation independent of cell death. In addition, low glutamine-induced DNA damage is abolished in ALKBH deficient cells. Importantly, we show that glutaminase inhibitors, 6-Diazo-5-oxo-L-norleucine (DON) or CB-839, hypersensitize cancer cells to alkylating agents both in vitro and in vivo. Together, the crosstalk between glutamine metabolism and the DNA repair pathway identified in this study highlights a potential role of metabolic stress in genomic instability and therapeutic response in cancer.

Glutamine deficiency induces DNA alkylation damage and sensitizes cancer cells to alkylating agents through inhibition of ALKBH enzymes

PubMed Central

Tran, Thai Q.; Ishak Gabra, Mari B.; Lowman, Xazmin H.; Yang, Ying; Reid, Michael A.; Pan, Min; O’Connor, Timothy R.

2017-01-01

Driven by oncogenic signaling, glutamine addiction exhibited by cancer cells often leads to severe glutamine depletion in solid tumors. Despite this nutritional environment that tumor cells often experience, the effect of glutamine deficiency on cellular responses to DNA damage and chemotherapeutic treatment remains unclear. Here, we show that glutamine deficiency, through the reduction of alpha-ketoglutarate, inhibits the AlkB homolog (ALKBH) enzymes activity and induces DNA alkylation damage. As a result, glutamine deprivation or glutaminase inhibitor treatment triggers DNA damage accumulation independent of cell death. In addition, low glutamine-induced DNA damage is abolished in ALKBH deficient cells. Importantly, we show that glutaminase inhibitors, 6-Diazo-5-oxo-L-norleucine (DON) or CB-839, hypersensitize cancer cells to alkylating agents both in vitro and in vivo. Together, the crosstalk between glutamine metabolism and the DNA repair pathway identified in this study highlights a potential role of metabolic stress in genomic instability and therapeutic response in cancer. PMID:29107960

DNA damage in cells exhibiting radiation-induced genomic instability

DOE PAGES

Keszenman, Deborah J.; Kolodiuk, Lucia; Baulch, Janet E.

2015-02-22

Cells exhibiting radiation induced genomic instability exhibit varied spectra of genetic and chromosomal aberrations. Even so, oxidative stress remains a common theme in the initiation and/or perpetuation of this phenomenon. Isolated oxidatively modified bases, abasic sites, DNA single strand breaks and clustered DNA damage are induced in normal mammalian cultured cells and tissues due to endogenous reactive oxygen species generated during normal cellular metabolism in an aerobic environment. While sparse DNA damage may be easily repaired, clustered DNA damage may lead to persistent cytotoxic or mutagenic events that can lead to genomic instability. In this study, we tested the hypothesismore » that DNA damage signatures characterised by altered levels of endogenous, potentially mutagenic, types of DNA damage and chromosomal breakage are related to radiation-induced genomic instability and persistent oxidative stress phenotypes observed in the chromosomally unstable progeny of irradiated cells. The measurement of oxypurine, oxypyrimidine and abasic site endogenous DNA damage showed differences in non-double-strand breaks (DSB) clusters among the three of the four unstable clones evaluated as compared to genomically stable clones and the parental cell line. These three unstable clones also had increased levels of DSB clusters. The results of this study demonstrate that each unstable cell line has a unique spectrum of persistent damage and lead us to speculate that alterations in DNA damage signaling and repair may be related to the perpetuation of genomic instability.« less

TRAIP promotes DNA damage response during genome replication and is mutated in primordial dwarfism

PubMed Central

Leitch, Andrea; Higgs, Martin R.; Bicknell, Louise S.; Yigit, Gökhan; Blackford, Andrew N.; Zlatanou, Anastasia; Mackenzie, Karen J.; Reddy, Kaalak; Halachev, Mihail; McGlasson, Sarah; Reijns, Martin A. M.; Fluteau, Adeline; Martin, Carol-Anne; Sabbioneda, Simone; Elcioglu, Nursel H.; Altmüller, Janine; Thiele, Holger; Greenhalgh, Lynn; Chessa, Luciana; Maghnie, Mohamad; Salim, Mahmoud; Bober, Michael B.; Nürnberg, Peter; Jackson, Stephen P.; Hurles, Matthew E.; Wollnik, Bernd; Stewart, Grant S.; Jackson, Andrew P.

2015-01-01

DNA lesions encountered by replicative polymerases threaten genome stability and cell cycle progression. Here we report the identification of mutations in TRAIP, encoding an E3 RING ubiquitin ligase, in patients with microcephalic primordial dwarfism/Seckel syndrome. We establish that TRAIP relocalizes to sites of DNA damage where it is required for optimal phosphorylation of H2AX and RPA2 during S-phase in response to UV irradiation, as well as fork progression through UV-induced DNA lesions. TRAIP is necessary for efficient cell cycle progression and mutations in TRAIP therefore limit cellular proliferation, providing a potential mechanism for microcephaly and dwarfism phenotypes. Human genetics thus identifies TRAIP as a novel component of the DNA damage response to replication-blocking DNA lesions. PMID:26595769

The human intra-S checkpoint response to UVC-induced DNA damage.

PubMed

Kaufmann, William K

2010-05-01

The intra-S checkpoint response to 254 nm light (UVC)-induced DNA damage appears to have dual functions to slow the rate of DNA synthesis and stabilize replication forks that become stalled at sites of UVC-induced photoproducts in DNA. These functions should provide more time for repair of damaged DNA before its replication and thereby reduce the frequencies of mutations and chromosomal aberrations in surviving cells. This review tries to summarize the history of discovery of the checkpoint, the current state of understanding of the biological features of intra-S checkpoint signaling and its mechanisms of action with a focus primarily on intra-S checkpoint responses in human cells. The differences in the intra-S checkpoint responses to UVC and ionizing radiation-induced DNA damage are emphasized. Evidence that [6-4]pyrimidine-pyrimidone photoproducts in DNA trigger the response is discussed and the relationships between cellular responses to UVC and the molecular dose of UVC-induced DNA damage are briefly summarized. The role of the intra-S checkpoint response in protecting against solar radiation carcinogenesis remains to be determined.

Protein Arginine Methyltransferase 7 Regulates Cellular Response to DNA Damage by Methylating Promoter Histones H2A and H4 of the Polymerase δ Catalytic Subunit Gene, POLD1*

PubMed Central

Karkhanis, Vrajesh; Wang, Li; Tae, Sookil; Hu, Yu-Jie; Imbalzano, Anthony N.; Sif, Saïd

2012-01-01

Covalent modification of histones by protein arginine methyltransferases (PRMTs) impacts genome organization and gene expression. In this report, we show that PRMT7 interacts with the BRG1-based hSWI/SNF chromatin remodeling complex and specifically methylates histone H2A Arg-3 (H2AR3) and histone H4 Arg-3 (H4R3). To elucidate the biological function of PRMT7, we knocked down its expression in NIH 3T3 cells and analyzed global gene expression. Our findings show that PRMT7 negatively regulates expression of genes involved in DNA repair, including ALKBH5, APEX2, POLD1, and POLD2. Chromatin immunoprecipitation (ChIP) revealed that PRMT7 and dimethylated H2AR3 and H4R3 are enriched at target DNA repair genes in parental cells, whereas PRMT7 knockdown caused a significant decrease in PRMT7 recruitment and H2AR3/H4R3 methylation. Decreased PRMT7 expression also resulted in derepression of target DNA repair genes and enhanced cell resistance to DNA-damaging agents. Furthermore, we show that BRG1 co-localizes with PRMT7 on target promoters and that expression of a catalytically inactive form of BRG1 results in derepression of PRMT7 target DNA repair genes. Remarkably, reducing expression of individual PRMT7 target DNA repair genes showed that only the catalytic subunit of DNA polymerase, POLD1, was able to resensitize PRMT7 knock-down cells to DNA-damaging agents. These results provide evidence for the important role played by PRMT7 in epigenetic regulation of DNA repair genes and cellular response to DNA damage. PMID:22761421

Identification of genotoxic compounds using isogenic DNA repair deficient DT40 cell lines on a quantitative high throughput screening platform

PubMed Central

Nishihara, Kana; Huang, Ruili; Zhao, Jinghua; Shahane, Sampada A.; Witt, Kristine L.; Smith-Roe, Stephanie L.; Tice, Raymond R.; Takeda, Shunichi; Xia, Menghang

2016-01-01

DNA repair pathways play a critical role in maintaining cellular homeostasis by repairing DNA damage induced by endogenous processes and xenobiotics, including environmental chemicals. Induction of DNA damage may lead to genomic instability, disruption of cellular homeostasis and potentially tumours. Isogenic chicken DT40 B-lymphocyte cell lines deficient in DNA repair pathways can be used to identify genotoxic compounds and aid in characterising the nature of the induced DNA damage. As part of the US Tox21 program, we previously optimised several different DT40 isogenic clones on a high-throughput screening platform and confirmed the utility of this approach for detecting genotoxicants by measuring differential cytotoxicity in wild-type and DNA repair-deficient clones following chemical exposure. In the study reported here, we screened the Tox21 10K compound library against two isogenic DNA repair-deficient DT40 cell lines (KU70 −/−/RAD54 −/− and REV3 −/−) and the wild-type cell line using a cell viability assay that measures intracellular adenosine triphosphate levels. KU70 and RAD54 are genes associated with DNA double-strand break repair processes, and REV3 is associated with translesion DNA synthesis pathways. Active compounds identified in the primary screening included many well-known genotoxicants (e.g. adriamycin, melphalan) and several compounds previously untested for genotoxicity. A subset of compounds was further evaluated by assessing their ability to induce micronuclei and phosphorylated H2AX. Using this comprehensive approach, three compounds with previously undefined genotoxicity—2-oxiranemethanamine, AD-67 and tetraphenylolethane glycidyl ether—were identified as genotoxic. These results demonstrate the utility of this approach for identifying and prioritising compounds that may damage DNA. PMID:26243743

Protein arginine methyltransferase 7 regulates cellular response to DNA damage by methylating promoter histones H2A and H4 of the polymerase δ catalytic subunit gene, POLD1.

PubMed

Karkhanis, Vrajesh; Wang, Li; Tae, Sookil; Hu, Yu-Jie; Imbalzano, Anthony N; Sif, Saïd

2012-08-24

Covalent modification of histones by protein arginine methyltransferases (PRMTs) impacts genome organization and gene expression. In this report, we show that PRMT7 interacts with the BRG1-based hSWI/SNF chromatin remodeling complex and specifically methylates histone H2A Arg-3 (H2AR3) and histone H4 Arg-3 (H4R3). To elucidate the biological function of PRMT7, we knocked down its expression in NIH 3T3 cells and analyzed global gene expression. Our findings show that PRMT7 negatively regulates expression of genes involved in DNA repair, including ALKBH5, APEX2, POLD1, and POLD2. Chromatin immunoprecipitation (ChIP) revealed that PRMT7 and dimethylated H2AR3 and H4R3 are enriched at target DNA repair genes in parental cells, whereas PRMT7 knockdown caused a significant decrease in PRMT7 recruitment and H2AR3/H4R3 methylation. Decreased PRMT7 expression also resulted in derepression of target DNA repair genes and enhanced cell resistance to DNA-damaging agents. Furthermore, we show that BRG1 co-localizes with PRMT7 on target promoters and that expression of a catalytically inactive form of BRG1 results in derepression of PRMT7 target DNA repair genes. Remarkably, reducing expression of individual PRMT7 target DNA repair genes showed that only the catalytic subunit of DNA polymerase, POLD1, was able to resensitize PRMT7 knock-down cells to DNA-damaging agents. These results provide evidence for the important role played by PRMT7 in epigenetic regulation of DNA repair genes and cellular response to DNA damage.

Imaging and radiation effects of gold nanoparticles in tumour cells

PubMed Central

McQuaid, Harold N.; Muir, Mark F.; Taggart, Laura E.; McMahon, Stephen J.; Coulter, Jonathan A.; Hyland, Wendy B.; Jain, Suneil; Butterworth, Karl T.; Schettino, Giuseppe; Prise, Kevin M.; Hirst, David G.; Botchway, Stanley W.; Currell, Fred J.

2016-01-01

Gold nanoparticle radiosensitization represents a novel technique in enhancement of ionising radiation dose and its effect on biological systems. Variation between theoretical predictions and experimental measurement is significant enough that the mechanism leading to an increase in cell killing and DNA damage is still not clear. We present the first experimental results that take into account both the measured biodistribution of gold nanoparticles at the cellular level and the range of the product electrons responsible for energy deposition. Combining synchrotron-generated monoenergetic X-rays, intracellular gold particle imaging and DNA damage assays, has enabled a DNA damage model to be generated that includes the production of intermediate electrons. We can therefore show for the first time good agreement between the prediction of biological outcomes from both the Local Effect Model and a DNA damage model with experimentally observed cell killing and DNA damage induction via the combination of X-rays and GNPs. However, the requirement of two distinct models as indicated by this mechanistic study, one for short-term DNA damage and another for cell survival, indicates that, at least for nanoparticle enhancement, it is not safe to equate the lethal lesions invoked in the local effect model with DNA damage events. PMID:26787230

Thyroid hormone-induced oxidative damage on lipids, glutathione and DNA in the mouse heart.

PubMed

Gredilla, R; Barja, G; López-Torres, M

2001-10-01

Oxygen radicals of mitochondrial origin are involved in oxidative damage. In order to analyze the possible relationship between metabolic rate, oxidative stress and oxidative damage, OF1 female mice were rendered hyper- and hypothyroid by chronic administration of 0.0012% L-thyroxine (T4) and 0.05% 6-n-propyl-2-thiouracil (PTU), respectively, in their drinking water for 5 weeks. Hyperthyroidism significantly increased the sensitivity to lipid peroxidation in the heart, although the endogenous levels of lipid peroxidation were not altered. Thyroid hormone-induced oxidative stress also resulted in higher levels of GSSG and GSSG/GSH ratio. Oxidative damage to mitochondrial DNA was greater than that to genomic DNA. Hyperthyroidism decreased oxidative damage to genomic DNA. Hypothyroidism did not modify oxidative damage in the lipid fraction but significantly decreased GSSG and GSSG/GSH ratio and oxidative damage to mitochondrial DNA. These results indicate that thyroid hormones modulate oxidative damage to lipids and DNA, and cellular redox potential in the mouse heart. A higher oxidative stress in the hyperthyroid group is presumably neutralized in the case of nuclear DNA by an increase in repair activity, thus protecting this key molecule. Treatment with PTU, a thyroid hormone inhibitor, reduced oxidative damage in the different cell compartments.

DNA Damage and Genomic Instability Induced by Inappropriate DNA Re-replication

DTIC Science & Technology

2007-04-01

Conway, A., Lockhart, D. J., Davis, R. W., Brewer , B. J., and Fangman, W. L. (2001). Replication dynamics of the yeast genome. Science 294, 115–121... Brewer , B. J. (2001). An origin-deficient yeast artificial chromosome triggers a cell cycle checkpoint. Mol. Cell 7, 705–713. Vas, A., Mok, W., and...replication in yeast cells. We have demonstrated that re-replication induces a rapid and significant decrease in cell viability and a cellular DNA damage

DNA Damage and Repair in Plants under Ultraviolet and Ionizing Radiations

PubMed Central

Gill, Sarvajeet S.; Gill, Ritu; Jha, Manoranjan; Tuteja, Narendra

2015-01-01

Being sessile, plants are continuously exposed to DNA-damaging agents present in the environment such as ultraviolet (UV) and ionizing radiations (IR). Sunlight acts as an energy source for photosynthetic plants; hence, avoidance of UV radiations (namely, UV-A, 315–400 nm; UV-B, 280–315 nm; and UV-C, <280 nm) is unpreventable. DNA in particular strongly absorbs UV-B; therefore, it is the most important target for UV-B induced damage. On the other hand, IR causes water radiolysis, which generates highly reactive hydroxyl radicals (OH•) and causes radiogenic damage to important cellular components. However, to maintain genomic integrity under UV/IR exposure, plants make use of several DNA repair mechanisms. In the light of recent breakthrough, the current minireview (a) introduces UV/IR and overviews UV/IR-mediated DNA damage products and (b) critically discusses the biochemistry and genetics of major pathways responsible for the repair of UV/IR-accrued DNA damage. The outcome of the discussion may be helpful in devising future research in the current context. PMID:25729769

DNA condensation by chiral alpha-methylated polyamine analogues and protection of cellular DNA from oxidative damage.

PubMed

Nayvelt, Irina; Hyvönen, Mervi T; Alhonen, Leena; Pandya, Ipsit; Thomas, Thresia; Khomutov, Alex R; Vepsäläinen, Jouko; Patel, Rajesh; Keinänen, Tuomo A; Thomas, T J

2010-01-11

Polyamines are essential molecules supporting the structure, conformation, and function of nucleic acids and proteins. We studied stereoisomers of alpha,alpha'-dimethylated spermine [(R,R)-Me(2)Spm, (S,S)-Me(2)Spm, (R,S)-Me(2)Spm] for their ability to provoke DNA condensation and protect DNA from damage. (R,R)- and (R,S)-Me(2)Spm displayed more efficient condensing ability than spermine, with significantly lower EC(50) (concentration for 50% compaction) values (p < or = 0.01). However, spermine exerted slightly more duplex stabilization than Me(2)Spm. Condensation resulted in nanoparticles with hydrodynamic radii between 39.6 and 48.4 nm, and electron microscopy showed the presence of toroids and spheroids. Natural polyamines and stereoisomers of Me(2)Spm protected DNA against DNase digestion and oxidative stress in vitro and against etoposide and oxidative stress in DU145 cells but afforded little protection against UV-C irradiation. Our findings indicate that Me(2)Spm stereoisomers are efficient DNA packaging agents with potential applications in gene delivery. Our study also reveals stereospecificity in DNA interaction and protection against cellular stress.

DNA damage induces nuclear actin filament assembly by Formin -2 and Spire-½ that promotes efficient DNA repair. [corrected].

PubMed

Belin, Brittany J; Lee, Terri; Mullins, R Dyche

2015-08-19

Actin filaments assemble inside the nucleus in response to multiple cellular perturbations, including heat shock, protein misfolding, integrin engagement, and serum stimulation. We find that DNA damage also generates nuclear actin filaments-detectable by phalloidin and live-cell actin probes-with three characteristic morphologies: (i) long, nucleoplasmic filaments; (ii) short, nucleolus-associated filaments; and (iii) dense, nucleoplasmic clusters. This DNA damage-induced nuclear actin assembly requires two biologically and physically linked nucleation factors: Formin-2 and Spire-1/Spire-2. Formin-2 accumulates in the nucleus after DNA damage, and depletion of either Formin-2 or actin's nuclear import factor, importin-9, increases the number of DNA double-strand breaks (DSBs), linking nuclear actin filaments to efficient DSB clearance. Nuclear actin filaments are also required for nuclear oxidation induced by acute genotoxic stress. Our results reveal a previously unknown role for nuclear actin filaments in DNA repair and identify the molecular mechanisms creating these nuclear filaments.

ATRX Dysfunction Induces Replication Defects in Primary Mouse Cells

PubMed Central

Clynes, David; Jelinska, Clare; Xella, Barbara; Ayyub, Helena; Taylor, Stephen; Mitson, Matthew; Bachrati, Csanád Z.; Higgs, Douglas R.; Gibbons, Richard J.

2014-01-01

The chromatin remodeling protein ATRX, which targets tandem repetitive DNA, has been shown to be required for expression of the alpha globin genes, for proliferation of a variety of cellular progenitors, for chromosome congression and for the maintenance of telomeres. Mutations in ATRX have recently been identified in tumours which maintain their telomeres by a telomerase independent pathway involving homologous recombination thought to be triggered by DNA damage. It is as yet unknown whether there is a central underlying mechanism associated with ATRX dysfunction which can explain the numerous cellular phenomena observed. There is, however, growing evidence for its role in the replication of various repetitive DNA templates which are thought to have a propensity to form secondary structures. Using a mouse knockout model we demonstrate that ATRX plays a direct role in facilitating DNA replication. Ablation of ATRX alone, although leading to a DNA damage response at telomeres, is not sufficient to trigger the alternative lengthening of telomere pathway in mouse embryonic stem cells. PMID:24651726

Modulation of proteostasis counteracts oxidative stress and affects DNA base excision repair capacity in ATM-deficient cells

PubMed Central

Yang, Di; Fletcher, Sally C.; Vendrell, Iolanda; Fischer, Roman; Legrand, Arnaud J.

2017-01-01

Abstract Ataxia telangiectasia (A-T) is a syndrome associated with loss of ATM protein function. Neurodegeneration and cancer predisposition, both hallmarks of A-T, are likely to emerge as a consequence of the persistent oxidative stress and DNA damage observed in this disease. Surprisingly however, despite these severe features, a lack of functional ATM is still compatible with early life, suggesting that adaptation mechanisms contributing to cell survival must be in place. Here we address this gap in our knowledge by analysing the process of human fibroblast adaptation to the lack of ATM. We identify profound rearrangement in cellular proteostasis occurring very early on after loss of ATM in order to counter protein damage originating from oxidative stress. Change in proteostasis, however, is not without repercussions. Modulating protein turnover in ATM-depleted cells also has an adverse effect on the DNA base excision repair pathway, the major DNA repair system that deals with oxidative DNA damage. As a consequence, the burden of unrepaired endogenous DNA lesions intensifies, progressively leading to genomic instability. Our study provides a glimpse at the cellular consequences of loss of ATM and highlights a previously overlooked role for proteostasis in maintaining cell survival in the absence of ATM function. PMID:28973444

Pulsewidth-dependent nature of laser-induced DNA damage in RPE cells

NASA Astrophysics Data System (ADS)

Hall, Rebecca M.; Glickman, Randolph D.; Rockwell, Benjamin A.; Kumar, Neeru; Noojin, Gary D.

2001-07-01

Ultrashort pulse laser radiation may produce cellular damage through unique mechanisms. Primary cultures of bovine retinal pigment epithelial (RPE) cells were exposed to the out put of a Ti:Sapphire laser producing 30 fs (mode-locked) pulses, 44 amplified fs pulses, or continuous wave exposures at 800 nm. Laser exposures at and below the damage threshold were studied. DNA damage was detected using single cell gel electrophoresis (comet assay). Unexposed (control) cells produced short tails with low tail moments. In contrast, all laser-exposed cells showed some degree of DNA fragmentation, but the size and shape of the resulting comets differed among the various modalities. CW-exposed cells produced generally light and relatively compact tails, suggesting fewer and larger DNA fragments, while mode-locked laser exposures (30 fs pulses) resulted in large and diffuse comets, indicating the DNA was fragmented into many very small pieces. Work is continuing to define the relationship of laser pulsewidth and intensity with the degree of DNA fragmentation. These results suggest that DNA damage may result from multiple mechanisms of laser-cell interaction, including multiphoton absorption.

Complex DNA Damage: A Route to Radiation-Induced Genomic Instability and Carcinogenesis

PubMed Central

Mavragani, Ifigeneia V.; Nikitaki, Zacharenia; Souli, Maria P.; Aziz, Asef; Nowsheen, Somaira; Aziz, Khaled; Rogakou, Emmy

2017-01-01

Cellular effects of ionizing radiation (IR) are of great variety and level, but they are mainly damaging since radiation can perturb all important components of the cell, from the membrane to the nucleus, due to alteration of different biological molecules ranging from lipids to proteins or DNA. Regarding DNA damage, which is the main focus of this review, as well as its repair, all current knowledge indicates that IR-induced DNA damage is always more complex than the corresponding endogenous damage resulting from endogenous oxidative stress. Specifically, it is expected that IR will create clusters of damage comprised of a diversity of DNA lesions like double strand breaks (DSBs), single strand breaks (SSBs) and base lesions within a short DNA region of up to 15–20 bp. Recent data from our groups and others support two main notions, that these damaged clusters are: (1) repair resistant, increasing genomic instability (GI) and malignant transformation and (2) can be considered as persistent “danger” signals promoting chronic inflammation and immune response, causing detrimental effects to the organism (like radiation toxicity). Last but not least, the paradigm shift for the role of radiation-induced systemic effects is also incorporated in this picture of IR-effects and consequences of complex DNA damage induction and its erroneous repair. PMID:28718816

Complex DNA Damage: A Route to Radiation-Induced Genomic Instability and Carcinogenesis.

PubMed

Mavragani, Ifigeneia V; Nikitaki, Zacharenia; Souli, Maria P; Aziz, Asef; Nowsheen, Somaira; Aziz, Khaled; Rogakou, Emmy; Georgakilas, Alexandros G

2017-07-18

Cellular effects of ionizing radiation (IR) are of great variety and level, but they are mainly damaging since radiation can perturb all important components of the cell, from the membrane to the nucleus, due to alteration of different biological molecules ranging from lipids to proteins or DNA. Regarding DNA damage, which is the main focus of this review, as well as its repair, all current knowledge indicates that IR-induced DNA damage is always more complex than the corresponding endogenous damage resulting from endogenous oxidative stress. Specifically, it is expected that IR will create clusters of damage comprised of a diversity of DNA lesions like double strand breaks (DSBs), single strand breaks (SSBs) and base lesions within a short DNA region of up to 15-20 bp. Recent data from our groups and others support two main notions, that these damaged clusters are: (1) repair resistant, increasing genomic instability (GI) and malignant transformation and (2) can be considered as persistent "danger" signals promoting chronic inflammation and immune response, causing detrimental effects to the organism (like radiation toxicity). Last but not least, the paradigm shift for the role of radiation-induced systemic effects is also incorporated in this picture of IR-effects and consequences of complex DNA damage induction and its erroneous repair.

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

PubMed Central

Proctor, Michael; Flaherty, Patrick; Jordan, Michael I; Arkin, Adam P; Davis, Ronald W; Nislow, Corey; Giaever, Guri

2005-01-01

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. PMID:16121259

Application of the CometChip platform to assess DNA damage in field-collected blood samples from turtles.

PubMed

Sykora, Peter; Chiari, Ylenia; Heaton, Andrew; Moreno, Nickolas; Glaberman, Scott; Sobol, Robert W

2018-05-01

DNA damage has been linked to genomic instability and the progressive breakdown of cellular and organismal homeostasis, leading to the onset of disease and reduced longevity. Insults to DNA from endogenous sources include base deamination, base hydrolysis, base alkylation, and metabolism-induced oxidative damage that can lead to single-strand and double-strand DNA breaks. Alternatively, exposure to environmental pollutants, radiation or ultra-violet light, can also contribute to exogenously derived DNA damage. We previously validated a novel, high through-put approach to measure levels of DNA damage in cultured mammalian cells. This new CometChip Platform builds on the classical single cell gel electrophoresis or comet methodology used extensively in environmental toxicology and molecular biology. We asked whether the CometChip Platform could be used to measure DNA damage in samples derived from environmental field studies. To this end, we determined that nucleated erythrocytes from multiple species of turtle could be successfully evaluated in the CometChip Platform to quantify levels of DNA damage. In total, we compared levels of DNA damage in 40 animals from two species: the box turtle (Terrapene carolina) and the red-eared slider (Trachemys scripta elegans). Endogenous levels of DNA damage were identical between the two species, yet we did discover some sex-linked differences and changes in DNA damage accumulation. Based on these results, we confirm that the CometChip Platform allows for the measurement of DNA damage in a large number of samples quickly and accurately, and is particularly adaptable to environmental studies using field-collected samples. Environ. Mol. Mutagen. 59:322-333, 2018. © 2018 Wiley Periodicals, Inc. © 2018 Wiley Periodicals, Inc.

Loss of cellular transformation efficiency induced by DNA irradiation with low-energy (10 eV) electrons.

PubMed

Kouass Sahbani, Saloua; Sanche, Leon; Cloutier, Pierre; Bass, Andrew D; Hunting, Darel J

2014-11-20

Low energy electrons (LEEs) of energies less than 20 eV are generated in large quantities by ionizing radiation in biological matter. While LEEs are known to induce single (SSBs) and double strand breaks (DSBs) in DNA, their ability to inactivate cells by inducing nonreparable lethal damage has not yet been demonstrated. Here we observe the effect of LEEs on the functionality of DNA, by measuring the efficiency of transforming Escherichia coli with a [pGEM-3Zf (-)] plasmid irradiated with 10 eV electrons. Highly ordered DNA films were prepared on pyrolitic graphite by molecular self-assembly using 1,3-diaminopropane ions (Dap(2+)). The uniformity of these films permits the inactivation of approximately 50% of the plasmids compared to <10% using previous methods, which is sufficient for the subsequent determination of their functionality. Upon LEE irradiation, the fraction of functional plasmids decreased exponentially with increasing electron fluence, while LEE-induced isolated base damage, frank DSB, and non DSB-cluster damage increased linearly with fluence. While DSBs can be toxic, their levels were too low to explain the loss of plasmid functionality observed upon LEE irradiation. Similarly, non-DSB cluster damage, revealed by transforming cluster damage into DSBs by digestion with repair enzymes, also occurred relatively infrequently. The exact nature of the lethal damage remains unknown, but it is probably a form of compact cluster damage in which the lesions are too close to be revealed by purified repair enzymes. In addition, this damage is either not repaired or is misrepaired by E. coli, since it results in plasmid inactivation, when they contain an average of three lesions. Comparison with previous results from a similar experiment performed with γ-irradiated plasmids indicates that the type of clustered DNA lesions, created directly on cellular DNA by LEEs, may be more difficult to repair than those produced by other species from radiolysis.

The DNA damage response during mitosis.

PubMed

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.

Current concepts for the combined treatment modality of ionizing radiation with anticancer agents.

PubMed

Oehler, Christoph; Dickinson, Daniel J; Broggini-Tenzer, Angela; Hofstetter, Barbara; Hollenstein, Andreas; Riesterer, Oliver; Vuong, Van; Pruschy, Martin

2007-01-01

In current applied radiobiology, there exists a tremendous effort in basic and translational research to identify novel treatment modalities combining ionizing radiation with anticancer agents. This is mainly due to the highly improved molecular understanding of intrinsic radioresistance and the profiling of cellular stress responses to irradiation during recent years. Ionizing radiation not only damages DNA but also affects multiple cellular components that induce a multi-layered stress response. The treatment responses can be restricted to the individual cell level but might also be part of an intercellular stress communication network. Both DNA damage-induced signaling (which results in cell cycle arrest and induction of the DNA-repair machinery) and also ionizing radiation-induced signal transduction cascades, which are generated at cellular sites distant from and independent of DNA-damage, represent interesting targets for anticancer treatment modalities to sensitize for ionizing radiation. Due to the lack of molecular knowledge classic radiobiology assembled the cellular and tissue responses into four groups (4 R's of radiotherapy) which describe biological factors influencing the treatment response to fractionated radiotherapy. These classic 4 R's are Repair, Reassortment, Repopulation and Reoxygenation. With the tremendous progress in molecular oncology we now begin to understand theses factors on the molecular level. At the same time this classification may guide modern molecular radiobiologists to identify novel pharmaceuticals and antisignaling agents which can modulate the treatment response to irradiation. In this review we describe current approaches to sensitize tumor cells with novel anticancer agents along the lines of these 4 R's.

Parvovirus infection-induced DNA damage response

PubMed Central

Luo, Yong; Qiu, Jianming

2014-01-01

Parvoviruses are a group of small DNA viruses with ssDNA genomes flanked by two inverted terminal structures. Due to a limited genetic resource they require host cellular factors and sometimes a helper virus for efficient viral replication. Recent studies have shown that parvoviruses interact with the DNA damage machinery, which has a significant impact on the life cycle of the virus as well as the fate of infected cells. In addition, due to special DNA structures of the viral genomes, parvoviruses are useful tools for the study of the molecular mechanisms underlying viral infection-induced DNA damage response (DDR). This review aims to summarize recent advances in parvovirus-induced DDR, with a focus on the diverse DDR pathways triggered by different parvoviruses and the consequences of DDR on the viral life cycle as well as the fate of infected cells. PMID:25429305

Response to DNA damage of CHEK2 missense mutations in familial breast cancer

PubMed Central

Roeb, Wendy; Higgins, Jake; King, Mary-Claire

2012-01-01

Comprehensive sequencing of tumor suppressor genes to evaluate inherited predisposition to cancer yields many individually rare missense alleles of unknown functional and clinical consequence. To address this problem for CHEK2 missense alleles, we developed a yeast-based assay to assess in vivo CHEK2-mediated response to DNA damage. Of 25 germline CHEK2 missense alleles detected in familial breast cancer patients, 12 alleles had complete loss of DNA damage response, 8 had partial loss and 5 exhibited a DNA damage response equivalent to that mediated by wild-type CHEK2. Variants exhibiting reduced response to DNA damage were found in all domains of the CHEK2 protein. Assay results were in agreement with epidemiologic assessments of breast cancer risk for those variants sufficiently common for case–control studies to have been undertaken. Assay results were largely concordant with consensus predictions of in silico tools, particularly for damaging alleles in the kinase domain. However, of the 25 variants, 6 were not consistently classifiable by in silico tools. An in vivo assay of cellular response to DNA damage by mutant CHEK2 alleles may complement and extend epidemiologic and genetic assessment of their clinical consequences. PMID:22419737

Response to DNA damage of CHEK2 missense mutations in familial breast cancer.

PubMed

Roeb, Wendy; Higgins, Jake; King, Mary-Claire

2012-06-15

Comprehensive sequencing of tumor suppressor genes to evaluate inherited predisposition to cancer yields many individually rare missense alleles of unknown functional and clinical consequence. To address this problem for CHEK2 missense alleles, we developed a yeast-based assay to assess in vivo CHEK2-mediated response to DNA damage. Of 25 germline CHEK2 missense alleles detected in familial breast cancer patients, 12 alleles had complete loss of DNA damage response, 8 had partial loss and 5 exhibited a DNA damage response equivalent to that mediated by wild-type CHEK2. Variants exhibiting reduced response to DNA damage were found in all domains of the CHEK2 protein. Assay results were in agreement with epidemiologic assessments of breast cancer risk for those variants sufficiently common for case-control studies to have been undertaken. Assay results were largely concordant with consensus predictions of in silico tools, particularly for damaging alleles in the kinase domain. However, of the 25 variants, 6 were not consistently classifiable by in silico tools. An in vivo assay of cellular response to DNA damage by mutant CHEK2 alleles may complement and extend epidemiologic and genetic assessment of their clinical consequences.

Assessment of DNA damage in a group of professional dancers during a 10-month dancing season.

PubMed

Esteves, Filipa; Teixeira, Eduardo; Amorim, Tânia; Costa, Carla; Pereira, Cristiana; Fraga, Sónia; De Andrade, Vanessa Moraes; Teixeira, João Paulo; Costa, Solange

2017-01-01

Despite the numerous health benefits of physical activity, some studies reported that increased intensity and duration may induce oxidative stress in several cellular components including DNA. The aim of this study was to assess the level of basal DNA damage as well as oxidative DNA damage in a group of professional dancers before and after a 10-month dancing season. A group of individuals from general population was also assessed as a control. The alkaline version of the comet assay was the method selected to measure both basal DNA damage and oxidative stress, since this method quantifies both endpoints. In order to measure oxidative stress, the comet assay was coupled with a lesion-specific endonuclease (formamidopyrimidine glycosylase) to detect oxidized purines. The levels of oxidative DNA damage in dancers were significantly increased after the dancing season. Pre-season levels of oxidative DNA damage were lower in dancers than those obtained from the general population, suggesting an adaptation of antioxidant system in dancers. Results of the present biomonitoring study indicate the need for more effective measures to protect ballet dancers from potentially occupational health risks related to regular intensive physical exercise.

DNA Repair and the Accumulation of Oxidatively Damaged DNA Are Affected by Fruit Intake in Mice

PubMed Central

Croteau, Deborah L.; de Souza-Pinto, Nadja C.; Harboe, Charlotte; Keijzers, Guido; Zhang, Yongqing; Becker, Kevin; Sheng, Shan

2010-01-01

AGING is associated with elevated oxidative stress and DNA damage. To achieve healthy aging, we must begin to understand how diet affects cellular processes. We postulated that fruit-enriched diets might initiate a program of enhanced DNA repair and thereby improve genome integrity. C57Bl/6 J mice were fed for 14 weeks a control diet or a diet with 8% peach or nectarine extract. The activities of DNA repair enzymes, the level of DNA damage, and gene expression changes were measured. Our study showed that repair of various oxidative DNA lesions was more efficient in liver extracts derived from mice fed fruit-enriched diets. In support of these findings, gas chromatography–mass spectrometry analysis revealed that there was a decrease in the levels of formamidopyrimidines in peach-fed mice compared with the controls. Additionally, microarray analysis revealed that NTH1 was upregulated in peach-fed mice. Taken together, these results suggest that an increased intake of fruits might modulate the efficiency of DNA repair, resulting in altered levels of DNA damage. PMID:20847039

Fanconi anemia: a disorder defective in the DNA damage response.

PubMed

Kitao, Hiroyuki; Takata, Minoru

2011-04-01

Fanconi anemia (FA) is a cancer predisposition disorder characterized by progressive bone marrow failure, congenital developmental defects, chromosomal abnormalities, and cellular hypersensitivity to DNA interstrand crosslink (ICL) agents. So far mutations in 14 FANC genes were identified in FA or FA-like patients. These gene products constitute a common ubiquitin-phosphorylation network called the "FA pathway" and cooperate with other proteins involved in DNA repair and cell cycle control to repair ICL lesions and to maintain genome stability. In this review, we summarize recent exciting discoveries that have expanded our view of the molecular mechanisms operating in DNA repair and DNA damage signaling.

The Regulatory Interactions of p21 and PCNA in Human Breast Cancer

DTIC Science & Technology

2002-07-01

Proliferating cell nuclear antigen (PCNA) is a multifunctional enzyme involved in multiple cellular processes including DNA replication and repair...During DNA replication , PCNA function as an accessory factor- for the DNA polymerases E arid and are part of a multiprotein DNA replication complex...a cyclin-dependent kinase inhibitor, p21WAF1 ability to inhibit DNA replication in response to DNA damage has been wall characterized. Interestingly

Inhibition of the Nedd8 system sensitizes cells to DNA Inter-strand crosslinking agents

PubMed Central

Kee, Younghoon; Huang, Min; Chang, Sophia; Moreau, Lisa A.; Park, Eunmi; Smith, Peter G.; D’Andrea, Alan D.

2012-01-01

The Fanconi Anemia (FA) pathway is required for repair of DNA interstrand crosslinks (ICLs). FA pathway-deficient cells are hypersensitive to DNA ICL-inducing drugs such as Cisplatin. Conversely, hyperactivation of the FA pathway is a mechanism that may underlie cellular resistance to DNA ICL agents. Modulating FANCD2 monoubiquitination, a key step in the FA pathway, may be an effective therapeutic approach to conferring cellular sensitivity to ICL agents. Here, we show that inhibition of the Nedd8 conjugation system increases cellular sensitivity to DNA ICL-inducing agents. Mechanistically, the Nedd8 inhibition, either by siRNA-mediated knockdown of Nedd8 conjugating enzymes or treatment with a Nedd8 activating enzyme inhibitor MLN4924, suppressed DNA damage-induced FANCD2 monoubiquitination and CHK1 phosphorylation. Our data indicate that inhibition of the FA pathway is largely responsible for the heightened cellular sensitivity to DNA ICLs upon Nedd8 inhibition. These results suggest that a combination of Nedd8 inhibition with ICL-inducing agents may be an effective strategy for sensitizing a subset of drug-resistant cancer cells. PMID:22219386

Important cellular targets for antimicrobial photodynamic therapy.

PubMed

Awad, Mariam M; Tovmasyan, Artak; Craik, James D; Batinic-Haberle, Ines; Benov, Ludmil T

2016-09-01

The persistent problem of antibiotic resistance has created a strong demand for new methods for therapy and disinfection. Photodynamic inactivation (PDI) of microbes has demonstrated promising results for eradication of antibiotic-resistant strains. PDI is based on the use of a photosensitive compound (photosensitizer, PS), which upon illumination with visible light generates reactive species capable of damaging and killing microorganisms. Since photogenerated reactive species are short lived, damage is limited to close proximity of the PS. It is reasonable to expect that the larger the number of damaged targets is and the greater their variety is, the higher the efficiency of PDI is and the lower the chances for development of resistance are. Exact molecular mechanisms and specific targets whose damage is essential for microbial inactivation have not been unequivocally established. Two main cellular components, DNA and plasma membrane, are regarded as the most important PDI targets. Using Zn porphyrin-based PSs and Escherichia coli as a model Gram-negative microorganism, we demonstrate that efficient photoinactivation of bacteria can be achieved without detectable DNA modification. Among the cellular components which are modified early during illumination and constitute key PDI targets are cytosolic enzymes, membrane-bound protein complexes, and the plasma membrane. As a result, membrane barrier function is lost, and energy and reducing equivalent production is disrupted, which in turn compromises cell defense mechanisms, thus augmenting the photoinduced oxidative injury. In conclusion, high PDI antimicrobial effectiveness does not necessarily require impairment of a specific critical cellular component and can be achieved by inducing damage to multiple cellular targets.

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

PubMed

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.

Chk1 Promotes DNA Damage Response Bypass following Oxidative Stress in a Model of Hydrogen Peroxide-Associated Ulcerative Colitis through JNK Inactivation and Chromatin Binding.

PubMed

Reissig, Kathrin; Silver, Andrew; Hartig, Roland; Schinlauer, Antje; Walluscheck, Diana; Guenther, Thomas; Siedentopf, Sandra; Ross, Jochen; Vo, Diep-Khanh; Roessner, Albert; Poehlmann-Nitsche, Angela

2017-01-01

Dysregulation of c-Jun N -terminal kinase (JNK) activation promoted DNA damage response bypass and tumorigenesis in our model of hydrogen peroxide-associated ulcerative colitis (UC) and in patients with quiescent UC (QUC), UC-related dysplasia, and UC-related carcinoma (UC-CRC), thereby adapting to oxidative stress. In the UC model, we have observed features of oncogenic transformation: increased proliferation, undetected DNA damage, and apoptosis resistance. Here, we show that Chk1 was downregulated but activated in the acute and quiescent chronic phases. In both phases, Chk1 was linked to DNA damage response bypass by suppressing JNK activation following oxidative stress, promoting cell cycle progression despite DNA damage. Simultaneously, activated Chk1 was bound to chromatin. This triggered histone acetylation and the binding of histone acetyltransferases and transcription factors to chromatin. Thus, chromatin-immobilized activated Chk1 executed a dual function by suppressing DNA damage response and simultaneously inducing chromatin modulation. This caused undetected DNA damage and increased cellular proliferation through failure to transmit the appropriate DNA damage signal. Findings in vitro were corroborated by chromatin accumulation of activated Chk1, Ac-H3, Ac-H4, and c-Jun in active UC (AUC) in vivo. Targeting chromatin-bound Chk1, GCN5, PCAF, and p300/CBP could be a novel therapeutic strategy to prevent UC-related tumor progression.

Mechanisms of sulfur mustard analog 2-chloroethyl ethyl sulfide-induced DNA damage in skin epidermal cells and fibroblasts.

PubMed

Inturi, Swetha; Tewari-Singh, Neera; Gu, Mallikarjuna; Shrotriya, Sangeeta; Gomez, Joe; Agarwal, Chapla; White, Carl W; Agarwal, Rajesh

2011-12-15

Employing mouse skin epidermal JB6 cells and dermal fibroblasts, here we examined the mechanisms of DNA damage by 2-chloroethyl ethyl sulfide (CEES), a monofunctional analog of sulfur mustard (SM). CEES exposure caused H2A.X and p53 phosphorylation as well as p53 accumulation in both cell types, starting at 1h, that was sustained for 24h, indicating a DNA-damaging effect of CEES, which was also confirmed and quantified by alkaline comet assay. CEES exposure also induced oxidative stress and oxidative DNA damage in both cell types, measured by an increase in mitochondrial and cellular reactive oxygen species and 8-hydroxydeoxyguanosine levels, respectively. In the studies distinguishing between oxidative and direct DNA damage, 1h pretreatment with glutathione (GSH) or the antioxidant Trolox showed a decrease in CEES-induced oxidative stress and oxidative DNA damage. However, only GSH pretreatment decreased CEES-induced total DNA damage measured by comet assay, H2A.X and p53 phosphorylation, and total p53 levels. This was possibly due to the formation of GSH-CEES conjugates detected by LC-MS analysis. Together, our results show that CEES causes both direct and oxidative DNA damage, suggesting that to rescue SM-caused skin injuries, pleiotropic agents (or cocktails) are needed that could target multiple pathways of mustard skin toxicities. Copyright © 2011 Elsevier Inc. All rights reserved.

Cellular defense against singlet oxygen-induced oxidative damage by cytosolic NADP+-dependent isocitrate dehydrogenase.

PubMed

Kim, Sun Yee; Park, Jeen-Woo

2003-03-01

Singlet oxygen (1O2) is a highly reactive form of molecular oxygen that may harm living systems by oxidizing critical cellular macromolecules. Recently, we have shown that NADP+-dependent isocitrate dehydrogenase is involved in the supply of NADPH needed for GSH production against cellular oxidative damage. In this study, we investigated the role of cytosolic form of NADP+-dependent isocitrate dehydrogenase (IDPc) against singlet oxygen-induced cytotoxicity by comparing the relative degree of cellular responses in three different NIH3T3 cells with stable transfection with the cDNA for mouse IDPc in sense and antisense orientations, where IDPc activities were 2.3-fold higher and 39% lower, respectively, than that in the parental cells carrying the vector alone. Upon exposure to singlet oxygen generated from photoactivated dye, the cells with low levels of IDPc became more sensitive to cell killing. Lipid peroxidation, protein oxidation, oxidative DNA damage and intracellular peroxide generation were higher in the cell-line expressing the lower level of IDPc. However, the cells with the highly over-expressed IDPc exhibited enhanced resistance against singlet oxygen, compared to the control cells. The data indicate that IDPc plays an important role in cellular defense against singlet oxygen-induced oxidative injury.

[Stress-induced cellular adaptive mutagenesis].

PubMed

Zhu, Linjiang; Li, Qi

2014-04-01

The adaptive mutations exist widely in the evolution of cells, such as antibiotic resistance mutations of pathogenic bacteria, adaptive evolution of industrial strains, and cancerization of human somatic cells. However, how these adaptive mutations are generated is still controversial. Based on the mutational analysis models under the nonlethal selection conditions, stress-induced cellular adaptive mutagenesis is proposed as a new evolutionary viewpoint. The hypothetic pathway of stress-induced mutagenesis involves several intracellular physiological responses, including DNA damages caused by accumulation of intracellular toxic chemicals, limitation of DNA MMR (mismatch repair) activity, upregulation of general stress response and activation of SOS response. These responses directly affect the accuracy of DNA replication from a high-fidelity manner to an error-prone one. The state changes of cell physiology significantly increase intracellular mutation rate and recombination activity. In addition, gene transcription under stress condition increases the instability of genome in response to DNA damage, resulting in transcription-associated DNA mutagenesis. In this review, we summarize these two molecular mechanisms of stress-induced mutagenesis and transcription-associated DNA mutagenesis to help better understand the mechanisms of adaptive mutagenesis.

Stress-induced DNA Damage biomarkers: Applications and limitations

NASA Astrophysics Data System (ADS)

Nikitaki, Zacharenia; Hellweg, Christine; Georgakilas, Alexandros; Ravanat, Jean-Luc

2015-06-01

A variety of environmental stresses like chemicals, UV and ionizing radiation and organism’s endogenous processes like replication stress and metabolism can lead to the generation of reactive oxygen and nitrogen species (ROS/RNS) that can attack cellular vital components like DNA, proteins and lipid membranes. Among them, much attention has been focused on DNA since DNA damages play a role in several biological disorders and aging processes. Thus, DNA damage can be used as a biomarker in a reliable and accurate way to quantify for example radiation exposure and can indicate its possible long term effects and cancer risk. Based on the type of DNA lesions detected one can hypothesize on the most probable mechanisms involved in the formation of these lesions for example in the case of UV and ionizing radiation (e.g. X- or α-, γ-rays, energetic ions, neutrons). In this review we describe the most accepted chemical pathways for DNA damage induction and the different types of DNA lesions, i.e. single, complex DNA lesions etc. that can be used as biomarkers. We critically compare DNA damage detection methods and their limitations. In addition to such DNA damage products, we suggest possible gene inductions that can be used to characterize responses to different types of stresses i.e. radiation, oxidative and replication stress, based on bioinformatic approaches and stringent meta-analysis of literature data.

Role of isolated and clustered DNA damage and the post-irradiating repair process in the effects of heavy ion beam irradiation.

PubMed

Tokuyama, Yuka; Furusawa, Yoshiya; Ide, Hiroshi; Yasui, Akira; Terato, Hiroaki

2015-05-01

Clustered DNA damage is a specific type of DNA damage induced by ionizing radiation. Any type of ionizing radiation traverses the target DNA molecule as a beam, inducing damage along its track. Our previous study showed that clustered DNA damage yields decreased with increased linear energy transfer (LET), leading us to investigate the importance of clustered DNA damage in the biological effects of heavy ion beam radiation. In this study, we analyzed the yield of clustered base damage (comprising multiple base lesions) in cultured cells irradiated with various heavy ion beams, and investigated isolated base damage and the repair process in post-irradiation cultured cells. Chinese hamster ovary (CHO) cells were irradiated by carbon, silicon, argon and iron ion beams with LETs of 13, 55, 90 and 200 keV µm(-1), respectively. Agarose gel electrophoresis of the cells with enzymatic treatments indicated that clustered base damage yields decreased as the LET increased. The aldehyde reactive probe procedure showed that isolated base damage yields in the irradiated cells followed the same pattern. To analyze the cellular base damage process, clustered DNA damage repair was investigated using DNA repair mutant cells. DNA double-strand breaks accumulated in CHO mutant cells lacking Xrcc1 after irradiation, and the cell viability decreased. On the other hand, mouse embryonic fibroblast (Mef) cells lacking both Nth1 and Ogg1 became more resistant than the wild type Mef. Thus, clustered base damage seems to be involved in the expression of heavy ion beam biological effects via the repair process. © The Author 2015. Published by Oxford University Press on behalf of The Japan Radiation Research Society and Japanese Society for Radiation Oncology.

Calculation on spectrum of direct DNA damage induced by low-energy electrons including dissociative electron attachment.

PubMed

Liu, Wei; Tan, Zhenyu; Zhang, Liming; Champion, Christophe

2017-03-01

In this work, direct DNA damage induced by low-energy electrons (sub-keV) is simulated using a Monte Carlo method. The characteristics of the present simulation are to consider the new mechanism of DNA damage due to dissociative electron attachment (DEA) and to allow determining damage to specific bases (i.e., adenine, thymine, guanine, or cytosine). The electron track structure in liquid water is generated, based on the dielectric response model for describing electron inelastic scattering and on a free-parameter theoretical model and the NIST database for calculating electron elastic scattering. Ionization cross sections of DNA bases are used to generate base radicals, and available DEA cross sections of DNA components are applied for determining DNA-strand breaks and base damage induced by sub-ionization electrons. The electron elastic scattering from DNA components is simulated using cross sections from different theoretical calculations. The resulting yields of various strand breaks and base damage in cellular environment are given. Especially, the contributions of sub-ionization electrons to various strand breaks and base damage are quantitatively presented, and the correlation between complex clustered DNA damage and the corresponding damaged bases is explored. This work shows that the contribution of sub-ionization electrons to strand breaks is substantial, up to about 40-70%, and this contribution is mainly focused on single-strand break. In addition, the base damage induced by sub-ionization electrons contributes to about 20-40% of the total base damage, and there is an evident correlation between single-strand break and damaged base pair A-T.

Cytotoxicity and inhibitory properties against topoisomerase II of doxorubicin and its formamidine derivatives.

PubMed

Kik, Krzysztof; Studzian, Kazimierz; Wasowska-Łukawska, Małgorzata; Oszczapowicz, Irena; Szmigiero, Leszek

2009-01-01

This work was undertaken to compare cytotoxicity, DNA damaging properties and effect on DNA cleavage by topoisomerase II of the anthracycline drug doxorubicin (DOX) and its two derivatives with a formamidino group containing a cyclic amine moiety such as morpholine (DOXM) or hexamethyleneimine (DOXH). The tetrazolium dye colorimetric assay was used to determine the cytotoxic activity of anthracyclines toward L1210 leukemia cells. DNA damage was measured by alkaline elution technique. The effect of anthracyclines on DNA cleavage was studied in a cell-free system containing supercoiled pBR322 DNA and purified human topoisomerase II. The cytotoxicity data and the results of studies on the mechanism of DNA break formation by anthracyclines at the cellular level and in the cell-free system showed that the presence of the formamidino group in the doxorubicin molecule reduced its ability to stimulate DNA cleavage by DNA topoisomerase II. DNA topoisomerase II is not a primary cellular target for DOXM or DOXH. An advantageous feature of formamidinoanthracyclines is their mechanism of cytotoxic action which is not related to the inhibition of DNA topoisomerase II. Therefore this class of anthracyclines seems to be a good source for selection of an anticancer drug directed toward cancer cells with the developed multidrug resistance attributed to the presence of altered DNA topoisomerase II.

The ATR Signaling Pathway Is Disabled during Infection with the Parvovirus Minute Virus of Mice

PubMed Central

Adeyemi, Richard O.

2014-01-01

ABSTRACT The ATR kinase has essential functions in maintenance of genome integrity in response to replication stress. ATR is recruited to RPA-coated single-stranded DNA at DNA damage sites via its interacting partner, ATRIP, which binds to the large subunit of RPA. ATR activation typically leads to activation of the Chk1 kinase among other substrates. We show here that, together with a number of other DNA repair proteins, both ATR and its associated protein, ATRIP, were recruited to viral nuclear replication compartments (autonomous parvovirus-associated replication [APAR] bodies) during replication of the single-stranded parvovirus minute virus of mice (MVM). Chk1, however, was not activated during MVM infection even though viral genomes bearing bound RPA, normally a potent trigger of ATR activation, accumulate in APAR bodies. Failure to activate Chk1 in response to MVM infection was likely due to our observation that Rad9 failed to associate with chromatin at MVM APAR bodies. Additionally, early in infection, prior to the onset of the virus-induced DNA damage response (DDR), stalling of the replication of MVM genomes with hydroxyurea (HU) resulted in Chk1 phosphorylation in a virus dose-dependent manner. However, upon establishment of full viral replication, MVM infection prevented activation of Chk1 in response to HU and various other drug treatments. Finally, ATR phosphorylation became undetectable upon MVM infection, and although virus infection induced RPA32 phosphorylation on serine 33, an ATR-associated phosphorylation site, this phosphorylation event could not be prevented by ATR depletion or inhibition. Together our results suggest that MVM infection disables the ATR signaling pathway. IMPORTANCE Upon infection, the parvovirus MVM activates a cellular DNA damage response that governs virus-induced cell cycle arrest and is required for efficient virus replication. ATM and ATR are major cellular kinases that coordinate the DNA damage response to diverse DNA damage stimuli. Although a significant amount has been discovered about ATM activation during parvovirus infection, involvement of the ATR pathway has been less studied. During MVM infection, Chk1, a major downstream target of ATR, is not detectably phosphorylated even though viral genomes bearing the bound cellular single-strand binding protein RPA, normally a potent trigger of ATR activation, accumulate in viral replication centers. ATR phosphorylation also became undetectable. In addition, upon establishment of full viral replication, MVM infection prevented activation of Chk1 in response to hydroxyurea and various other drug treatments. Our results suggest that MVM infection disables this important cellular signaling pathway. PMID:24965470

The ATR signaling pathway is disabled during infection with the parvovirus minute virus of mice.

PubMed

Adeyemi, Richard O; Pintel, David J

2014-09-01

The ATR kinase has essential functions in maintenance of genome integrity in response to replication stress. ATR is recruited to RPA-coated single-stranded DNA at DNA damage sites via its interacting partner, ATRIP, which binds to the large subunit of RPA. ATR activation typically leads to activation of the Chk1 kinase among other substrates. We show here that, together with a number of other DNA repair proteins, both ATR and its associated protein, ATRIP, were recruited to viral nuclear replication compartments (autonomous parvovirus-associated replication [APAR] bodies) during replication of the single-stranded parvovirus minute virus of mice (MVM). Chk1, however, was not activated during MVM infection even though viral genomes bearing bound RPA, normally a potent trigger of ATR activation, accumulate in APAR bodies. Failure to activate Chk1 in response to MVM infection was likely due to our observation that Rad9 failed to associate with chromatin at MVM APAR bodies. Additionally, early in infection, prior to the onset of the virus-induced DNA damage response (DDR), stalling of the replication of MVM genomes with hydroxyurea (HU) resulted in Chk1 phosphorylation in a virus dose-dependent manner. However, upon establishment of full viral replication, MVM infection prevented activation of Chk1 in response to HU and various other drug treatments. Finally, ATR phosphorylation became undetectable upon MVM infection, and although virus infection induced RPA32 phosphorylation on serine 33, an ATR-associated phosphorylation site, this phosphorylation event could not be prevented by ATR depletion or inhibition. Together our results suggest that MVM infection disables the ATR signaling pathway. Upon infection, the parvovirus MVM activates a cellular DNA damage response that governs virus-induced cell cycle arrest and is required for efficient virus replication. ATM and ATR are major cellular kinases that coordinate the DNA damage response to diverse DNA damage stimuli. Although a significant amount has been discovered about ATM activation during parvovirus infection, involvement of the ATR pathway has been less studied. During MVM infection, Chk1, a major downstream target of ATR, is not detectably phosphorylated even though viral genomes bearing the bound cellular single-strand binding protein RPA, normally a potent trigger of ATR activation, accumulate in viral replication centers. ATR phosphorylation also became undetectable. In addition, upon establishment of full viral replication, MVM infection prevented activation of Chk1 in response to hydroxyurea and various other drug treatments. Our results suggest that MVM infection disables this important cellular signaling pathway. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

ATM directs DNA damage responses and proteostasis via genetically separable pathways.

PubMed

Lee, Ji-Hoon; Mand, Michael R; Kao, Chung-Hsuan; Zhou, Yi; Ryu, Seung W; Richards, Alicia L; Coon, Joshua J; Paull, Tanya T

2018-01-09

The protein kinase ATM is a master regulator of the DNA damage response but also responds directly to oxidative stress. Loss of ATM causes ataxia telangiectasia, a neurodegenerative disorder with pleiotropic symptoms that include cerebellar dysfunction, cancer, diabetes, and premature aging. We genetically separated the activation of ATM by DNA damage from that by oxidative stress using separation-of-function mutations. We found that deficient activation of ATM by the Mre11-Rad50-Nbs1 complex and DNA double-strand breaks resulted in loss of cell viability, checkpoint activation, and DNA end resection in response to DNA damage. In contrast, loss of oxidative activation of ATM had minimal effects on DNA damage-related outcomes but blocked ATM-mediated initiation of checkpoint responses after oxidative stress and resulted in deficiencies in mitochondrial function and autophagy. In addition, expression of a variant ATM incapable of activation by oxidative stress resulted in widespread protein aggregation. These results indicate a direct relationship between the mechanism of ATM activation and its effects on cellular metabolism and DNA damage responses in human cells and implicate ATM in the control of protein homeostasis. Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

Destabilization of the MutSα's protein-protein interface due to binding to the DNA adduct induced by anticancer agent carboplatin via molecular dynamics simulations.

PubMed

Negureanu, Lacramioara; Salsbury, Freddie R

2013-11-01

DNA mismatch repair (MMR) proteins maintain genetic integrity in all organisms by recognizing and repairing DNA errors. Such alteration of hereditary information can lead to various diseases, including cancer. Besides their role in DNA repair, MMR proteins detect and initiate cellular responses to certain type of DNA damage. Its response to the damaged DNA has made the human MMR pathway a useful target for anticancer agents such as carboplatin. This study indicates that strong, specific interactions at the interface of MutSα in response to the mismatched DNA recognition are replaced by weak, non-specific interactions in response to the damaged DNA recognition. Data suggest a severe impairment of the dimerization of MutSα in response to the damaged DNA recognition. While the core of MutSα is preserved in response to the damaged DNA recognition, the loss of contact surface and the rearrangement of contacts at the protein interface suggest a different packing in response to the damaged DNA recognition. Coupled in response to the mismatched DNA recognition, interaction energies, hydrogen bonds, salt bridges, and solvent accessible surface areas at the interface of MutSα and within the subunits are uncoupled or asynchronously coupled in response to the damaged DNA recognition. These pieces of evidence suggest that the loss of a synchronous mode of response in the MutSα's surveillance for DNA errors would possibly be one of the mechanism(s) of signaling the MMR-dependent programed cell death much wanted in anticancer therapies. The analysis was drawn from dynamics simulations.

SV40 Utilizes ATM Kinase Activity to Prevent Non-homologous End Joining of Broken Viral DNA Replication Products

PubMed Central

Sowd, Gregory A.; Mody, Dviti; Eggold, Joshua; Cortez, David; Friedman, Katherine L.; Fanning, Ellen

2014-01-01

Simian virus 40 (SV40) and cellular DNA replication rely on host ATM and ATR DNA damage signaling kinases to facilitate DNA repair and elicit cell cycle arrest following DNA damage. During SV40 DNA replication, ATM kinase activity prevents concatemerization of the viral genome whereas ATR activity prevents accumulation of aberrant genomes resulting from breakage of a moving replication fork as it converges with a stalled fork. However, the repair pathways that ATM and ATR orchestrate to prevent these aberrant SV40 DNA replication products are unclear. Using two-dimensional gel electrophoresis and Southern blotting, we show that ATR kinase activity, but not DNA-PKcs kinase activity, facilitates some aspects of double strand break (DSB) repair when ATM is inhibited during SV40 infection. To clarify which repair factors associate with viral DNA replication centers, we examined the localization of DSB repair proteins in response to SV40 infection. Under normal conditions, viral replication centers exclusively associate with homology-directed repair (HDR) and do not colocalize with non-homologous end joining (NHEJ) factors. Following ATM inhibition, but not ATR inhibition, activated DNA-PKcs and KU70/80 accumulate at the viral replication centers while CtIP and BLM, proteins that initiate 5′ to 3′ end resection during HDR, become undetectable. Similar to what has been observed during cellular DSB repair in S phase, these data suggest that ATM kinase influences DSB repair pathway choice by preventing the recruitment of NHEJ factors to replicating viral DNA. These data may explain how ATM prevents concatemerization of the viral genome and promotes viral propagation. We suggest that inhibitors of DNA damage signaling and DNA repair could be used during infection to disrupt productive viral DNA replication. PMID:25474690

ATM Protein Physically and Functionally Interacts with Proliferating Cell Nuclear Antigen to Regulate DNA Synthesis*

PubMed Central

Gamper, Armin M.; Choi, Serah; Matsumoto, Yoshihiro; Banerjee, Dibyendu; Tomkinson, Alan E.; Bakkenist, Christopher J.

2012-01-01

Ataxia telangiectasia (A-T) is a pleiotropic disease, with a characteristic hypersensitivity to ionizing radiation that is caused by biallelic mutations in A-T mutated (ATM), a gene encoding a protein kinase critical for the induction of cellular responses to DNA damage, particularly to DNA double strand breaks. A long known characteristic of A-T cells is their ability to synthesize DNA even in the presence of ionizing radiation-induced DNA damage, a phenomenon termed radioresistant DNA synthesis. We previously reported that ATM kinase inhibition, but not ATM protein disruption, blocks sister chromatid exchange following DNA damage. We now show that ATM kinase inhibition, but not ATM protein disruption, also inhibits DNA synthesis. Investigating a potential physical interaction of ATM with the DNA replication machinery, we found that ATM co-precipitates with proliferating cell nuclear antigen (PCNA) from cellular extracts. Using bacterially purified ATM truncation mutants and in vitro translated PCNA, we showed that the interaction is direct and mediated by the C terminus of ATM. Indeed, a 20-amino acid region close to the kinase domain is sufficient for strong binding to PCNA. This binding is specific to ATM, because the homologous regions of other PIKK members, including the closely related kinase A-T and Rad3-related (ATR), did not bind PCNA. ATM was found to bind two regions in PCNA. To examine the functional significance of the interaction between ATM and PCNA, we tested the ability of ATM to stimulate DNA synthesis by DNA polymerase δ, which is implicated in both DNA replication and DNA repair processes. ATM was observed to stimulate DNA polymerase activity in a PCNA-dependent manner. PMID:22362778

Non linear processes modulated by low doses of radiation exposure

NASA Astrophysics Data System (ADS)

Mariotti, Luca; Ottolenghi, Andrea; Alloni, Daniele; Babini, Gabriele; Morini, Jacopo; Baiocco, Giorgio

The perturbation induced by radiation impinging on biological targets can stimulate the activation of several different pathways, spanning from the DNA damage processing to intra/extra -cellular signalling. In the mechanistic investigation of radiobiological damage this complex “system” response (e.g. omics, signalling networks, micro-environmental modifications, etc.) has to be taken into account, shifting from a focus on the DNA molecule solely to a systemic/collective view. An additional complication comes from the finding that the individual response of each of the involved processes is often not linear as a function of the dose. In this context, a systems biology approach to investigate the effects of low dose irradiations on intra/extra-cellular signalling will be presented, where low doses of radiation act as a mild perturbation of a robustly interconnected network. Results obtained through a multi-level investigation of both DNA damage repair processes (e.g. gamma-H2AX response) and of the activation kinetics for intra/extra cellular signalling pathways (e.g. NFkB activation) show that the overall cell response is dominated by non-linear processes - such as negative feedbacks - leading to possible non equilibrium steady states and to a poor signal-to-noise ratio. Together with experimental data of radiation perturbed pathways, different modelling approaches will be also discussed.

Human Papillomavirus Types 16 and 18 Early-expressed Proteins Differentially Modulate the Cellular Redox State and DNA Damage

PubMed Central

Cruz-Gregorio, Alfredo; Manzo-Merino, Joaquín; Gonzaléz-García, María Cecilia; Pedraza-Chaverri, José; Medina-Campos, Omar Noel; Valverde, Mahara; Rojas, Emilio; Rodríguez-Sastre, María Alexandra; García-Cuellar, Claudia María; Lizano, Marcela

2018-01-01

Oxidative stress has been proposed as a risk factor for cervical cancer development. However, few studies have evaluated the redox state associated with human papillomavirus (HPV) infection. The aim of this work was to determine the role of the early expressed viral proteins E1, E2, E6 and E7 from HPV types 16 and 18 in the modulation of the redox state in an integral form. Therefore, generation of reactive oxygen species (ROS), concentration of reduced glutathione (GSH), levels and activity of the antioxidant enzymes catalase and superoxide dismutase (SOD) and deoxyribonucleic acid (DNA) damage, were analysed in epithelial cells ectopically expressing the viral proteins. Our research shows that E6 oncoproteins decreased GSH and catalase protein levels, as well as its enzymatic activity, which was associated with an increase in ROS production and DNA damage. In contrast, E7 oncoproteins increased GSH, as well as catalase protein levels and its activity, which correlated with a decrease in ROS without affecting DNA integrity. The co-expression of both E6 and E7 oncoproteins neutralized the effects that were independently observed for each of the viral proteins. Additionally, the combined expression of E1 and E2 proteins increased ROS levels with the subsequent increase in the marker for DNA damage phospho-histone 2AX (γH2AX). A decrease in GSH, as well as SOD2 levels and activity were also detected in the presence of E1 and E2, even though catalase activity increased. This study demonstrates that HPV early expressed proteins differentially modulate cellular redox state and DNA damage. PMID:29483822

Characterization of UVC-induced DNA damage in bloodstains: forensic implications.

PubMed

Hall, Ashley; Ballantyne, Jack

2004-09-01

The ability to detect DNA polymorphisms using molecular genetic techniques has revolutionized the forensic analysis of biological evidence. DNA typing now plays a critical role within the criminal justice system, but one of the limiting factors with the technology is that DNA isolated from biological stains recovered from the crime scene is sometimes so damaged as to be intractable to analysis. Potential remedies for damaged DNA are likely to be dependent upon the precise nature of the DNA damage present in any particular sample but, unfortunately, current knowledge of the biochemical nature, and the extent, of such DNA damage in dried biological stains is rudimentary. As a model for DNA damage assessment in biological stains recovered from crime scenes, we have subjected human bloodstains and naked DNA in the hydrated and dehydrated states to varying doses of UVC radiation. It was possible to damage the DNA sufficiently in a bloodstain to cause a standard autosomal short tandem repeat (STR) profile to be lost. However, a detailed analysis of the process, based upon assays developed to detect bipyrimidine photoproducts (BPPPs), single- and double-strand breaks, and DNA-DNA crosslinks, produced some unexpected findings. Contrary to the situation with living tissues or cells in culture, the predominant UVC-induced damage to DNA in bloodstains appears not to be pyrimidine dimers. Although some evidence for the presence of BPPPs and DNA crosslinks was obtained, the major form of UVC damage causing genetic profile loss appeared to be single-strand breaks. It was not possible, however, to preclude the possibility that a combination of damage types was responsible for the profile loss observed. We demonstrate here that a significant measure of protection against UVC-mediated genetic profile loss in dried biological stain material is afforded by the dehydrated state of the DNA and, to a lesser extent, the DNA cellular milieu.

Formation of Clustered DNA Damage after High-LET Irradiation: A Review

NASA Technical Reports Server (NTRS)

Hada, Megumi; Georgakilas, Alexandros G.

2008-01-01

Radiation can cause as well as cure cancer. The risk of developing radiation-induced cancer has traditionally been estimated from cancer incidence among survivors of the atomic bombs in Hiroshima and Nagasaki. These data provide the best estimate of human cancer risk over the dose range for low linear energy transfer (LET) radiations, such as X- or gamma-rays. The situation of estimating the real biological effects becomes even more difficult in the case of high LET particles encountered in space or as the result of domestic exposure to particles from radon gas emitters or other radioactive emitters like uranium-238. Complex DNA damage, i.e., the signature of high-LET radiations comprises by closely spaced DNA lesions forming a cluster of DNA damage. The two basic groups of complex DNA damage are double strand breaks (DSBs) and non-DSB oxidative clustered DNA lesions (OCDL). Theoretical analysis and experimental evidence suggest there is increased complexity and severity of complex DNA damage with increasing LET (linear energy transfer) and a high mutagenic or carcinogenic potential. Data available on the formation of clustered DNA damage (DSBs and OCDL) by high-LET radiations are often controversial suggesting a variable response to dose and type of radiation. The chemical nature and cellular repair mechanisms of complex DNA damage have been much less characterized than those of isolated DNA lesions like an oxidized base or a single strand break especially in the case of high-LET radiation. This review will focus on the induction of clustered DNA damage by high-LET radiations presenting the earlier and recent relative data.

Molecular Clues to Physiological and Premature Ageing Revealed | Center for Cancer Research

Cancer.gov

There are many theories about the molecular basis of ageing. One of the most popular ones postulates that organisms age by accumulating damage to their tissues, cells, and molecules. On the cellular level, ageing is associated with progressive changes in chromatin (a combination of DNA and proteins that makes up chromosomes). These changes include loss of chromatin structure, loss and/or modification of essential proteins, and accumulation of DNA damage.

DNA Damage and Oxidative Stress in Dyskeratosis Congenita: Analysis of Pathways and Therapeutic Stategies Using CPISPR and iPSC Model Systems

DTIC Science & Technology

2016-06-01

telomeres and characterized by a classical clinical triad of leukoplakia, skin dyspigmentation and nail dystrophy with concomitant marrow failure...DC symptomology, to a degree, corresponds to critically shortened telomeres that limits cellular replicative potential and thus prematurely exhausts...stem cell pools. Our previous findings support a hypothesis whereby shortened telomeres increase DNA damage responses within the cell leading to

Maintenance of cellular ATP level by caloric restriction correlates chronological survival of budding yeast

DOE Office of Scientific and Technical Information (OSTI.GOV)

Choi, Joon-Seok; Lee, Cheol-Koo, E-mail: cklee2005@korea.ac.kr

Highlights: •CR decreases total ROS and mitochondrial superoxide during the chronological aging. •CR does not affect the levels of oxidative damage on protein and DNA. •CR contributes extension of chronological lifespan by maintenance of ATP level -- Abstract: The free radical theory of aging emphasizes cumulative oxidative damage in the genome and intracellular proteins due to reactive oxygen species (ROS), which is a major cause for aging. Caloric restriction (CR) has been known as a representative treatment that prevents aging; however, its mechanism of action remains elusive. Here, we show that CR extends the chronological lifespan (CLS) of budding yeastmore » by maintaining cellular energy levels. CR reduced the generation of total ROS and mitochondrial superoxide; however, CR did not reduce the oxidative damage in proteins and DNA. Subsequently, calorie-restricted yeast had higher mitochondrial membrane potential (MMP), and it sustained consistent ATP levels during the process of chronological aging. Our results suggest that CR extends the survival of the chronologically aged cells by improving the efficiency of energy metabolism for the maintenance of the ATP level rather than reducing the global oxidative damage of proteins and DNA.« less

Proteomic profiling reveals DNA damage, nucleolar and ribosomal stress are the main responses to oxaliplatin treatment in cancer cells.

PubMed

Ozdian, Tomas; Holub, Dusan; Maceckova, Zuzana; Varanasi, Lakshman; Rylova, Gabriela; Rehulka, Jiri; Vaclavkova, Jana; Slavik, Hanus; Moudry, Pavel; Znojek, Pawel; Stankova, Jarmila; de Sanctis, Juan Bautista; Hajduch, Marian; Dzubak, Petr

2017-06-06

Oxaliplatin is widely used to treat colorectal cancer in both palliative and adjuvant settings. It is also being tested for use in treating hematological, esophageal, biliary tract, pancreatic, gastric, and hepatocellular cancers. Despite its routine clinical use, little is known about the responses it induces in cancer cells. Therefore the whole-cell proteomics study was conducted to characterize the cellular response induced by oxaliplatin. Chemosensitive CCRF-CEM cells were treated with oxaliplatin at 29.3μM (5×IC 50 ) for 240min (half-time to caspase activation). The proteomes of un-/treated cells were then compared by high-resolution mass spectrometry, revealing 4049 proteins expressed over 3 biological replicates. Among these proteins, 76 were significantly downregulated and 31 significantly upregulated in at least two replicates. In agreement with the DNA-damaging effects of platinum drugs, proteins involved in DNA damage responses were present in both the upregulated and downregulated groups. The downregulated proteins were divided into three subgroups; i) centrosomal proteins, ii) RNA processing and iii) ribosomal proteins, which indicates nucleolar and ribosomal stress. In conclusion, our data supported by further validation experiments indicate the initial cellular response to oxaliplatin is the activation of DNA damage response, which in turn or in parallel triggers nucleolar and ribosomal stress. We have performed a whole-cell proteomic study of cellular response to oxaliplatin treatment, which is the drug predominantly used in the treatment of colorectal cancer. Compared to its predecessors, cisplatin and carboplatin, there is only a small fraction of studies dedicated to oxaliplatin. From those studies, most of them are focused on modification of treatment regimens or study of oxaliplatin in new cancer diagnoses. Cellular response hasn't been studied deeply and to our best knowledge, this is the first whole-cell proteomics study focused exclusively to this important topic, which can help to understand molecular mechanisms of action. Copyright © 2017 Elsevier B.V. All rights reserved.

The cellular Mre11 protein interferes with adenovirus E4 mutant DNA replication.

PubMed

Mathew, Shomita S; Bridge, Eileen

2007-09-01

Adenovirus type 5 (Ad5) relocalizes and degrades the host DNA repair protein Mre11, and efficiently initiates viral DNA replication. Mre11 associates with Ad E4 mutant DNA replication centers and is important for concatenating viral genomes. We have investigated the role of Mre11 in the E4 mutant DNA replication defect. RNAi-mediated knockdown of Mre11 dramatically rescues E4 mutant DNA replication in cells that do or do not concatenate viral genomes, suggesting that Mre11 inhibits DNA replication independent of genome concatenation. The mediator of DNA damage checkpoint 1 (Mdc1) protein is involved in recruiting and sustaining Mre11 at sites of DNA damage following ionizing radiation. We observe foci formation by Mdc1 in response to viral infection, indicating that this damage response protein is activated. However, knockdown of Mdc1 does not prevent Mre11 from localizing at viral DNA replication foci or rescue E4 mutant DNA replication. Our results are consistent with a model in which Mre11 interferes with DNA replication when it is localized at viral DNA replication foci.

DNA Damage Signaling Is Induced in the Absence of Epstein-Barr Virus (EBV) Lytic DNA Replication and in Response to Expression of ZEBRA.

PubMed

Wang'ondu, Ruth; Teal, Stuart; Park, Richard; Heston, Lee; Delecluse, Henri; Miller, George

2015-01-01

Epstein Barr virus (EBV), like other oncogenic viruses, modulates the activity of cellular DNA damage responses (DDR) during its life cycle. Our aim was to characterize the role of early lytic proteins and viral lytic DNA replication in activation of DNA damage signaling during the EBV lytic cycle. Our data challenge the prevalent hypothesis that activation of DDR pathways during the EBV lytic cycle occurs solely in response to large amounts of exogenous double stranded DNA products generated during lytic viral DNA replication. In immunofluorescence or immunoblot assays, DDR activation markers, specifically phosphorylated ATM (pATM), H2AX (γH2AX), or 53BP1 (p53BP1), were induced in the presence or absence of viral DNA amplification or replication compartments during the EBV lytic cycle. In assays with an ATM inhibitor and DNA damaging reagents in Burkitt lymphoma cell lines, γH2AX induction was necessary for optimal expression of early EBV genes, but not sufficient for lytic reactivation. Studies in lytically reactivated EBV-positive cells in which early EBV proteins, BGLF4, BGLF5, or BALF2, were not expressed showed that these proteins were not necessary for DDR activation during the EBV lytic cycle. Expression of ZEBRA, a viral protein that is necessary for EBV entry into the lytic phase, induced pATM foci and γH2AX independent of other EBV gene products. ZEBRA mutants deficient in DNA binding, Z(R183E) and Z(S186E), did not induce foci of pATM. ZEBRA co-localized with HP1β, a heterochromatin associated protein involved in DNA damage signaling. We propose a model of DDR activation during the EBV lytic cycle in which ZEBRA induces ATM kinase phosphorylation, in a DNA binding dependent manner, to modulate gene expression. ATM and H2AX phosphorylation induced prior to EBV replication may be critical for creating a microenvironment of viral and cellular gene expression that enables lytic cycle progression.

Phytochemical Ginkgolide B Attenuates Amyloid-β1-42 Induced Oxidative Damage and Altered Cellular Responses in Human Neuroblastoma SH-SY5Y Cells.

PubMed

Gill, Iqbal; Kaur, Sukhchain; Kaur, Navrattan; Dhiman, Monisha; Mantha, Anil K

2017-01-01

Oxidative stress is an upsurge in reactive oxygen/nitrogen species (ROS/RNS), which aggravates damage to cellular components viz. lipids, proteins, and nucleic acids resulting in impaired cellular functions and neurological pathologies including Alzheimer's disease (AD). In the present study, we have examined amyloid-β (Aβ)-induced oxidative stress responses, a major cause for AD, in the undifferentiated and differentiated human neuroblastoma SH-SY5Y cells. Aβ1-42-induced oxidative damage was evaluated on lipids by lipid peroxidation; proteins by protein carbonyls; antioxidant status by SOD and GSH enzyme activities; and DNA and RNA damage levels by evaluating the number of AP sites and 8-OHG base damages produced. In addition, the neuro-protective role of the phytochemical ginkgolide B (GB) in countering Aβ1-42-induced oxidative stress was assessed. We report that the differentiated cells are highly vulnerable to Aβ1-42-induced oxidative stress events as exerted by the deposition of Aβ in AD. Results of the current study suggest that the pre-treatment of GB, followed by Aβ1-42 treatment for 24 h, displayed neuro-protective potential, which countered Aβ1-42-induced oxidative stress responses in both undifferentiated and differentiated SH-SY5Y neuronal cells by: 1) hampering production of ROS and RNS; 2) reducing lipid peroxidation; 3) decreasing protein carbonyl content; 4) restoring antioxidant activities of SOD and GSH enzymes; and 5) maintaining genome integrity by reducing the oxidative DNA and RNA base damages. In conclusion, Aβ1-42 induces oxidative damage to the cellular biomolecules, which are associated with AD pathology, and are protected by the pre-treatment of GB against Aβ-toxicity. Taken together, this study advocates for phytochemical-based therapeutic interventions against AD.

PPARGC1A variation associated with DNA damage, diabetes, and cardiovascular diseases: the Boston Puerto Rican Health Study.

PubMed

Lai, Chao-Qiang; Tucker, Katherine L; Parnell, Laurence D; Adiconis, Xian; García-Bailo, Bibiana; Griffith, John; Meydani, Mohsen; Ordovás, José M

2008-04-01

Individuals with type 2 diabetes exhibit higher DNA damage and increased risk of cardiovascular disease (CVD). However, mechanisms underlying the association between DNA damage and development of type 2 diabetes and CVD are not understood. We sought to link peroxisome proliferator-activated receptor-gamma coactivator-1 alpha (PPARGC1A), a master transcriptional regulator of mitochondrial oxidative phosphorylation and cellular energy metabolism, with DNA damage, type 2 diabetes, and CVD. We measured DNA damage as urinary 8-hydroxydeoxyguanosine (8-OHdG) concentration and examined the relationship between nine PPARGC1A genetic variants, DNA damage, type 2 diabetes, and self-reported CVD in 959 participants of the Boston Puerto Rican Health Study. With respect to urinary 8-OHdG, PPARGC1A variants showed significant association, and PPARGC1A haplotypes exhibited significant association after correction for multiple testing. Two independent PPARGC1A variants associated significantly with type 2 diabetes (odds ratios [ORs] 1.35 and 2.46; P = 0.045 and <0.001). Carriers of minor alleles of two other PPARGC1A variants, both in strong linkage disequilibrium and associated with lower DNA damage, showed lower prevalence of CVD (ORs 0.53 and 0.65; P = 0.030 and 0.175). Moreover, we found that physical activity correlated negatively with DNA damage. It is plausible that low physical activity combined with risk haplotyes contribute to the high prevalence of type 2 diabetes in this population. We propose that PPARGC1A influences development of type 2 diabetes and CVD via DNA damage. Increasing physical activity, which induces PPARGC1A expression, is a potential strategy to slow DNA damage, thereby decreasing the risk of CVD for individuals with type 2 diabetes.

Damage pattern as a function of radiation quality and other factors.

PubMed

Burkart, W; Jung, T; Frasch, G

1999-01-01

An understanding of damage pattern in critical cellular structures such as DNA is an important prerequisite for a mechanistic assessment of primary radiation damage, its possible repair, and the propagation of residual changes in somatic and germ cells as potential contributors to disease or ageing. Important quantitative insights have been made recently on the distribution in time and space of critical lesions from direct and indirect action of ionizing radiation on mammalian cells. When compared to damage from chemicals or from spontaneous degradation, e.g. depurination or base deamination in DNA, the potential of even low-LET radiation to create local hot spots of damage from single particle tracks is of utmost importance. This has important repercussions on inferences from critical biological effects at high dose and dose rate exposure situations to health risks at chronic, low-level exposures as experienced in environmental and controlled occupational settings. About 10,000 DNA lesions per human cell nucleus and day from spontaneous degradation and chemical attack cause no apparent effect, but a dose of 4 Gy translating into a similar number of direct and indirect DNA breaks induces acute lethality. Therefore, single lesions cannot explain the high efficiency of ionizing radiation in the induction of mutation, transformation and loss of proliferative capacity. Clustered damage leading to poorly repairable double-strand breaks or even more complex local DNA degradation, correlates better with fixed damage and critical biological endpoints. A comparison with other physical, chemical and biological agents indicates that ionizing radiation is indeed set apart from these by its unique micro- and nano-dosimetric traits. Only a few other agents such as bleomycin have a similar potential to cause complex damage from single events. However, in view of the multi-stage mechanism of carcinogenesis, it is still an open question whether dose-effect linearity for complex primary DNA damage and resulting fixed critical cellular lesions translate into linearity for radiation-induced cancer. To solve this enigma, a quantitative assessment of all genotoxic and harmful non-genotoxic agents affecting the human body would be needed.

Quantitative Profiling of DNA Damage and Apoptotic Pathways in UV Damaged Cells Using PTMScan Direct

PubMed Central

Stokes, Matthew P.; Silva, Jeffrey C.; Jia, Xiaoying; Lee, Kimberly A.; Polakiewicz, Roberto D.; Comb, Michael J.

2013-01-01

Traditional methods for analysis of peptides using liquid chromatography and tandem mass spectrometry (LC-MS/MS) lack the specificity to comprehensively monitor specific biological processes due to the inherent duty cycle limitations of the MS instrument and the stochastic nature of the analytical platform. PTMScan Direct is a novel, antibody-based method that allows quantitative LC-MS/MS profiling of specific peptides from proteins that reside in the same signaling pathway. New PTMScan Direct reagents have been produced that target peptides from proteins involved in DNA Damage/Cell Cycle and Apoptosis/Autophagy pathways. Together, the reagents provide access to 438 sites on 237 proteins in these signaling cascades. These reagents have been used to profile the response to UV damage of DNA in human cell lines. UV damage was shown to activate canonical DNA damage response pathways through ATM/ATR-dependent signaling, stress response pathways and induce the initiation of apoptosis, as assessed by an increase in the abundance of peptides corresponding to cleaved, activated caspases. These data demonstrate the utility of PTMScan Direct as a multiplexed assay for profiling specific cellular responses to various stimuli, such as UV damage of DNA. PMID:23344034

Modulation of proteostasis counteracts oxidative stress and affects DNA base excision repair capacity in ATM-deficient cells.

PubMed

Poletto, Mattia; Yang, Di; Fletcher, Sally C; Vendrell, Iolanda; Fischer, Roman; Legrand, Arnaud J; Dianov, Grigory L

2017-09-29

Ataxia telangiectasia (A-T) is a syndrome associated with loss of ATM protein function. Neurodegeneration and cancer predisposition, both hallmarks of A-T, are likely to emerge as a consequence of the persistent oxidative stress and DNA damage observed in this disease. Surprisingly however, despite these severe features, a lack of functional ATM is still compatible with early life, suggesting that adaptation mechanisms contributing to cell survival must be in place. Here we address this gap in our knowledge by analysing the process of human fibroblast adaptation to the lack of ATM. We identify profound rearrangement in cellular proteostasis occurring very early on after loss of ATM in order to counter protein damage originating from oxidative stress. Change in proteostasis, however, is not without repercussions. Modulating protein turnover in ATM-depleted cells also has an adverse effect on the DNA base excision repair pathway, the major DNA repair system that deals with oxidative DNA damage. As a consequence, the burden of unrepaired endogenous DNA lesions intensifies, progressively leading to genomic instability. Our study provides a glimpse at the cellular consequences of loss of ATM and highlights a previously overlooked role for proteostasis in maintaining cell survival in the absence of ATM function. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Human T-cell leukemia virus-I tax oncoprotein functionally targets a subnuclear complex involved in cellular DNA damage-response.

PubMed

Haoudi, Abdelali; Daniels, Rodney C; Wong, Eric; Kupfer, Gary; Semmes, O John

2003-09-26

The virally encoded oncoprotein Tax has been implicated in HTLV-1-mediated cellular transformation. The exact mechanism by which this protein contributes to the oncogenic process is not known. However, it has been hypothesized that Tax induces genomic instability via repression of cellular DNA repair. We examined the effect of de novo Tax expression upon the cell cycle, because appropriate activation of cell cycle checkpoints is essential to a robust damage-repair response. Upon induction of tax expression, Jurkat T-cells displayed a pronounced accumulation in G2/M that was reversible by caffeine. We examined the G2-specific checkpoint signaling response in these cells and found activation of the ATM/chk2-mediated pathway, whereas the ATR/chk1-mediated response was unaffected. Immunoprecipitation with anti-chk2 antibody results in co-precipitation of Tax demonstrating a direct interaction of Tax with a chk2-containing complex. We also show that Tax targets a discrete nuclear site and co-localizes with chk2 and not chk1. This nuclear site, previously identified as Tax Speckled Structures (TSS), also contains the early damage response factor 53BP1. The recruitment of 53BP1 to TSS is dependent upon ATM signaling and requires expression of Tax. Specifically, Tax expression induces redistribution of diffuse nuclear 53BP1 to the TSS foci. Taken together these data suggest that the TSS describe a unique nuclear site involved in DNA damage recognition, repair response, and cell cycle checkpoint activation. We suggest that association of Tax with this multifunctional subnuclear site results in disruption of a subset of the site-specific activities and contributes to cellular genomic instability.

Genome-Wide Functional and Stress Response Profiling Reveals Toxic Mechanism and Genes Required for Tolerance to Benzo[a]pyrene in S. cerevisiae

PubMed Central

O’Connor, Sean Timothy Francis; Lan, Jiaqi; North, Matthew; Loguinov, Alexandre; Zhang, Luoping; Smith, Martyn T.; Gu, April Z.; Vulpe, Chris

2012-01-01

Benzo[a]pyrene (BaP) is a ubiquitous, potent, and complete carcinogen resulting from incomplete organic combustion. BaP can form DNA adducts but other mechanisms may play a role in toxicity. We used a functional toxicology approach in S. cerevisiae to assess the genetic requirements for cellular resistance to BaP. In addition, we examined translational activities of key genes involved in various stress response pathways. We identified multiple genes and processes involved in modulating BaP toxicity in yeast which support DNA damage as a primary mechanism of toxicity, but also identify other potential toxicity pathways. Gene ontology enrichment analysis indicated that DNA damage and repair as well as redox homeostasis and oxidative stress are key processes in cellular response to BaP suggesting a similar mode of action of BaP in yeast and mammals. Interestingly, toxicant export is also implicated as a potential novel modulator of cellular susceptibility. In particular, we identified several transporters with human orthologs (solute carrier family 22) which may play a role in mammalian systems. PMID:23403841

Eccentric localization of catalase to protect chromosomes from oxidative damages during meiotic maturation in mouse oocytes.

PubMed

Park, Yong Seok; You, Seung Yeop; Cho, Sungrae; Jeon, Hyuk-Joon; Lee, Sukchan; Cho, Dong-Hyung; Kim, Jae-Sung; Oh, Jeong Su

2016-09-01

The maintenance of genomic integrity and stability is essential for the survival of every organism. Unfortunately, DNA is vulnerable to attack by a variety of damaging agents. Oxidative stress is a major cause of DNA damage because reactive oxygen species (ROS) are produced as by-products of normal cellular metabolism. Cells have developed eloquent antioxidant defense systems to protect themselves from oxidative damage along with aerobic metabolism. Here, we show that catalase (CAT) is present in mouse oocytes to protect the genome from oxidative damage during meiotic maturation. CAT was expressed in the nucleus to form unique vesicular structures. However, after nuclear envelope breakdown, CAT was redistributed in the cytoplasm with particular focus at the chromosomes. Inhibition of CAT activity increased endogenous ROS levels, but did not perturb meiotic maturation. In addition, CAT inhibition produced chromosomal defects, including chromosome misalignment and DNA damage. Therefore, our data suggest that CAT is required not only to scavenge ROS, but also to protect DNA from oxidative damage during meiotic maturation in mouse oocytes.

Mitochondria damage checkpoint in apoptosis and genome stability.

PubMed

Singh, Keshav K

2004-11-01

Mitochondria perform multiple cellular functions including energy production, cell proliferation and apoptosis. Studies described in this paper suggest a role for mitochondria in maintaining genomic stability. Genomic stability appears to be dependent on mitochondrial functions involved in maintenance of proper intracellular redox status, ATP-dependent transcription, DNA replication, DNA repair and DNA recombination. To further elucidate the role of mitochondria in genomic stability, I propose a mitochondria damage checkpoint (mitocheckpoint) that monitors and responds to damaged mitochondria. Mitocheckpoint can coordinate and maintain proper balance between apoptotic and anti-apoptotic signals. When mitochondria are damaged, mitocheckpoint can be activated to help cells repair damaged mitochondria, to restore normal mitochondrial function and avoid production of mitochondria-defective cells. If mitochondria are severely damaged, mitocheckpoint may not be able to repair the damage and protect cells. Such an event triggers apoptosis. If damage to mitochondria is continuous or persistent such as damage to mitochondrial DNA resulting in mutations, mitocheckpoint may fail which can lead to genomic instability and increased cell survival in yeast. In human it can cause cancer. In support of this proposal we provide evidence that mitochondrial genetic defects in both yeast and mammalian systems lead to impaired DNA repair, increased genomic instability and increased cell survival. This study reveals molecular genetic mechanisms underlying a role for mitochondria in carcinogenesis in humans.

DNA Excision Repair at Telomeres

PubMed Central

Jia, Pingping; Her, Chengtao; Chai, Weihang

2015-01-01

DNA damage is caused by either endogenous cellular metabolic processes such as hydrolysis, oxidation, alkylation, and DNA base mismatches, or exogenous sources including ultraviolet (UV) light, ionizing radiation, and chemical agents. Damaged DNA that is not properly repaired can lead to genomic instability, driving tumorigenesis. To protect genomic stability, mammalian cells have evolved highly conserved DNA repair mechanisms to remove and repair DNA lesions. Telomeres are composed of long tandem TTAGGG repeats located at the ends of chromosomes. Maintenance of functional telomeres is critical for preventing genome instability. The telomeric sequence possesses unique features that predispose telomeres to a variety of DNA damage induced by environmental genotoxins. This review briefly describes the relevance of excision repair pathways in telomere maintenance, with the focus on base excision repair (BER), nucleotide excision repair (NER), and mismatch repair (MMR). By summarizing current knowledge on excision repair of telomere damage and outlining many unanswered questions, it is our hope to stimulate further interest in a better understanding of excision repair processes at telomeres and in how these processes contribute to telomere maintenance. PMID:26422132

Repair of DNA-polypeptide crosslinks by human excision nuclease

NASA Astrophysics Data System (ADS)

Reardon, Joyce T.; Sancar, Aziz

2006-03-01

DNA-protein crosslinks are relatively common DNA lesions that form during the physiological processing of DNA by replication and recombination proteins, by side reactions of base excision repair enzymes, and by cellular exposure to bifunctional DNA-damaging agents such as platinum compounds. The mechanism by which pathological DNA-protein crosslinks are repaired in humans is not known. In this study, we investigated the mechanism of recognition and repair of protein-DNA and oligopeptide-DNA crosslinks by the human excision nuclease. Under our assay conditions, the human nucleotide excision repair system did not remove a 16-kDa protein crosslinked to DNA at a detectable level. However, 4- and 12-aa-long oligopeptides crosslinked to the DNA backbone were recognized by some of the damage recognition factors of the human excision nuclease with moderate selectivity and were excised from DNA at relatively efficient rates. Our data suggest that, if coupled with proteolytic degradation of the crosslinked protein, the human excision nuclease may be the major enzyme system for eliminating protein-DNA crosslinks from the genome. damage recognition | nucleotide excision repair

Inhibition of Ku70 acetylation by INHAT subunit SET/TAF-Iβ regulates Ku70-mediated DNA damage response.

PubMed

Kim, Kee-Beom; Kim, Dong-Wook; Park, Jin Woo; Jeon, Young-Joo; Kim, Daehwan; Rhee, Sangmyung; Chae, Jung-Il; Seo, Sang-Beom

2014-07-01

DNA double-strand breaks (DSBs) can cause either cell death or genomic instability. The Ku heterodimer Ku70/80 is required for the NHEJ (non-homologous end-joining) DNA DSB repair pathway. The INHAT (inhibitor of histone acetyltransferases) complex subunit, SET/TAF-Iβ, can inhibit p300- and PCAF-mediated acetylation of both histone and p53, thereby repressing general transcription and that of p53 target genes. Here, we show that SET/TAF-Iβ interacts with Ku70/80, and that this interaction inhibits CBP- and PCAF-mediated Ku70 acetylation in an INHAT domain-dependent manner. Notably, DNA damage by UV disrupted the interaction between SET/TAF-Iβ and Ku70. Furthermore, we demonstrate that overexpressed SET/TAF-Iβ inhibits recruitment of Ku70/80 to DNA damage sites. We propose that dysregulation of SET/TAF-Iβ expression prevents repair of damaged DNA and also contributes to cellular proliferation. All together, our findings indicate that SET/TAF-Iβ interacts with Ku70/80 in the nucleus and inhibits Ku70 acetylation. Upon DNA damage, SET/TAF-Iβ dissociates from the Ku complex and releases Ku70/Ku80, which are then recruited to DNA DSB sites via the NHEJ DNA repair pathway.

Edaravone protects human peripheral blood lymphocytes from γ-irradiation-induced apoptosis and DNA damage.

PubMed

Chen, Liming; Liu, Yinghui; Dong, Liangliang; Chu, Xiaoxia

2015-03-01

Radiation-induced cellular injury is attributed primarily to the harmful effects of free radicals, which play a key role in irradiation-induced apoptosis. In this study, we investigated the radioprotective efficacy of edaravone, a licensed clinical drug and a powerful free radical scavenger that has been tested against γ-irradiation-induced cellular damage in cultured human peripheral blood lymphocytes in studies of various diseases. Edaravone was pre-incubated with lymphocytes for 2 h prior to γ-irradiation. It was found that pretreatment with edaravone increased cell viability and inhibited generation of γ-radiation-induced reactive oxygen species (ROS) in lymphocytes exposed to 3 Gy γ-radiation. In addition, γ-radiation decreased antioxidant enzymatic activity, such as superoxide dismutase and glutathione peroxidase, as well as the level of reduced glutathione. Conversely, treatment with 100 μM edaravone prior to irradiation improved antioxidant enzyme activity and increased reduced glutathione levels in irradiated lymphocytes. Importantly, we also report that edaravone reduced γ-irradiation-induced apoptosis through downregulation of Bax, upregulation of Bcl-2, and consequent reduction of the Bax:Bcl-2 ratio. The current study shows edaravone to be an effective radioprotector against γ-irradiation-induced cellular damage in lymphocytes in vitro. Finally, edaravone pretreatment significantly reduced DNA damage in γ-irradiated lymphocytes, as measured by comet assay (% tail DNA, tail length, tail moment, and olive tail moment) (p < 0.05). Thus, the current study indicates that edaravone offers protection from radiation-induced cytogenetic alterations.

Ionizing Radiation: The issue of radiation quality

NASA Astrophysics Data System (ADS)

Prise, Kevin; Schettino, Giuseppe

Types of Ionising radiations are differentiated from each other by fundamental characteristics of their energy deposition patterns when they interact with biological materials. At the level of the DNA these non-random patterns drive differences in the yields and distributions of DNA damage patterns and specifically the production of clustered damage or complex lesions. The complex radiation fields found in space bring significant challenges for developing a mechanistic understanding of radiation effects from the perspective of radiation quality as these consist of a diverse range of particle and energy types unique to the space environment. Linear energy transfer, energy deposited per unit track length in units of keV per micron, has long been used as a comparator for different types of radiation but has limitations in that it is an average value. Difference in primary core ionizations relative to secondary delta ray ranges vary significantly with particle mass and energy leading to complex interrelationships with damage production at the cellular level. At the cellular level a greater mechanistic understanding is necessary, linking energy deposition patterns to DNA damage patterns and cellular response, to build appropriate biophysical models that are predictive for different radiation qualities and mixed field exposures. Defined studies using monoenergetic beams delivered under controlled conditions are building quantitative data sets of both initial and long term changes in cells as a basis for a great mechanistic understanding of radiation quality effects of relevance to not only space exposures but clinical application of ion-beams.

Multifunctional Ebselen drug functions through the activation of DNA damage response and alterations in nuclear proteins.

PubMed

Azad, Gajendra K; Balkrishna, Shah Jaimin; Sathish, Narayanan; Kumar, Sangit; Tomar, Raghuvir S

2012-01-15

Several studies have demonstrated that Ebselen is an anti-inflammatory and anti-oxidative agent. Contrary to this, studies have also shown a high degree of cellular toxicity associated with Ebselen usage, the underlying mechanism of which remains less understood. In this study we have attempted to identify a possible molecular mechanism behind the above by investigating the effects of Ebselen on Saccharomyces cerevisiae. Significant growth arrest was documented in yeast cells exposed to Ebselen similar to that seen in presence of DNA damaging agents (including methyl methane sulfonate [MMS] and hydroxy urea [HU]). Furthermore, mutations in specific lysine residues in the histone H3 tail (H3 K56R) resulted in increased sensitivity of yeast cells to Ebselen presumably due to alterations in post-translational modifications of histone proteins towards regulating replication and DNA damage repair. Our findings suggest that Ebselen functions through activation of DNA damage response, alterations in histone modifications, activation of checkpoint kinase pathway and derepression of ribonucleotide reductases (DNA repair genes) which to the best of our knowledge is being reported for the first time. Interestingly subsequent to Ebselen exposure there were changes in global yeast protein expression and specific histone modifications, identification of which is expected to reveal a fundamental cellular mechanism underlying the action of Ebselen. Taken together these observations will help to redesign Ebselen-based therapy in clinical trials. Copyright © 2011 Elsevier Inc. All rights reserved.

Chk1 Promotes DNA Damage Response Bypass following Oxidative Stress in a Model of Hydrogen Peroxide-Associated Ulcerative Colitis through JNK Inactivation and Chromatin Binding

PubMed Central

Silver, Andrew; Guenther, Thomas; Siedentopf, Sandra; Ross, Jochen; Vo, Diep-Khanh; Roessner, Albert

2017-01-01

Dysregulation of c-Jun N-terminal kinase (JNK) activation promoted DNA damage response bypass and tumorigenesis in our model of hydrogen peroxide-associated ulcerative colitis (UC) and in patients with quiescent UC (QUC), UC-related dysplasia, and UC-related carcinoma (UC-CRC), thereby adapting to oxidative stress. In the UC model, we have observed features of oncogenic transformation: increased proliferation, undetected DNA damage, and apoptosis resistance. Here, we show that Chk1 was downregulated but activated in the acute and quiescent chronic phases. In both phases, Chk1 was linked to DNA damage response bypass by suppressing JNK activation following oxidative stress, promoting cell cycle progression despite DNA damage. Simultaneously, activated Chk1 was bound to chromatin. This triggered histone acetylation and the binding of histone acetyltransferases and transcription factors to chromatin. Thus, chromatin-immobilized activated Chk1 executed a dual function by suppressing DNA damage response and simultaneously inducing chromatin modulation. This caused undetected DNA damage and increased cellular proliferation through failure to transmit the appropriate DNA damage signal. Findings in vitro were corroborated by chromatin accumulation of activated Chk1, Ac-H3, Ac-H4, and c-Jun in active UC (AUC) in vivo. Targeting chromatin-bound Chk1, GCN5, PCAF, and p300/CBP could be a novel therapeutic strategy to prevent UC-related tumor progression. PMID:28751935

Assessment of Multiple Types of DNA Damage in Human Placentas from Smoking and Non-smoking Women in the Czech Republic

PubMed Central

Margaret Pratt, M.; King, Leon C.; Adams, Linda D.; John, Kaarthik; Sirajuddin, Paul; Olivero, Ofelia A.; Manchester, David K.; Sram, Radim J.; DeMarini, David M.; Poirier, Miriam C.

2010-01-01

Three classes of DNA damage were assessed in human placentas collected (in 2000-4) from 51 women living in the Teplice region of the Czech Republic, a mining area considered to have some of the worst environmental pollution in Europe in the 1980s. Polycyclic aromatic hydrocarbon (PAH)-DNA adducts were localized and semiquantified using immunohistochemistry (IHC) and the Automated Cellular Imaging System (ACIS). More generalized DNA damage was measured both by 32P-postlabeling and by abasic (AB) site analysis. Placenta stained with antiserum elicited against DNA modified with r7, t8-dihydroxy-t-9, 10-oxy-7,8,9,10-tetrahydro-benzo[a]pyrene (BPDE) revealed PAH-DNA adduct localization in nuclei of the cytotrophoblast (CT) cells and syncytiotrophoblast (ST) knots lining the chorionic villi. The highest levels of DNA damage, 49–312 PAH-DNA adducts/108 nucleotides, were found by IHC/ACIS in 14 immediately-fixed placenta samples. An additional 37 placenta samples were stored frozen before fixation and embedding, and because PAH-DNA adducts were largely undetectable in these samples, freezing was implicated in the loss of IHC signal. The same placentas (n = 37) contained 1.7 – 8.6 stable/bulky DNA adducts/108 nucleotides and 0.6 – 47.2 AB sites/105 nucleotides. For all methods there was no correlation among types of DNA damage and no difference in extent of DNA damage between smokers and non-smokers. Therefore, the data show that DNA from placentas obtained in Teplice contained multiple types of DNA damage, which likely arose from various environmental exposures. In addition, PAH-DNA adducts were present at high concentrations in the CT cells and ST knots of the chorionic villi. PMID:20839217

Assessment of multiple types of DNA damage in human placentas from smoking and nonsmoking women in the Czech Republic.

PubMed

Pratt, M Margaret; King, Leon C; Adams, Linda D; John, Kaarthik; Sirajuddin, Paul; Olivero, Ofelia A; Manchester, David K; Sram, Radim J; DeMarini, David M; Poirier, Miriam C

2011-01-01

Three classes of DNA damage were assessed in human placentas collected (2000-2004) from 51 women living in the Teplice region of the Czech Republic, a mining area considered to have some of the worst environmental pollution in Europe in the 1980s. Polycyclic aromatic hydrocarbon (PAH)-DNA adducts were localized and semiquantified using immunohistochemistry (IHC) and the Automated Cellular Imaging System (ACIS). More generalized DNA damage was measured both by (32)P-postlabeling and by abasic (AB) site analysis. Placenta stained with antiserum elicited against DNA modified with 7β,8α-dihydroxy-9α,10α-epoxy-7,8,9,10-tetrahydro-benzo[a]pyrene (BPDE) revealed PAH-DNA adduct localization in nuclei of the cytotrophoblast (CT) cells and syncytiotrophoblast (ST) knots lining the chorionic villi. The highest levels of DNA damage, 49-312 PAH-DNA adducts/10(8) nucleotides, were found by IHC/ACIS in 14 immediately fixed placenta samples. An additional 37 placenta samples were stored frozen before fixation and embedding, and because PAH-DNA adducts were largely undetectable in these samples, freezing was implicated in the loss of IHC signal. The same placentas (n = 37) contained 1.7-8.6 stable/bulky DNA adducts/10(8) nucleotides and 0.6-47.2 AB sites/10(5) nucleotides. For all methods, there was no correlation among types of DNA damage and no difference in extent of DNA damage between smokers and nonsmokers. Therefore, the data show that DNA from placentas obtained in Teplice contained multiple types of DNA damage, which likely arose from various environmental exposures. In addition, PAH-DNA adducts were present at high concentrations in the CT cells and ST knots of the chorionic villi. Copyright © 2010 Wiley-Liss, Inc.

Systems Biology Model of Interactions between Tissue Growth Factors and DNA Damage Pathways: Low Dose Response and Cross-Talk in TGFβ and ATM Signaling

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cucinotta, Francis A

The etiology of radiation carcinogenesis has been described in terms of aberrant changes that span several levels of biological organization. Growth factors regulate many important cellular and tissue functions including apoptosis, differentiation and proliferation. A variety of genetic and epigenetic changes of growth factors have been shown to contribute to cancer initiation and progression. It is known that cellular and tissue damage to ionizing radiation is in part initiated by the production of reactive oxygen species, which can activate cytokine signaling, and the DNA damage response pathways, most notably the ATM signaling pathway. Recently, the transforming growth factor β (TGFβ)more » pathway has been shown to regulate or directly interact with the ATM pathway in the response to radiation. The relevance of this interaction with the ATM pathway is not known although p53 becomes phosphorylated and DNA damage responses are involved. However, growth factor interactions with DNA damage responses have not been elucidated particularly at low doses, and further characterization of their relationship to cancer processes is warranted. Our goal will be to use a systems biology approach to mathematically and experimentally describe the low-dose responses and cross-talk between the ATM and TGFβ pathways initiated by low- and high-LET radiation. We will characterize ATM and TGFβ signaling in epithelial and fibroblast cells using 2D models and ultimately extending to 3D organotypic cell culture models to begin to elucidate possible differences that may occur for different cell types and/or inter-cellular communication. We will investigate the roles of the Smad and Activating transcription factor 2 (ATF2) proteins as the potential major contributors to crosstalk between the TGFβ and ATM pathways, and links to cell cycle control and/or the DNA damage response, and potential differences in their responses at low and high doses. We have developed various experimental approaches to apply to these problems using confocal microscopy and flow cytometry to detail changes at low dose/dose-rate in order to understand individual cell responses, and will establish our mathematical models based on the experimental findings resulting from changes in DNA repair, apoptosis and proliferation.« less

Systems Biology Model of Interactions Between Tissue Growth Factors and DNA Damage Pathways: Low Dose Response and Cross-Talk in TGFbeta and ATM Signaling

DOE Office of Scientific and Technical Information (OSTI.GOV)

O'Neill, Peter; Anderson, Jennifer

The etiology of radiation carcinogenesis has been described in terms of aberrant changes that span several levels of biological organization. Growth factors regulate many important cellular and tissue functions including apoptosis, differentiation and proliferation. A variety of genetic and epigenetic changes of growth factors have been shown to contribute to cancer initiation and progression. It is known that cellular and tissue damage to ionizing radiation is in part initiated by the production of reactive oxygen species, which can activate cytokine signaling, and the DNA damage response pathways, most notably the ATM signaling pathway. Recently the transforming growth factor β (TGFβ)more » pathway has been shown to regulate or directly interact with the ATM pathway in the response to radiation. The relevance of this interaction with the ATM pathway is not known although p53 becomes phosphorylated and DNA damage responses are involved. However, growth factor interactions with DNA damage responses have not been elucidated particularly at low doses and further characterization of their relationship to cancer processes is warranted. Our goal will be to use a systems biology approach to mathematically and experimentally describe the low dose responses and cross-talk between the ATM and TGFβ pathways initiated by low and high LET radiation. We will characterize ATM and TGFβ signaling in epithelial and fibroblast cells using 2D models and ultimately extending to 3D organotypic cell culture models to begin to elucidate possible differences that may occur for different cell types and/or inter-cellular communication. We will investigate the roles of the Smad and Activating transcription factor 2 (ATF2) proteins as the potential major contributors to cross- talk between the TGFβ and ATM pathways, and links to cell cycle control and/or the DNA damage response, and potential differences in their responses at low and high doses. We have developed various experimental approaches to apply to these problems using confocal microscopy and flow cytometry to detail changes at low dose/dose-rate in order to understand individual cell responses, and will establish our mathematical models based on the experimental findings resulting from changes in DNA repair, apoptosis and proliferation.« less

Shedding Light on the Role of UV Exposure in Melanoma | Center for Cancer Research

Cancer.gov

When a cell is exposed to UV radiation, the chemical makeup of its DNA is changed in a specific manner, resulting in a recognizable modification that can be measured by scientists. These changes are normally detected and fixed by cellular mechanisms for DNA repair. However, if the damage is extensive or if a cell has defective DNA repair machinery, permanent mutations can be produced with harmful consequences for the cell. If the mutation occurs within the gene for a protein that regulates cellular growth, the development of cancer is possible.

Single-molecule live-cell imaging of bacterial DNA repair and damage tolerance.

PubMed

Ghodke, Harshad; Ho, Han; van Oijen, Antoine M

2018-02-19

Genomic DNA is constantly under threat from intracellular and environmental factors that damage its chemical structure. Uncorrected DNA damage may impede cellular propagation or even result in cell death, making it critical to restore genomic integrity. Decades of research have revealed a wide range of mechanisms through which repair factors recognize damage and co-ordinate repair processes. In recent years, single-molecule live-cell imaging methods have further enriched our understanding of how repair factors operate in the crowded intracellular environment. The ability to follow individual biochemical events, as they occur in live cells, makes single-molecule techniques tremendously powerful to uncover the spatial organization and temporal regulation of repair factors during DNA-repair reactions. In this review, we will cover practical aspects of single-molecule live-cell imaging and highlight recent advances accomplished by the application of these experimental approaches to the study of DNA-repair processes in prokaryotes. © 2018 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.

Toxicoproteomic analysis of human lung epithelial cells exposed to steel industry ambient particulate matter (PM) reveals possible mechanism of PM related carcinogenesis.

PubMed

Senthil Kumar, S; Muthuselvam, P; Pugalenthi, V; Subramanian, N; Ramkumar, K M; Suresh, T; Suzuki, T; Rajaguru, P

2018-08-01

Toxicoproteomic analysis of steel industry ambient particulate matter (PM) that contain high concentrations of PAHs and metals was done by treating human lung cancer cell-line, A549 and the cell lysates were analysed using quantitative label-free nano LC-MS/MS. A total of 18,562 peptides representing 1576 proteins were identified and quantified, with 196 proteins had significantly altered expression in the treated cells. Enrichment analyses revealed that proteins associated to redox homeostsis, metabolism, and cellular energy generation were inhibited while, proteins related to DNA damage and repair and other stresses were over expressed. Altered activities of several tumor associated proteins were observed. Protein-protein interaction network and biological pathway analysis of these differentially expressed proteins were carried out to obtain a systems level view of proteome changes. Together it could be inferred that PM exposure induced oxidative stress which could have lead into DNA damage and tumor related changes. However, lowering of cellular metabolism, and energy production could reduce its ability to overcome these stress. This kind of disequilibrium between the DNA damage and ability of the cells to repair the DNA damage may lead into genomic instability that is capable of acting as the driving force during PM induced carcinogenesis. Copyright © 2018 Elsevier Ltd. All rights reserved.

Destabilization of the MutSα’s protein-protein interface due to binding to the DNA adduct induced by anticancer agent Carboplatin via molecular dynamics simulations

PubMed Central

Negureanu, Lacramioara; Salsbury, Freddie R

2013-01-01

DNA mismatch repair (MMR) proteins maintain genetic integrity in all organisms by recognizing and repairing DNA errors. Such alteration of hereditary information can lead to various diseases, including cancer. Besides their role in DNA repair, MMR proteins detect and initiate cellular responses to certain type of DNA damage. Its response to the damaged DNA has made the human MMR pathway a useful target for anticancer agents such as carboplatin. This study indicates that strong, specific interactions at the interface of MutSα in response to the mismatched DNA recognition are replaced by weak, non-specific interactions in response to the damaged DNA recognition. Data suggest a severe impairment of the dimerization of MutSα in response to the damaged DNA recognition. While the core of MutSα is preserved in response to the damaged DNA recognition, the loss of contact surface and the rearrangement of contacts at the protein interface suggest a different packing in response to the damaged DNA recognition. Coupled in response to the mismatched DNA recognition, interaction energies, hydrogen bonds, salt bridges, and solvent accessible surface areas at the interface of MutSα and within the subunits are uncoupled or asynchronously coupled in response to the damaged DNA recognition. These pieces of evidence suggest that the loss of a synchronous mode of response in the MutSα’s surveillance for DNA errors would possible be one of the mechanism(s) of signaling the MMR-dependent programed cell death much wanted in anticancer therapies. The analysis was drawn from dynamics simulations. PMID:24061854

A novel regulation mechanism of DNA repair by damage-induced and RAD23-dependent stabilization of xeroderma pigmentosum group C protein

PubMed Central

Ng, Jessica M.Y.; Vermeulen, Wim; van der Horst, Gijsbertus T.J.; Bergink, Steven; Sugasawa, Kaoru; Vrieling, Harry; Hoeijmakers, Jan H.J.

2003-01-01

Primary DNA damage sensing in mammalian global genome nucleotide excision repair (GG-NER) is performed by the xeroderma pigmentosum group C (XPC)/HR23B protein complex. HR23B and HR23A are human homologs of the yeast ubiquitin-domain repair factor RAD23, the function of which is unknown. Knockout mice revealed that mHR23A and mHR23B have a fully redundant role in NER, and a partially redundant function in embryonic development. Inactivation of both genes causes embryonic lethality, but appeared still compatible with cellular viability. Analysis of mHR23A/B double-mutant cells showed that HR23 proteins function in NER by governing XPC stability via partial protection against proteasomal degradation. Interestingly, NER-type DNA damage further stabilizes XPC and thereby enhances repair. These findings resolve the primary function of RAD23 in repair and reveal a novel DNA-damage-dependent regulation mechanism of DNA repair in eukaryotes, which may be part of a more global damage-response circuitry. PMID:12815074

Preserved DNA Damage Checkpoint Pathway Protects against Complications in Long-Standing Type 1 Diabetes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bhatt, Shweta; Gupta, Manoj K.; Khamaisi, Mogher

The mechanisms underlying the development of complications in type 1 diabetes (T1D) are poorly understood. Disease modeling of induced pluripotent stem cells (iPSCs) from patients with longstanding T1D (disease duration ≥ 50 years) with severe (Medalist +C) or absent to mild complications (Medalist -C) revealed impaired growth, reprogramming, and differentiation in Medalist +C. Genomics and proteomics analyses suggested differential regulation of DNA damage checkpoint proteins favoring protection from cellular apoptosis in Medalist -C. In silico analyses showed altered expression patterns of DNA damage checkpoint factors among the Medalist groups to be targets of miR200, whose expression was significantly elevated inmore » Medalist +C serum. Notably, neurons differentiated from Medalist +C iPSCs exhibited enhanced susceptibility to genotoxic stress that worsened upon miR200 overexpression. Furthermore, knockdown of miR200 in Medalist +C fibroblasts and iPSCs rescued checkpoint protein expression and reduced DNA damage. Lastly, we propose miR200-regulated DNA damage checkpoint pathway as a potential therapeutic target for treating complications of diabetes.« less

Preserved DNA Damage Checkpoint Pathway Protects against Complications in Long-Standing Type 1 Diabetes

DOE PAGES

Bhatt, Shweta; Gupta, Manoj K.; Khamaisi, Mogher; ...

2015-08-04

The mechanisms underlying the development of complications in type 1 diabetes (T1D) are poorly understood. Disease modeling of induced pluripotent stem cells (iPSCs) from patients with longstanding T1D (disease duration ≥ 50 years) with severe (Medalist +C) or absent to mild complications (Medalist -C) revealed impaired growth, reprogramming, and differentiation in Medalist +C. Genomics and proteomics analyses suggested differential regulation of DNA damage checkpoint proteins favoring protection from cellular apoptosis in Medalist -C. In silico analyses showed altered expression patterns of DNA damage checkpoint factors among the Medalist groups to be targets of miR200, whose expression was significantly elevated inmore » Medalist +C serum. Notably, neurons differentiated from Medalist +C iPSCs exhibited enhanced susceptibility to genotoxic stress that worsened upon miR200 overexpression. Furthermore, knockdown of miR200 in Medalist +C fibroblasts and iPSCs rescued checkpoint protein expression and reduced DNA damage. Lastly, we propose miR200-regulated DNA damage checkpoint pathway as a potential therapeutic target for treating complications of diabetes.« less

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

PubMed

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 to alterations to DNA damage signaling pathways that balance cell survival versus death. Our results have general implications for the design of safer paramyxovirus-based vectors that cannot establish persistent infection, the repurposing of drugs that target cellular IAPs as antivirals, and the combined use of DNA-damaging chemotherapy agents in conjunction with oncolytic RNA virus vectors. Copyright © 2018 American Society for Microbiology.

Regulation of replicative senescence by NADP+ -dependent isocitrate dehydrogenase.

PubMed

Kil, In Sup; Huh, Tae Lin; Lee, Young Sup; Lee, You Mie; Park, Jeen-Woo

2006-01-01

The free radical hypothesis of aging postulates that senescence is due to an accumulation of cellular oxidative damage, caused largely by reactive oxygen species that are produced as by-products of normal metabolic processes. Recently, we demonstrated that the control of cytosolic and mitochondrial redox balance and the cellular defense against oxidative damage is one of the primary functions of cytosolic (IDPc) and mitochondrial NADP+ -dependent isocitrate dehydrogenase (IDPm) by supplying NADPH for antioxidant systems. In this paper, we demonstrate that modulation of IDPc or IDPm activity in IMR-90 cells regulates cellular redox status and replicative senescence. When we examined the regulatory role of IDPc and IDPm against the aging process with IMR-90 cells transfected with cDNA for IDPc or IDPm in sense and antisense orientations, a clear inverse relationship was observed between the amount of IDPc or IDPm expressed in target cells and their susceptibility to senescence, which was reflected by changes in replicative potential, cell cycle, senescence-associated beta-galactosidase activity, expression of p21 and p53, and morphology of cells. Furthermore, lipid peroxidation, oxidative DNA damage, and intracellular peroxide generation were higher and cellular redox status shifted to a prooxidant condition in the cell lines expressing the lower level of IDPc or IDPm. The results suggest that IDPc and IDPm play an important regulatory role in cellular defense against oxidative stress and in the senescence of IMR-90 cells.

miR-300 regulates cellular radiosensitivity through targeting p53 and apaf1 in human lung cancer cells.

PubMed

He, Jinpeng; Feng, Xiu; Hua, Junrui; Wei, Li; Lu, Zhiwei; Wei, Wenjun; Cai, Hui; Wang, Bing; Shi, Wengui; Ding, Nan; Li, He; Zhang, Yanan; Wang, Jufang

2017-10-18

microRNAs (miRNAs) play a crucial role in mediation of the cellular sensitivity to ionizing radiation (IR). Previous studies revealed that miR-300 was involved in the cellular response to IR or chemotherapy drug. However, whether miR-300 could regulate the DNA damage responses induced by extrinsic genotoxic stress in human lung cancer and the underlying mechanism remain unknown. In this study, the expression of miR-300 was examined in lung cancer cells treated with IR, and the effects of miR-300 on DNA damage repair, cell cycle arrest, apoptosis and senescence induced by IR were investigated. It was found that IR induced upregulation of endogenous miR-300, and ectopic expression of miR-300 by transfected with miR-300 mimics not only greatly enhanced the cellular DNA damage repair ability but also substantially abrogated the G2 cell cycle arrest and apoptosis induced by IR. Bioinformatic analysis predicted that p53 and apaf1 were potential targets of miR-300, and the luciferase reporter assay showed that miR-300 significantly suppressed the luciferase activity through binding to the 3'-UTR of p53 or apaf1 mRNA. In addition, overexpression of miR-300 significantly reduced p53/apaf1 and/or IR-induced p53/apaf1 protein expression levels. Flow cytomertry analysis and colony formation assay showed that miR-300 desensitized lung cancer cells to IR by suppressing p53-dependent G2 cell cycle arrest, apoptosis and senescence. These data demonstrate that miR-300 regulates the cellular sensitivity to IR through targeting p53 and apaf1 in lung cancer cells.

The Molecular Origin of the MMR-dependent Apoptosis Pathway from Dynamics Analysis of MutSα-DNA Complexes

PubMed Central

Negureanu, Lacramioara; Salsbury, Freddie R.

2012-01-01

The cellular response to DNA damage signaling by MMR proteins is incompletely understood. It is generally accepted that MMR-dependent apoptosis pathway in response to DNA damage detection is independent of MMR's DNA repair function. In this study we investigate correlated motions in response to the binding of mismatched and PCL DNA fragments by MutSα, as derived from 50 ns molecular dynamics simulations. The protein dynamics in response to the mismatched and damaged DNA recognition suggests that MutSα signals their recognition through independent pathways providing evidence for the molecular origin of the MMR-dependent apoptosis. MSH2 subunit is indicated to play a key role in signaling both mismatched and damaged DNA recognition; localized and collective motions within the protein allow identifying sites on the MSH2 surface possible involved in recruiting proteins responsible for downstream events. Unlike in the mismatch complex, predicted key communication sites specific for the damage recognition are on the list of known cancer causing mutations or deletions. This confirms MSH2's role in signaling DNA-damage induced apoptosis and suggests that defects in MMR alone is sufficient to trigger tumorigenesis, supporting the experimental evidence that MMR-damage response function could protect from the early occurrence of tumors. Identifying these particular communication sites may have implications for the treatment of cancers that are not defective for MMR, but are unable to function optimally for MMR-dependent responses following DNA damage such as the case of resistance to cisplatin. PMID:22712459

Biomarkers of oxidative stress and DNA damage in agricultural workers: A pilot study

DOE Office of Scientific and Technical Information (OSTI.GOV)

Muniz, Juan F.; McCauley, Linda; Scherer, J.

Oxidative stress and DNA damage have been proposed as mechanisms linking pesticide exposure to health effects such as cancer and neurological diseases. A study of pesticide applicators and farmworkers was conducted to examine the relationship between organophosphate pesticide exposure and biomarkers of oxidative stress and DNA damage. Urine samples were analyzed for OP metabolites and 8-hydroxy-2'-deoxyguanosine (8-OH-dG). Lymphocytes were analyzed for oxidative DNA repair activity and DNA damage (Comet assay), and serum was analyzed for lipid peroxides (i.e., malondialdehyde, MDA). Cellular damage in agricultural workers was validated using lymphocyte cell cultures. Urinary OP metabolites were significantly higher in farmworkers andmore » applicators (p < 0.001) when compared to controls. 8-OH-dG levels were 8.5 times and 2.3 times higher in farmworkers or applicators (respectively) than in controls. Serum MDA levels were 4.9 times and 24 times higher in farmworkers or applicators (respectively) than in controls. DNA damage (Comet assay) and oxidative DNA repair were significantly greater in lymphocytes from applicators and farmworkers when compared with controls. Markers of oxidative stress (i.e., increased reactive oxygen species and reduced glutathione levels) and DNA damage were also observed in lymphocyte cell cultures treated with an OP. The findings from these in vivo and in vitro studies indicate that organophosphate pesticides induce oxidative stress and DNA damage in agricultural workers. These biomarkers may be useful for increasing our understanding of the link between pesticides and a number of health effects.« less

Chronic Oxidative Damage together with Genome Repair Deficiency in the Neurons is a Double Whammy for Neurodegeneration: Is Damage Response Signaling a Potential Therapeutic Target?

PubMed Central

Wang, Haibo; Dharmalingam, Prakash; Vasquez, Velmarini; Mitra, Joy; Boldogh, Istvan; Rao, K. S.; Kent, Thomas A.; Mitra, Sankar; Hegde, Muralidhar L.

2016-01-01

A foremost challenge for the neurons, which are among the most oxygenated cells, is the genome damage caused by chronic exposure to endogenous reactive oxygen species (ROS), formed as cellular respiratory byproducts. Strong metabolic activity associated with high transcriptional levels in these long lived post-mitotic cells render them vulnerable to oxidative genome damage, including DNA strand breaks and mutagenic base lesions. There is growing evidence for the accumulation of unrepaired DNA lesions in the central nervous system (CNS) during accelerated ageing and progressive neurodegeneration. Several germ line mutations in DNA repair or DNA damage response (DDR) signaling genes are uniquely manifested in the phenotype of neuronal dysfunction and are etiologically linked to many neurodegenerative disorders. Studies in our lab and elsewhere revealed that pro-oxidant metals, ROS and misfolded amyloidogenic proteins not only contribute to genome damage in CNS, but also impede their repair/DDR signaling leading to persistent damage accumulation, a common feature in sporadic neurodegeneration. Here, we have reviewed recent advances in our understanding of the etiological implications of DNA damage vs. repair imbalance, abnormal DDR signaling in triggering neurodegeneration and potential of DDR as a target for the amelioration of neurodegenerative diseases. PMID:27663141

Copper-mediated DNA damage by the neurotransmitter dopamine and L-DOPA: A pro-oxidant mechanism.

PubMed

Rehmani, Nida; Zafar, Atif; Arif, Hussain; Hadi, Sheikh Mumtaz; Wani, Altaf A

2017-04-01

Oxidative DNA damage has been implicated in the pathogenesis of neurological disorders, cancer and ageing. Owing to the established link between labile copper concentrations and neurological diseases, it is critical to explore the interactions of neurotransmitters and drug supplements with copper. Herein, we investigate the pro-oxidant DNA damage induced by the interaction of L-DOPA and dopamine (DA) with copper. The DNA binding affinity order of the compounds has been determined by in silico molecular docking. Agarose gel electrophoresis reveals that L-DOPA and DA are able to induce strand scission in plasmid pcDNA3.1 (+/-) in a copper dependent reaction. These metabolites also cause cellular DNA breakage in human lymphocytes by mobilizing endogenous copper, as assessed by comet assay. Further, L-DOPA and DA-mediated DNA breaks were detected by the appearance of post-DNA damage sensitive marker γH2AX in cancer cell lines accumulating high copper. Immunofluorescence demonstrated the co-localization of downstream repair factor 53BP1 at the damaged induced γH2AX foci in cancer cells. The present study corroborates and provides a mechanism to the hypothesis that suggests metal-mediated oxidation of catecholamines contributes to the pathogenesis of neurodegenerative diseases. Copyright © 2017 Elsevier Ltd. All rights reserved.

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

PubMed

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.

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

PubMed Central

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. PMID:25892555

Roles of PCNA ubiquitination and TLS polymerases κ and η in the bypass of methyl methanesulfonate-induced DNA damage

PubMed Central

Wit, Niek; Buoninfante, Olimpia Alessandra; van den Berk, Paul C.M.; Jansen, Jacob G.; Hogenbirk, Marc A.; de Wind, Niels; Jacobs, Heinz

2015-01-01

Translesion synthesis (TLS) provides a highly conserved mechanism that enables DNA synthesis on a damaged template. TLS is performed by specialized DNA polymerases of which polymerase (Pol) κ is important for the cellular response to DNA damage induced by benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE), ultraviolet (UV) light and the alkylating agent methyl methanesulfonate (MMS). As TLS polymerases are intrinsically error-prone, tight regulation of their activity is required. One level of control is provided by ubiquitination of the homotrimeric DNA clamp PCNA at lysine residue 164 (PCNA-Ub). We here show that Polκ can function independently of PCNA modification and that Polη can function as a backup during TLS of MMS-induced lesions. Compared to cell lines deficient for PCNA modification (PcnaK164R) or Polκ, double mutant cell lines display hypersensitivity to MMS but not to BPDE or UV-C. Double mutant cells also displayed delayed post-replicative TLS, accumulate higher levels of replication stress and delayed S-phase progression. Furthermore, we show that Polη and Polκ are redundant in the DNA damage bypass of MMS-induced DNA damage. Taken together, we provide evidence for PCNA-Ub-independent activation of Polκ and establish Polη as an important backup polymerase in the absence of Polκ in response to MMS-induced DNA damage. PMID:25505145

Homeodomain-Interacting Protein Kinase-2: A Critical Regulator of the DNA Damage Response and the Epigenome

PubMed Central

Kuwano, Yuki; Nishida, Kensei; Akaike, Yoko; Kurokawa, Ken; Nishikawa, Tatsuya; Masuda, Kiyoshi; Rokutan, Kazuhito

2016-01-01

Homeodomain-interacting protein kinase 2 (HIPK2) is a serine/threonine kinase that phosphorylates and activates the apoptotic program through interaction with diverse downstream targets including tumor suppressor p53. HIPK2 is activated by genotoxic stimuli and modulates cell fate following DNA damage. The DNA damage response (DDR) is triggered by DNA lesions or chromatin alterations. The DDR regulates DNA repair, cell cycle checkpoint activation, and apoptosis to restore genome integrity and cellular homeostasis. Maintenance of the DDR is essential to prevent development of diseases caused by genomic instability, including cancer, defects of development, and neurodegenerative disorders. Recent studies reveal a novel HIPK2-mediated pathway for DDR through interaction with chromatin remodeling factor homeodomain protein 1γ. In this review, we will highlight the molecular mechanisms of HIPK2 and show its functions as a crucial DDR regulator. PMID:27689990

Helicase-inactivating mutations as a basis for dominant negative phenotypes

PubMed Central

Wu, Yuliang

2010-01-01

There is ample evidence from studies of both unicellular and multicellular organisms that helicase-inactivating mutations lead to cellular dysfunction and disease phenotypes. In this review, we will discuss the mechanisms underlying the basis for abnormal phenotypes linked to mutations in genes encoding DNA helicases. Recent evidence demonstrates that a clinically relevant patient missense mutation in Fanconi Anemia Complementation Group J exerts detrimental effects on the biochemical activities of the FANC J helicase, and these molecular defects are responsible for aberrant genomic stability and a poor DNA damage response. The ability of FANC J to use the energy from AT P hydrolysis to produce the force required to unwind duplex or G-quadruplex DNA structures or destabilize protein bound to DNA is required for its DNA repair functions in vivo. Strikingly, helicase-inactivating mutations can exert a spectrum of dominant negative phenotypes, indicating that expression of the mutant helicase protein potentially interferes with normal DNA metabolism and has an effect on basic cellular processes such as DNA replication, the DNA damage response and protein trafficking. This review emphasizes that future studies of clinically relevant mutations in helicase genes will be important to understand the molecular pathologies of the associated diseases and their impact on heterozygote carriers. PMID:20980836

SIRT3 Links Oxidative Stress with Aging and Cancer | Center for Cancer Research

Cancer.gov

When cells produce energy, they also form reactive oxygen molecules capable of damaging proteins and DNA. Normally, these molecules are neutralized by a protein called superoxide dismutase, or SOD. However, as a cell ages, oxidative damage accumulates. The increase in oxidative cellular damage as people age may provide a mechanistic connection between aging and carcinogenesis.

Assessing the Fidelity of Ancient DNA Sequences Amplified From Nuclear Genes

PubMed Central

Binladen, Jonas; Wiuf, Carsten; Gilbert, M. Thomas P.; Bunce, Michael; Barnett, Ross; Larson, Greger; Greenwood, Alex D.; Haile, James; Ho, Simon Y. W.; Hansen, Anders J.; Willerslev, Eske

2006-01-01

To date, the field of ancient DNA has relied almost exclusively on mitochondrial DNA (mtDNA) sequences. However, a number of recent studies have reported the successful recovery of ancient nuclear DNA (nuDNA) sequences, thereby allowing the characterization of genetic loci directly involved in phenotypic traits of extinct taxa. It is well documented that postmortem damage in ancient mtDNA can lead to the generation of artifactual sequences. However, as yet no one has thoroughly investigated the damage spectrum in ancient nuDNA. By comparing clone sequences from 23 fossil specimens, recovered from environments ranging from permafrost to desert, we demonstrate the presence of miscoding lesion damage in both the mtDNA and nuDNA, resulting in insertion of erroneous bases during amplification. Interestingly, no significant differences in the frequency of miscoding lesion damage are recorded between mtDNA and nuDNA despite great differences in cellular copy numbers. For both mtDNA and nuDNA, we find significant positive correlations between total sequence heterogeneity and the rates of type 1 transitions (adenine → guanine and thymine → cytosine) and type 2 transitions (cytosine → thymine and guanine → adenine), respectively. Type 2 transitions are by far the most dominant and increase relative to those of type 1 with damage load. The results suggest that the deamination of cytosine (and 5-methyl cytosine) to uracil (and thymine) is the main cause of miscoding lesions in both ancient mtDNA and nuDNA sequences. We argue that the problems presented by postmortem damage, as well as problems with contamination from exogenous sources of conserved nuclear genes, allelic variation, and the reliance on single nucleotide polymorphisms, call for great caution in studies relying on ancient nuDNA sequences. PMID:16299392

Oxidation of DNA bases, deoxyribonucleosides and homopolymers by peroxyl radicals.

PubMed Central

Simandan, T; Sun, J; Dix, T A

1998-01-01

DNA base oxidation is considered to be a key event associated with disease initiation and progression in humans. Peroxyl radicals (ROO. ) are important oxidants found in cells whose ability to react with the DNA bases has not been characterized extensively. In this paper, the products resulting from ROO. oxidation of the DNA bases are determined by gas chromatography/MS in comparison with authentic standards. ROO. radicals oxidize adenine and guanine to their 8-hydroxy derivatives, which are considered biomarkers of hydroxyl radical (HO.) oxidations in cells. ROO. radicals also oxidize adenine to its hydroxylamine, a previously unidentified product. ROO. radicals oxidize cytosine and thymine to the monohydroxy and dihydroxy derivatives that are formed by oxidative damage in cells. Identical ROO. oxidation profiles are observed for each base when exposed as deoxyribonucleosides, monohomopolymers and base-paired dihomopolymers. These results have significance for the development, utilization and interpretation of DNA base-derived biomarkers of oxidative damage associated with disease initiation and propagation, and support the idea that the mutagenic potential of N-oxidized bases, when generated in cellular DNA, will require careful evaluation. Adenine hydroxylamine is proposed as a specific molecular probe for the activity of ROO. in cellular systems. PMID:9761719

Deciphering the Acute Cellular Phosphoproteome Response to Irradiation with X-rays, Protons and Carbon Ions*

PubMed Central

Winter, Martin; Dokic, Ivana; Schlegel, Julian; Warnken, Uwe; Debus, Jürgen; Abdollahi, Amir; Schnölzer, Martina

2017-01-01

Radiotherapy is a cornerstone of cancer therapy. The recently established particle therapy with raster-scanning protons and carbon ions landmarks a new era in the field of high-precision cancer medicine. However, molecular mechanisms governing radiation induced intracellular signaling remain elusive. Here, we present the first comprehensive proteomic and phosphoproteomic study applying stable isotope labeling by amino acids in cell culture (SILAC) in combination with high-resolution mass spectrometry to decipher cellular response to irradiation with X-rays, protons and carbon ions. At protein expression level limited alterations were observed 2 h post irradiation of human lung adenocarcinoma cells. In contrast, 181 phosphorylation sites were found to be differentially regulated out of which 151 sites were not hitherto attributed to radiation response as revealed by crosscheck with the PhosphoSitePlus database. Radiation-induced phosphorylation of the p(S/T)Q motif was the prevailing regulation pattern affecting proteins involved in DNA damage response signaling. Because radiation doses were selected to produce same level of cell kill and DNA double-strand breakage for each radiation quality, DNA damage responsive phosphorylation sites were regulated to same extent. However, differential phosphorylation between radiation qualities was observed for 55 phosphorylation sites indicating the existence of distinct signaling circuitries induced by X-ray versus particle (proton/carbon) irradiation beyond the canonical DNA damage response. This unexpected finding was confirmed in targeted spike-in experiments using synthetic isotope labeled phosphopeptides. Herewith, we successfully validated uniform DNA damage response signaling coexisting with altered signaling involved in apoptosis and metabolic processes induced by X-ray and particle based treatments. In summary, the comprehensive insight into the radiation-induced phosphoproteome landscape is instructive for the design of functional studies aiming to decipher cellular signaling processes in response to radiotherapy, space radiation or ionizing radiation per se. Further, our data will have a significant impact on the ongoing debate about patient treatment modalities. PMID:28302921

Deciphering the Acute Cellular Phosphoproteome Response to Irradiation with X-rays, Protons and Carbon Ions.

PubMed

Winter, Martin; Dokic, Ivana; Schlegel, Julian; Warnken, Uwe; Debus, Jürgen; Abdollahi, Amir; Schnölzer, Martina

2017-05-01

Radiotherapy is a cornerstone of cancer therapy. The recently established particle therapy with raster-scanning protons and carbon ions landmarks a new era in the field of high-precision cancer medicine. However, molecular mechanisms governing radiation induced intracellular signaling remain elusive. Here, we present the first comprehensive proteomic and phosphoproteomic study applying stable isotope labeling by amino acids in cell culture (SILAC) in combination with high-resolution mass spectrometry to decipher cellular response to irradiation with X-rays, protons and carbon ions. At protein expression level limited alterations were observed 2 h post irradiation of human lung adenocarcinoma cells. In contrast, 181 phosphorylation sites were found to be differentially regulated out of which 151 sites were not hitherto attributed to radiation response as revealed by crosscheck with the PhosphoSitePlus database.Radiation-induced phosphorylation of the p(S/T)Q motif was the prevailing regulation pattern affecting proteins involved in DNA damage response signaling. Because radiation doses were selected to produce same level of cell kill and DNA double-strand breakage for each radiation quality, DNA damage responsive phosphorylation sites were regulated to same extent. However, differential phosphorylation between radiation qualities was observed for 55 phosphorylation sites indicating the existence of distinct signaling circuitries induced by X-ray versus particle (proton/carbon) irradiation beyond the canonical DNA damage response. This unexpected finding was confirmed in targeted spike-in experiments using synthetic isotope labeled phosphopeptides. Herewith, we successfully validated uniform DNA damage response signaling coexisting with altered signaling involved in apoptosis and metabolic processes induced by X-ray and particle based treatments.In summary, the comprehensive insight into the radiation-induced phosphoproteome landscape is instructive for the design of functional studies aiming to decipher cellular signaling processes in response to radiotherapy, space radiation or ionizing radiation per se Further, our data will have a significant impact on the ongoing debate about patient treatment modalities. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Diphenylpyrazoles as Replication Protein A inhibitors

DOE PAGES

Waterson, Alex G.; Kennedy, J. Phillip; Patrone, James D.; ...

2014-11-11

Replication Protein A is the primary eukaryotic ssDNA binding protein that has a central role in initiating the cellular response to DNA damage. RPA recruits multiple proteins to sites of DNA damage via the N-terminal domain of the 70 kDa subunit (RPA70N). Here we describe the optimization of a diphenylpyrazole carboxylic acid series of inhibitors of these RPA–protein interactions. Lastly, we evaluated substituents on the aromatic rings as well as the type and geometry of the linkers used to combine fragments, ultimately leading to submicromolar inhibitors of RPA70N protein–protein interactions.

Application of an in vitro DNA protection assay to visualize stress mediation properties of the Dps protein.

PubMed

Karas, Vlad O; Westerlaken, Ilja; Meyer, Anne S

2013-05-31

Oxidative stress is an unavoidable byproduct of aerobic life. Molecular oxygen is essential for terrestrial metabolism, but it also takes part in many damaging reactions within living organisms. The combination of aerobic metabolism and iron, which is another vital compound for life, is enough to produce radicals through Fenton chemistry and degrade cellular components. DNA degradation is arguably the most damaging process involving intracellular radicals, as DNA repair is far from trivial. The assay presented in this article offers a quantitative technique to measure and visualize the effect of molecules and enzymes on radical-mediated DNA damage. The DNA protection assay is a simple, quick, and robust tool for the in vitro characterization of the protective properties of proteins or chemicals. It involves exposing DNA to a damaging oxidative reaction and adding varying concentrations of the compound of interest. The reduction or increase of DNA damage as a function of compound concentration is then visualized using gel electrophoresis. In this article we demonstrate the technique of the DNA protection assay by measuring the protective properties of the DNA-binding protein from starved cells (Dps). Dps is a mini-ferritin that is utilized by more than 300 bacterial species to powerfully combat environmental stressors. Here we present the Dps purification protocol and the optimized assay conditions for evaluating DNA protection by Dps.

Protective role of integrin-linked kinase against oxidative stress and in maintenance of genomic integrity

PubMed Central

Im, Michelle; Dagnino, Lina

2018-01-01

The balance between the production of reactive oxygen species and activation of antioxidant pathways is essential to maintain a normal redox state in all tissues. Oxidative stress caused by excessive oxidant species generation can cause damage to DNA and other macromolecules, affecting cell function and viability. Here we show that integrin-linked kinase (ILK) plays a key role in eliciting a protective response to oxidative damage in epidermal cells. Inactivation of the Ilk gene causes elevated levels of intracellular oxidant species (IOS) and DNA damage in the absence of exogenous oxidative insults. In ILK-deficient cells, excessive IOS production can be prevented through inhibition of NADPH oxidase activity, with a concomitant reduction in DNA damage. Additionally, ILK is necessary for DNA repair processes following UVB-induced damage, as ILK-deficient cells show a significantly impaired ability to remove cyclobutane pyrimidine dimers following irradiation. Thus, ILK is essential to maintain cellular redox balance and, in its absence, epidermal cells become more susceptible to oxidative damage through mechanisms that involve IOS production by NADPH oxidase activity. PMID:29568383

Protective role of integrin-linked kinase against oxidative stress and in maintenance of genomic integrity.

PubMed

Im, Michelle; Dagnino, Lina

2018-03-02

The balance between the production of reactive oxygen species and activation of antioxidant pathways is essential to maintain a normal redox state in all tissues. Oxidative stress caused by excessive oxidant species generation can cause damage to DNA and other macromolecules, affecting cell function and viability. Here we show that integrin-linked kinase (ILK) plays a key role in eliciting a protective response to oxidative damage in epidermal cells. Inactivation of the Ilk gene causes elevated levels of intracellular oxidant species (IOS) and DNA damage in the absence of exogenous oxidative insults. In ILK-deficient cells, excessive IOS production can be prevented through inhibition of NADPH oxidase activity, with a concomitant reduction in DNA damage. Additionally, ILK is necessary for DNA repair processes following UVB-induced damage, as ILK-deficient cells show a significantly impaired ability to remove cyclobutane pyrimidine dimers following irradiation. Thus, ILK is essential to maintain cellular redox balance and, in its absence, epidermal cells become more susceptible to oxidative damage through mechanisms that involve IOS production by NADPH oxidase activity.

Real Estate in the DNA Damage Response: Ubiquitin and SUMO Ligases Home in on DNA Double-Strand Breaks.

PubMed

Dantuma, Nico P; Pfeiffer, Annika

2016-01-01

Ubiquitin and the ubiquitin-like modifier SUMO are intimately connected with the cellular response to various types of DNA damage. A striking feature is the local accumulation of these proteinaceous post-translational modifications in the direct vicinity to DNA double-strand breaks, which plays a critical role in the formation of ionizing radiation-induced foci. The functional significance of these modifications is the coordinated recruitment and removal of proteins involved in DNA damage signaling and repair in a timely manner. The central orchestrators of these processes are the ubiquitin and SUMO ligases that are responsible for accurately tagging a broad array of chromatin and chromatin-associated proteins thereby changing their behavior or destination. Despite many differences in the mode of action of these enzymes, they share some striking features that are of direct relevance for their function in the DNA damage response. In this review, we outline the molecular mechanisms that are responsible for the recruitment of ubiquitin and SUMO ligases and discuss the importance of chromatin proximity in this process.

Cellular responses and gene expression profile changes due to bleomycin-induced DNA damage in human fibroblasts in space

PubMed Central

Kidane, Yared; Feiveson, Alan; Stodieck, Louis; Karouia, Fathi; Ramesh, Govindarajan; Rohde, Larry; Wu, Honglu

2017-01-01

Living organisms in space are constantly exposed to radiation, toxic chemicals or reactive oxygen species generated due to increased levels of environmental and psychological stresses. Understanding the impact of spaceflight factors, microgravity in particular, on cellular responses to DNA damage is essential for assessing the radiation risk for astronauts and the mutation rate in microorganisms. In a study conducted on the International Space Station, confluent human fibroblasts in culture were treated with bleomycin for three hours in the true microgravity environment. The degree of DNA damage was quantified by immunofluorescence staining for γ-H2AX, which is manifested in three types of staining patterns. Although similar percentages of these types of patterns were found between flight and ground cells, there was a slight shift in the distribution of foci counts in the flown cells with countable numbers of γ-H2AX foci. Comparison of the cells in confluent and in exponential growth conditions indicated that the proliferation rate between flight and the ground may be responsible for such a shift. We also performed a microarray analysis of gene expressions in response to bleomycin treatment. A qualitative comparison of the responsive pathways between the flown and ground cells showed similar responses with the p53 network being the top upstream regulator. The microarray data was confirmed with a PCR array analysis containing a set of genes involved in DNA damage signaling; with BBC3, CDKN1A, PCNA and PPM1D being significantly upregulated in both flight and ground cells after bleomycin treatment. Our results suggest that whether microgravity affects DNA damage response in space can be dependent on the cell type and cell growth condition. PMID:28248986

A role for nuclear translocation of tripeptidyl-peptidase II in reactive oxygen species-dependent DNA damage responses

DOE Office of Scientific and Technical Information (OSTI.GOV)

Preta, Giulio; Klark, Rainier de; Glas, Rickard, E-mail: rickard.glas@ki.se

2009-11-27

Responses to DNA damage are influenced by cellular metabolism through the continuous production of reactive oxygen species (ROS), of which most are by-products of mitochondrial respiration. ROS have a strong influence on signaling pathways during responses to DNA damage, by relatively unclear mechanisms. Previous reports have shown conflicting data on a possible role for tripeptidyl-peptidase II (TPPII), a large cytosolic peptidase, within the DNA damage response. Here we show that TPPII translocated into the nucleus in a p160-ROCK-dependent fashion in response to {gamma}-irradiation, and that nuclear expression of TPPII was present in most {gamma}-irradiated transformed cell lines. We used amore » panel of nine cell lines of diverse tissue origin, including four lymphoma cell lines (T, B and Hodgkins lymphoma), a melanoma, a sarcoma, a colon and two breast carcinomas, where seven out of nine cell lines showed nuclear TPPII expression after {gamma}-irradiation. Further, this required cellular production of ROS; treatment with either N-acetyl-Cysteine (anti-oxidant) or Rotenone (inhibitor of mitochondrial respiration) inhibited nuclear accumulation of TPPII. The local density of cells was important for nuclear accumulation of TPPII at early time-points following {gamma}-irradiation (at 1-4 h), indicating a bystander effect. Further, we showed that the peptide-based inhibitor Z-Gly-Leu-Ala-OH, but not its analogue Z-Gly-(D)-Leu-Ala-OH, excluded TPPII from the nucleus. This correlated with reduced nuclear expression of p53 as well as caspase-3 and -9 activation in {gamma}-irradiated lymphoma cells. Our data suggest a role for TPPII in ROS-dependent DNA damage responses, through alteration of its localization from the cytosol into the nucleus.« less

A role for nuclear translocation of tripeptidyl-peptidase II in reactive oxygen species-dependent DNA damage responses.

PubMed

Preta, Giulio; de Klark, Rainier; Glas, Rickard

2009-11-27

Responses to DNA damage are influenced by cellular metabolism through the continuous production of reactive oxygen species (ROS), of which most are by-products of mitochondrial respiration. ROS have a strong influence on signaling pathways during responses to DNA damage, by relatively unclear mechanisms. Previous reports have shown conflicting data on a possible role for tripeptidyl-peptidase II (TPPII), a large cytosolic peptidase, within the DNA damage response. Here we show that TPPII translocated into the nucleus in a p160-ROCK-dependent fashion in response to gamma-irradiation, and that nuclear expression of TPPII was present in most gamma-irradiated transformed cell lines. We used a panel of nine cell lines of diverse tissue origin, including four lymphoma cell lines (T, B and Hodgkins lymphoma), a melanoma, a sarcoma, a colon and two breast carcinomas, where seven out of nine cell lines showed nuclear TPPII expression after gamma-irradiation. Further, this required cellular production of ROS; treatment with either N-acetyl-Cysteine (anti-oxidant) or Rotenone (inhibitor of mitochondrial respiration) inhibited nuclear accumulation of TPPII. The local density of cells was important for nuclear accumulation of TPPII at early time-points following gamma-irradiation (at 1-4h), indicating a bystander effect. Further, we showed that the peptide-based inhibitor Z-Gly-Leu-Ala-OH, but not its analogue Z-Gly-(D)-Leu-Ala-OH, excluded TPPII from the nucleus. This correlated with reduced nuclear expression of p53 as well as caspase-3 and -9 activation in gamma-irradiated lymphoma cells. Our data suggest a role for TPPII in ROS-dependent DNA damage responses, through alteration of its localization from the cytosol into the nucleus.

Developing Master Keys to Brain Pathology, Cancer and Aging from the Structural Biology of Proteins Controlling Reactive Oxygen Species and DNA Repair

PubMed Central

Perry, J. Jefferson P.; Fan, Li; Tainer, John A.

2007-01-01

This review is focused on proteins with key roles in pathways controlling either reactive oxygen species or DNA damage responses, both of which are essential for preserving the nervous system. An imbalance of reactive oxygen species or inappropriate DNA damage response likely causes mutational or cytotoxic outcomes, which may lead to cancer and/or aging phenotypes. Moreover, individuals with hereditary disorders in proteins of these cellular pathways have significant neurological abnormalities. Mutations in a superoxide dismutase, which removes oxygen free radicals, may cause the neurodegenerative disease amyotrophic lateral sclerosis. Additionally, DNA repair disorders that affect the brain to varying extents include ataxia-telangiectasia-like disorder, Cockayne syndrome or Werner syndrome. Here, we highlight recent advances gained through structural biochemistry studies on enzymes linked to these disorders and other related enzymes acting within the same cellular pathways. We describe the current understanding of how these vital proteins coordinate chemical steps and integrate cellular signaling and response events. Significantly, these structural studies may provide a set of master keys to developing a unified understanding of the survival mechanisms utilized after insults by reactive oxygen species and genotoxic agents, and also provide a basis for developing an informed intervention in brain tumor and neurodegenerative disease progression. PMID:17174478

Doxorubicin-induced necrosis is mediated by poly-(ADP-ribose) polymerase 1 (PARP1) but is independent of p53.

PubMed

Shin, Hyeon-Jun; Kwon, Hyuk-Kwon; Lee, Jae-Hyeok; Gui, Xiangai; Achek, Asma; Kim, Jae-Ho; Choi, Sangdun

2015-11-02

Necrosis, unregulated cell death, is characterized by plasma membrane rupture as well as nuclear and cellular swelling. However, it has recently been reported that necrosis is a regulated form of cell death mediated by poly-(ADP-ribose) polymerase 1 (PARP1). PARP1 is thought to mediate necrosis by inducing DNA damage, although this remains unconfirmed. In this study, we examined the mechanisms of PARP1-mediated necrosis following doxorubicin (DOX)-induced DNA damage in human kidney proximal tubular (HK-2) cells. DOX initiated DNA damage response (DDR) and upregulated PARP1 and p53 expression, resulting in morphological changes similar to those observed during necrosis. Additionally, DOX induced mitochondrial hyper-activation, as evidenced by increased mitochondrial respiration and cytosolic ATP (cATP) production. However, DOX affected mitochondrial mass. DOX-induced DNA damage, cytosolic reactive oxygen species (cROS) generation, and mitochondrial hyper-activation decreased in cells with inhibited PARP1 expression, while generation of nitric oxide (NO) and mitochondrial ROS (mROS) remained unaffected. Moreover, DOX-induced DNA damage, cell cycle changes, and oxidative stress were not affected by p53 inhibition. These findings suggest that DNA damage induced necrosis through a PARP1-dependent and p53-independent pathway.

Doxorubicin-induced necrosis is mediated by poly-(ADP-ribose) polymerase 1 (PARP1) but is independent of p53

PubMed Central

Shin, Hyeon-Jun; Kwon, Hyuk-Kwon; Lee, Jae-Hyeok; Gui, Xiangai; Achek, Asma; Kim, Jae-Ho; Choi, Sangdun

2015-01-01

Necrosis, unregulated cell death, is characterized by plasma membrane rupture as well as nuclear and cellular swelling. However, it has recently been reported that necrosis is a regulated form of cell death mediated by poly-(ADP-ribose) polymerase 1 (PARP1). PARP1 is thought to mediate necrosis by inducing DNA damage, although this remains unconfirmed. In this study, we examined the mechanisms of PARP1-mediated necrosis following doxorubicin (DOX)-induced DNA damage in human kidney proximal tubular (HK-2) cells. DOX initiated DNA damage response (DDR) and upregulated PARP1 and p53 expression, resulting in morphological changes similar to those observed during necrosis. Additionally, DOX induced mitochondrial hyper-activation, as evidenced by increased mitochondrial respiration and cytosolic ATP (cATP) production. However, DOX affected mitochondrial mass. DOX-induced DNA damage, cytosolic reactive oxygen species (cROS) generation, and mitochondrial hyper-activation decreased in cells with inhibited PARP1 expression, while generation of nitric oxide (NO) and mitochondrial ROS (mROS) remained unaffected. Moreover, DOX-induced DNA damage, cell cycle changes, and oxidative stress were not affected by p53 inhibition. These findings suggest that DNA damage induced necrosis through a PARP1-dependent and p53-independent pathway. PMID:26522181

Poly(GR) in C9ORF72-Related ALS/FTD Compromises Mitochondrial Function and Increases Oxidative Stress and DNA Damage in iPSC-Derived Motor Neurons.

PubMed

Lopez-Gonzalez, Rodrigo; Lu, Yubing; Gendron, Tania F; Karydas, Anna; Tran, Helene; Yang, Dejun; Petrucelli, Leonard; Miller, Bruce L; Almeida, Sandra; Gao, Fen-Biao

2016-10-19

GGGGCC repeat expansions in C9ORF72 are the most common genetic cause of both ALS and FTD. To uncover underlying pathogenic mechanisms, we found that DNA damage was greater, in an age-dependent manner, in motor neurons differentiated from iPSCs of multiple C9ORF72 patients than control neurons. Ectopic expression of the dipeptide repeat (DPR) protein (GR) 80 in iPSC-derived control neurons increased DNA damage, suggesting poly(GR) contributes to DNA damage in aged C9ORF72 neurons. Oxidative stress was also increased in C9ORF72 neurons in an age-dependent manner. Pharmacological or genetic reduction of oxidative stress partially rescued DNA damage in C9ORF72 neurons and control neurons expressing (GR) 80 or (GR) 80 -induced cellular toxicity in flies. Moreover, interactome analysis revealed that (GR) 80 preferentially bound to mitochondrial ribosomal proteins and caused mitochondrial dysfunction. Thus, poly(GR) in C9ORF72 neurons compromises mitochondrial function and causes DNA damage in part by increasing oxidative stress, revealing another pathogenic mechanism in C9ORF72-related ALS and FTD. Copyright © 2016 Elsevier Inc. All rights reserved.

Stem cells: Balancing resistance and sensitivity to DNA damage

PubMed Central

Liu, Julia C.; Lerou, Paul H.; Lahav, Galit

2015-01-01

Embryonic stem cells are known to be very sensitive to DNA damage and undergo rapid apoptosis even after low damage doses. In contrast, adult stem cells show variable sensitivity to damage. Here we describe the multiple pathways that have been proposed to affect the sensitivity of stem cells to damage, including proximity to the apoptotic threshold (mitochondrial priming) and the p53 signaling pathway, through activation of transcription or direct interaction with pro apoptotic proteins in the cytoplasm. We also discuss which cellular factors might connect mitochondrial priming with pluripotency and the potential therapeutic advances that can be achieved by better understanding the molecular mechanisms leading to sensitivity or resistance of embryonic or adult stem cells from different tissues. PMID:24721782

CEP152 is a genome maintenance protein disrupted in Seckel syndrome

PubMed Central

Kalay, Ersan; Yigit, Gökhan; Aslan, Yakup; Brown, Karen E; Pohl, Esther; Bicknell, Louise S; Kayserili, Hülya; Li, Yun; Tüysüz, Beyhan; Nürnberg, Gudrun; Kiess, Wieland; Koegl, Manfred; Baessmann, Ingelore; Buruk, Kurtulus; Toraman, Bayram; Kayipmaz, Saadettin; Kul, Sibel; Ikbal, Mevlit; Turner, Daniel J; Taylor, Martin S; Aerts, Jan; Scott, Carol; Milstein, Karen; Dollfus, Helene; Wieczorek, Dagmar; Brunner, Han G; Hurles, Matthew; Jackson, Andrew P; Rauch, Anita; Nürnberg, Peter; Karagüzel, Ahmet; Wollnik, Bernd

2012-01-01

Functional impairment of DNA damage response pathways leads to increased genomic instability. Here we describe the centrosomal protein CEP152 as a new regulator of genomic integrity and cellular response to DNA damage. Using homozygosity mapping and exome sequencing, we identified CEP152 mutations in Seckel syndrome and showed that impaired CEP152 function leads to accumulation of genomic defects resulting from replicative stress through enhanced activation of ATM signaling and increased H2AX phosphorylation. PMID:21131973

Human cytomegalovirus inhibits a DNA damage response by mislocalizing checkpoint proteins

NASA Astrophysics Data System (ADS)

Gaspar, Miguel; Shenk, Thomas

2006-02-01

The DNA damage checkpoint pathway responds to DNA damage and induces a cell cycle arrest to allow time for DNA repair. Several viruses are known to activate or modulate this cellular response. Here we show that the ataxia-telangiectasia mutated checkpoint pathway, which responds to double-strand breaks in DNA, is activated in response to human cytomegalovirus DNA replication. However, this activation does not propagate through the pathway; it is blocked at the level of the effector kinase, checkpoint kinase 2 (Chk2). Late after infection, several checkpoint proteins, including ataxia-telangiectasia mutated and Chk2, are mislocalized to a cytoplasmic virus assembly zone, where they are colocalized with virion structural proteins. This colocalization was confirmed by immunoprecipitation of virion proteins with an antibody that recognizes Chk2. Virus replication was resistant to ionizing radiation, which causes double-strand breaks in DNA. We propose that human CMV DNA replication activates the checkpoint response to DNA double-strand breaks, and the virus responds by altering the localization of checkpoint proteins to the cytoplasm and thereby inhibiting the signaling pathway. ionizing radiation | ataxia-telangiectasia mutated pathway

Improvement of the Immunogenicity of Porcine Circovirus Type 2 DNA Vaccine by Recombinant ORF2 Gene and CpG Motifs.

PubMed

Li, Jun; Shi, Jian-Li; Wu, Xiao-Yan; Fu, Fang; Yu, Jiang; Yuan, Xiao-Yuan; Peng, Zhe; Cong, Xiao-Yan; Xu, Shao-Jian; Sun, Wen-Bo; Cheng, Kai-Hui; Du, Yi-Jun; Wu, Jia-Qiang; Wang, Jin-Bao; Huang, Bao-Hua

2015-06-01

Nowadays, adjuvant is still important for boosting immunity and improving resistance in animals. In order to boost the immunity of porcine circovirus type 2 (PCV2) DNA vaccine, CpG motifs were inserted. In this study, the dose-effect was studied, and the immunity of PCV2 DNA vaccines by recombinant open reading frame 2 (ORF2) gene and CpG motifs was evaluated. Three-week-old Changbai piglets were inoculated intramuscularly with 200 μg, 400 μg, and 800 μg DNA vaccines containing 14 and 18 CpG motifs, respectively. Average gain and rectum temperature were recorded everyday during the experiments. Blood was collected from the piglets after vaccination to detect the changes of specific antibodies, interleukin-2, and immune cells every week. Tissues were collected for histopathology and polymerase chain reaction. The results indicated that compared to those of the control piglets, all concentrations of two DNA vaccines could induce PCV2-specific antibodies. A cellular immunity test showed that PCV2-specific lymphocytes proliferated the number of TH, TC, and CD3+ positive T-cells raised in the blood of DNA vaccine immune groups. There was no distinct pathological damage and viremia occurring in pigs that were inoculated with DNA vaccines, but there was some minor pathological damage in the control group. The results demonstrated that CpG motifs as an adjuvant could boost the humoral and cellular immunity of pigs to PCV2, especially in terms of cellular immunity. Comparing two DNA vaccines that were constructed, the one containing 18 CpG motifs was more effective. This is the first report that CpG motifs as an adjuvant insert to the PCV2 DNA vaccine could boost immunity.

Genotoxic effect of ethacrynic acid and impact of antioxidants

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ward, William M.; Hoffman, Jared D.; Loo, George, E-mail: g_loo@uncg.edu

It is known that ethacrynic acid (EA) decreases the intracellular levels of glutathione. Whether the anticipated oxidative stress affects the structural integrity of DNA is unknown. Therefore, DNA damage was assessed in EA-treated HCT116 cells, and the impact of several antioxidants was also determined. EA caused both concentration-dependent and time-dependent DNA damage that eventually resulted in cell death. Unexpectedly, the DNA damage caused by EA was intensified by either ascorbic acid or trolox. In contrast, EA-induced DNA damage was reduced by N-acetylcysteine and by the iron chelator, deferoxamine. In elucidating the DNA damage, it was determined that EA increased themore » production of reactive oxygen species, which was inhibited by N-acetylcysteine and deferoxamine but not by ascorbic acid and trolox. Also, EA decreased glutathione levels, which were inhibited by N-acetylcysteine. But, ascorbic acid, trolox, and deferoxamine neither inhibited nor enhanced the capacity of EA to decrease glutathione. Interestingly, the glutathione synthesis inhibitor, buthionine sulfoxime, lowered glutathione to a similar degree as EA, but no noticeable DNA damage was found. Nevertheless, buthionine sulfoxime potentiated the glutathione-lowering effect of EA and intensified the DNA damage caused by EA. Additionally, in examining redox-sensitive stress gene expression, it was found that EA increased HO-1, GADD153, and p21mRNA expression, in association with increased nuclear localization of Nrf-2 and p53 proteins. In contrast to ascorbic acid, trolox, and deferoxamine, N-acetylcysteine suppressed the EA-induced upregulation of GADD153, although not of HO-1. Overall, it is concluded that EA has genotoxic properties that can be amplified by certain antioxidants. - Highlights: • Ethacrynic acid (EA) caused cellular DNA damage. • EA-induced DNA damage was potentiated by ascorbic acid or trolox. • EA increased ROS production, not inhibited by ascorbic acid or trolox. • EA decreased glutathione levels, not prevented by ascorbic acid or trolox. • Buthionine sulfoxime intensified the DNA damage caused by EA.« less

Adrenergic Receptor Stimulation Prevents Radiation-Induced DNA Strand Breaks, Apoptosis and Gene Expression in Simulated Microgravity

NASA Technical Reports Server (NTRS)

Moreno-Villanueva, Maria; Krieger, Stephanie; Feiveson, Alan; Kovach, Annie Marie; Buerkle, Alexander; Wu, Honglu

2017-01-01

Under Earth gravity conditions cellular damage can be counteracted by activation of the physiological defense mechanisms or through medical interventions. The mode of action of both, physiological response and medical interventions can be affected by microgravity leading to failure in repairing the damage. There are many studies reporting the effects of microgravity and/or radiation on cellular functions. However, little is known about the synergistic effects on cellular response to radiation when other endogenous cellular stress-response pathways are previously activated. Here, we investigated whether previous stimulation of the adrenergic receptor, which modulates immune response, affects radiation-induced apoptosis in immune cells under simulated microgravity conditions. Peripheral blood mononuclear cells (PBMCs) were stimulated with isoproterenol (a sympathomimetic drug) and exposed to 0.8 or 2Gy gamma-radiation in simulated microgravity versus Earth gravity. Expression of genes involved in adrenergic receptor pathways, DNA repair and apoptosis as well as the number of apoptotic cells and DNA strand breaks were determined. Our results showed that, under simulated microgravity conditions, previous treatment with isoproterenol prevented radiation-induced i) gene down regulation, ii) DNA strand breaks formation and iii) apoptosis induction. Interestedly, we found a radiation-induced increase of adrenergic receptor gene expression, which was also abolished in simulated microgravity. Understanding the mechanisms of isoproterenol-mediated radioprotection in simulated microgravity can help to develop countermeasures for space-associated health risks as well as radio-sensitizers for cancer therapy.

Milk thistle and olive extract: old substances with a new mission against sun-induced skin damage.

PubMed

DI Caprio, Roberta; Monfrecola, Giuseppe; Gasparri, Franco; Micillo, Raffaella; Balato, Anna; Lembo, Serena

2017-11-30

Natural antioxidants represent an effective option in the prevention and/or improvement of ultraviolet radiations (UVR)-induced/aggravated skin conditions. UVR cause DNA damage in keratinocytes, directly, in the form of cyclobutane pyrimidine dimers (CPDs), or indirectly, through oxidative stress production. Failure of the repair system can result in genetic mutations primarily responsible for the initiation of NMSCs. The aim of our study was to evaluate the in vitro protective effect of milk thistle and olive purified extracts on cultured keratinocytes after solar simulator irradiations (SSR). Immortalized keratinocytes were pre-incubated with different concentrations of milk thistle and olive purified extracts, and irradiated with increasing doses of SSR. Thereafter, CPDs and p53 expression were evaluated to assess DNA damage, whereas cellular antioxidants consumption and lipid membranes peroxidation were measured to analyse oxidative stress. The study substances were well tolerated by cells and displayed good cytoprotective and anti-oxidant activities, being milk thistle dry extract more effective in limiting the direct DNA damage, and olive extract particularly able to reduce lipid membrane peroxidation and to increase cellular antioxidants. Both study substances can be defined as safe compounds, showing differential cytoprotective and anti-oxidant activities and might represent interesting options for NMSCs chemoprevention.

Laboratory and clinical studies of cancer chemoprevention by antioxidants in berries.

PubMed

Stoner, Gary David; Wang, Li-Shu; Casto, Bruce Cordell

2008-09-01

Reactive oxygen species (ROS) are a major cause of cellular injury in an increasing number of diseases, including cancer. Most ROS are created in the cell through normal cellular metabolism. They can be produced by environmental insults such as ultraviolet light and toxic chemicals, as well as by the inflammatory process. Interception of ROS or limiting their cellular effects is a major role of antioxidants. Due to their content of phenolic and flavonoid compounds, berries exhibit high antioxidant potential, exceeding that of many other foodstuffs. Through their ability to scavenge ROS and reduce oxidative DNA damage, stimulate antioxidant enzymes, inhibit carcinogen-induced DNA adduct formation and enhance DNA repair, berry compounds have been shown to inhibit mutagenesis and cancer initiation. Berry constituents also influence cellular processes associated with cancer progression including signaling pathways associated with cell proliferation, differentiation, apoptosis and angiogenesis. This review article summarizes laboratory and human studies, demonstrating the protective effects of berries and berry constituents on oxidative and other cellular processes leading to cancer development.

Laboratory and clinical studies of cancer chemoprevention by antioxidants in berries

PubMed Central

Stoner, Gary David; Wang, Li-Shu; Casto, Bruce Cordell

2008-01-01

Reactive oxygen species (ROS) are a major cause of cellular injury in an increasing number of diseases, including cancer. Most ROS are created in the cell through normal cellular metabolism. They can be produced by environmental insults such as ultraviolet light and toxic chemicals, as well as by the inflammatory process. Interception of ROS or limiting their cellular effects is a major role of antioxidants. Due to their content of phenolic and flavonoid compounds, berries exhibit high antioxidant potential, exceeding that of many other foodstuffs. Through their ability to scavenge ROS and reduce oxidative DNA damage, stimulate antioxidant enzymes, inhibit carcinogen-induced DNA adduct formation and enhance DNA repair, berry compounds have been shown to inhibit mutagenesis and cancer initiation. Berry constituents also influence cellular processes associated with cancer progression including signaling pathways associated with cell proliferation, differentiation, apoptosis and angiogenesis. This review article summarizes laboratory and human studies, demonstrating the protective effects of berries and berry constituents on oxidative and other cellular processes leading to cancer development. PMID:18544560

Blood micronutrients and DNA damage in children.

PubMed

Milne, Elizabeth; Greenop, Kathryn R; Ramankutty, Padmaja; Miller, Margaret; de Klerk, Nicholas H; Armstrong, Bruce K; Almond, Theodora; O'Callaghan, Nathan J; Fenech, Michael

2015-10-01

Maintenance of normal cellular phenotype depends largely on accurate DNA replication and repair. DNA damage causes gene mutations and predisposes to cancer and other chronic diseases. Growing evidence indicates that nutritional factors are associated with DNA damage in adults; here, we investigate these associations in children. We conducted a cross-sectional study among 462 healthy children 3, 6, and 9 years of age. Blood was collected and micronutrient levels were measured. The cytokinesis-block micronucleus cytome assay was used to measure chromosomal DNA damage (micronuclei, nucleoplasmic bridges, and nuclear buds) in lymphocytes. Cell apoptosis, necrosis, and the nuclear division index were also measured. Nine loci in genes involved in folate metabolism and DNA repair were genotyped. Data were analyzed using linear regression with adjustment for potential confounders. Plasma calcium was positively associated with micronuclei and necrosis, and α-tocopherol negatively associated with apoptosis, nuclear division index, and nucleoplasmic bridges; lutein was positively associated with nucleoplasmic bridges. α-tocopherol was positively associated with necrosis. DNA damage in healthy children may be influenced by blood micronutrient levels and certain genotypes. Further investigation of associations between nutritional status and genomic integrity in children is needed to shed additional light on potential mechanisms. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Hippo pathway and protection of genome stability in response to DNA damage.

PubMed

Pefani, Dafni E; O'Neill, Eric

2016-04-01

The integrity of DNA is constantly challenged by exposure to the damaging effects of chemical and physical agents. Elucidating the cellular mechanisms that maintain genomic integrity via DNA repair and cell growth control is vital because errors in these processes lead to genomic damage and the development of cancer. By gaining a deep molecular understanding of the signaling pathways regulating genome integrity it is hoped to uncover new therapeutics and treatment designs to combat cancer. Components of the Hippo pathway, a tumor-suppressor cascade, have recently been defined to limit cancer transformation in response to DNA damage. In this review, we briefly introduce the Hippo signaling cascade in mammals and discuss in detail how the Hippo pathway has been established as part of the DNA damage response, activated by apical signaling kinases that recognize breaks in DNA. We also highlight the significance of the Hippo pathway activator RASSF1A tumor suppressor, a direct target of ataxia telangiectasia mutated and ataxia telangiectasia and Rad3 related ATR. Furthermore we discuss how Hippo pathway in response DNA lesions can induce cell death via Yes-associated protein (YAP) (the canonical Hippo pathway effector) or promote maintenance of genome integrity in a YAP-independent manner. © 2015 FEBS.

SPOC1 modulates DNA repair by regulating key determinants of chromatin compaction and DNA damage response

PubMed Central

Mund, Andreas; Schubert, Tobias; Staege, Hannah; Kinkley, Sarah; Reumann, Kerstin; Kriegs, Malte; Fritsch, Lauriane; Battisti, Valentine; Ait-Si-Ali, Slimane; Hoffbeck, Anne-Sophie; Soutoglou, Evi; Will, Hans

2012-01-01

Survival time-associated plant homeodomain (PHD) finger protein in Ovarian Cancer 1 (SPOC1, also known as PHF13) is known to modulate chromatin structure and is essential for testicular stem-cell differentiation. Here we show that SPOC1 is recruited to DNA double-strand breaks (DSBs) in an ATM-dependent manner. Moreover, SPOC1 localizes at endogenous repair foci, including OPT domains and accumulates at large DSB repair foci characteristic for delayed repair at heterochromatic sites. SPOC1 depletion enhances the kinetics of ionizing radiation-induced foci (IRIF) formation after γ-irradiation (γ-IR), non-homologous end-joining (NHEJ) repair activity, and cellular radioresistance, but impairs homologous recombination (HR) repair. Conversely, SPOC1 overexpression delays IRIF formation and γH2AX expansion, reduces NHEJ repair activity and enhances cellular radiosensitivity. SPOC1 mediates dose-dependent changes in chromatin association of DNA compaction factors KAP-1, HP1-α and H3K9 methyltransferases (KMT) GLP, G9A and SETDB1. In addition, SPOC1 interacts with KAP-1 and H3K9 KMTs, inhibits KAP-1 phosphorylation and enhances H3K9 trimethylation. These findings provide the first evidence for a function of SPOC1 in DNA damage response (DDR) and repair. SPOC1 acts as a modulator of repair kinetics and choice of pathways. This involves its dose-dependent effects on DNA damage sensors, repair mediators and key regulators of chromatin structure. PMID:23034801

DNA-damage response associated with occupational exposure, age and chronic inflammation in workers in the automotive industry.

PubMed

Savina, Natalya V; Smal, Marharyta P; Kuzhir, Tatyana D; Ershova-Pavlova, Alla A; Goncharova, Roza I

2012-10-09

The evaluation of genome integrity in populations occupationally exposed to combine industrial factors is of medical importance. In the present study, the DNA-damage response was estimated by means of the alkaline comet assay in a sizeable cohort of volunteers recruited among workers in the automotive industry. For this purpose, freshly collected lymphocytes were treated with hydrogen peroxide (100μM, 1min, 4°C) in vitro, and the levels of basal and H(2)O(2)-induced DNA damage, and the kinetics and efficiency of DNA repair were measured during a 180-min interval after exposure. The parameters studied in the total cohort of workers were in a range of values prescribed for healthy adult residents of Belarus. Based on the 95th percentiles, individuals possessing enhanced cellular sensitivity to DNA damage were present in different groups, but the frequency was significantly higher among elderly persons and among individuals with chronic inflammatory diseases. The results indicate that the inter-individual variations in DNA-damage response should be taken into account to estimate adequately the environmental genotoxic effects and to identify individuals with an enhanced DNA-damage response due to the influence of some external factors or intrinsic properties of the organism. Underling mechanisms need to be further explored. © 2012 Elsevier B.V. All rights reserved.

Analyzing the dose-dependence of the Saccharomyces cerevisiae global transcriptional response to methyl methanesulfonate and ionizing radiation.

PubMed

Benton, Michael G; Somasundaram, Swetha; Glasner, Jeremy D; Palecek, Sean P

2006-12-01

One of the most crucial tasks for a cell to ensure its long term survival is preserving the integrity of its genetic heritage via maintenance of DNA structure and sequence. While the DNA damage response in the yeast Saccharomyces cerevisiae, a model eukaryotic organism, has been extensively studied, much remains to be elucidated about how the organism senses and responds to different types and doses of DNA damage. We have measured the global transcriptional response of S. cerevisiae to multiple doses of two representative DNA damaging agents, methyl methanesulfonate (MMS) and gamma radiation. Hierarchical clustering of genes with a statistically significant change in transcription illustrated the differences in the cellular responses to MMS and gamma radiation. Overall, MMS produced a larger transcriptional response than gamma radiation, and many of the genes modulated in response to MMS are involved in protein and translational regulation. Several clusters of coregulated genes whose responses varied with DNA damaging agent dose were identified. Perhaps the most interesting cluster contained four genes exhibiting biphasic induction in response to MMS dose. All of the genes (DUN1, RNR2, RNR4, and HUG1) are involved in the Mec1p kinase pathway known to respond to MMS, presumably due to stalled DNA replication forks. The biphasic responses of these genes suggest that the pathway is induced at lower levels as MMS dose increases. The genes in this cluster with a threefold or greater transcriptional response to gamma radiation all showed an increased induction with increasing gamma radiation dosage. Analyzing genome-wide transcriptional changes to multiple doses of external stresses enabled the identification of cellular responses that are modulated by magnitude of the stress, providing insights into how a cell deals with genotoxicity.

Ameliorating reactive oxygen species-induced in vitro lipid peroxidation in brain, liver, mitochondria and DNA damage by Zingiber officinale Roscoe.

PubMed

Ajith, T A

2010-01-01

Iron is an essential nutrient for a number of cellular activities. However, excess cellular iron can be toxic by producing reactive oxygen species (ROS) such as superoxide anion (O(2) (-)) and hydroxyl radical (HO(·)) that damage proteins, lipids and DNA. Mutagenic and genotoxic end products of lipid peroxidation can induce the decline of mitochondrial respiration and are associated with various human ailments including aging, neurodegenerative disorders, cancer etc. Zingiber officinale Roscoe (ginger) is a widely used spice around the world. The protective effect of aqueous ethanol extract of Z. officinale against ROS-induced in vitro lipid peroxidation and DNA damage was evaluated in this study. The lipid peroxidation was induced by hydroxyl radical generated from Fenton's reaction in rat liver and brain homogenates and mitochondrial fraction (isolated from rat liver). The DNA protection was evaluated using H(2)O(2)-induced changes in pBR-322 plasmid and Fenton reaction-induced DNA fragmentation in rat liver. The results indicated that Z. officinale significantly (P<0.001) protected the lipid peroxidation in all the tissue homogenate/mitochondria. The extract at 2 and 0.5 mg/ml could protect 92 % of the lipid peroxidation in brain homogenate and liver mitochondria respectively. The percent inhibition of lipid peroxidation at 1mg/ml of Z. officinale in the liver homogenate was 94 %. However, the extract could partially alleviate the DNA damage. The protective mechanism can be correlated to the radical scavenging property of Z. officinale. The results of the study suggest the possible nutraceutical role of Z. officinale against the oxidative stress induced human ailments.

Deregulation of vital mitotic kinase-phosphatase signaling in hematopoietic stem/progenitor compartment leads to cellular catastrophe in experimental aplastic anemia.

PubMed

Chatterjee, Ritam; Chattopadhyay, Sukalpa; Law, Sujata

2016-11-01

Aplastic anemia, the paradigm of bone marrow failure, is characterized by pancytopenic peripheral blood and hypoplastic bone marrow. Among various etiologies, inappropriate use of DNA alkylating drugs like cyclophosphamide and busulfan often causes the manifestation of the dreadful disease. Cell cycle impairment in marrow hematopoietic stem/progenitor compartment together with cellular apoptosis has been recognized as culpable factors behind aplastic pathophysiologies. However, the intricate molecular mechanisms remain unrevealed till date. In the present study, we have dealt with the mechanistic intervention of the disease by peripheral blood hemogram, bone marrow histopathology, cytopathology, hematopoietic kinetic study, scanning electron microscopy, DNA damage assessment and flowcytometric analysis of cellular proliferation and apoptosis in hematopoietic stem/progenitor cell (HSPC) rich marrow compartment using busulfan and cyclophosphamidemediated mouse model. To unveil the molecular mechanisms behind aplastic pathophysiology, we further investigated the role of some crucial mitotic and apoptotic regulators like Protein kinase-B (PKB), Gsk-3β, Cyclin-D1, PP2A, Cdc25c, Plk-1, Aurora kinase-A, Chk-1 regarding the hematopoietic catastrophe. Our observations revealed that the alteration of PKB-GSK-3β axis, Plk-1, and Aurora kinase-A expressions in HSPC compartment due to DNA damage response was associated with the proliferative impairment and apoptosis during aplastic anemia. The study established the correlation between the accumulation of DNA damage and alteration of the mentioned molecules in aplastic HSPCs that lead to the hematopoietic catastrophe. We anticipate that our findings will be beneficial for developing better therapeutic strategies for the dreadful disease concerned.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kao, Shyan-Yuan, E-mail: shyan-yuan_kao@meei.harvard.edu

Mutation of parkin is one of the most prevalent causes of autosomal recessive Parkinson's disease (PD). Parkin is an E3 ubiquitin ligase that acts on a variety of substrates, resulting in polyubiquitination and degradation by the proteasome or monoubiquitination and regulation of biological activity. However, the cellular functions of parkin that relate to its pathological involvement in PD are not well understood. Here we show that parkin is essential for optimal repair of DNA damage. Parkin-deficient cells exhibit reduced DNA excision repair that can be restored by transfection of wild-type parkin, but not by transfection of a pathological parkin mutant.more » Parkin also protects against DNA damage-induced cell death, an activity that is largely lost in the pathological mutant. Moreover, parkin interacts with the proliferating cell nuclear antigen (PCNA), a protein that coordinates DNA excision repair. These results suggest that parkin promotes DNA repair and protects against genotoxicity, and implicate DNA damage as a potential pathogenic mechanism in PD.« less

Individual variations in the correlation between erythemal threshold, UV-induced DNA damage and sun-burn cell formation.

PubMed

Heenen, M; Giacomoni, P U; Golstein, P

2001-10-01

A linear correlation between erythema intensity and DNA damage upon exposure to UV has not been firmly established. Many of the deleterious effects of UV exposure do occur after exposure to suberythemal doses. After DNA damage, cells undergo DNA repair. It is commonly accepted that when the burden of damage is beyond the repair capacities, the cell undergoes programmed cell death or apoptosis. The aim of this study is to quantify the amount of UV-induced DNA damage (estimated via the measurement of DNA repair or unscheduled DNA synthesis or UDS) and cellular damage (estimated via the determination of the density of sunburn cells or SBC). If DNA damage and erythema are correlated, similar intensity of UDS and similar density of SBC should be found in volunteers irradiated with a UV dose equal to two minimal erythema doses (MED). Our results show that in 15 different individuals the same relative dose (2 MEDs) provokes UDS values, which vary within a factor of 4. An even larger variability affects SBC counts after the same relative dose. When DNA damage or SBC are plotted versus the absolute dose (i.e. the dose expressed in J/m(2)), there is a rough correlation (with several exceptions) between dose and extent of UDS and SBC counts. It seems possible to divide the volunteers into two subpopulations with different susceptibilities to UV damage. It is well known that UDS and SBC measurements are often affected by large experimental indeterminacy, yet, the analysis of our results makes it plausible to suggest that for the triggering of erythema, a common threshold value for DNA damage or for SBC count are not to be found. In conclusion, the erythema response seems to be loosely correlated with DNA damage. This suggests that the protection offered by the sunscreens against DNA damage, the molecular basis of UV-induced mutagenesis, might not be related to the sun protection factor (SPF) indicated on the label of sunscreens, which is evaluated using the erythema as an endpoint.

Cellular response to low dose radiation: Role of phosphatidylinositol-3 kinase like kinases

DOE Office of Scientific and Technical Information (OSTI.GOV)

Balajee, A.S.; Meador, J.A.; Su, Y.

It is increasingly realized that human exposure either to an acute low dose or multiple chronic low doses of low LET radiation has the potential to cause different types of cancer. Therefore, the central theme of research for DOE and NASA is focused on understanding the molecular mechanisms and pathways responsible for the cellular response to low dose radiation which would not only improve the accuracy of estimating health risks but also help in the development of predictive assays for low dose radiation risks associated with tissue degeneration and cancer. The working hypothesis for this proposal is that the cellularmore » mechanisms in terms of DNA damage signaling, repair and cell cycle checkpoint regulation are different for low and high doses of low LET radiation and that the mode of action of phosphatidylinositol-3 kinase like kinases (PIKK: ATM, ATR and DNA-PK) determines the dose dependent cellular responses. The hypothesis will be tested at two levels: (I) Evaluation of the role of ATM, ATR and DNA-PK in cellular response to low and high doses of low LET radiation in simple in vitro human cell systems and (II) Determination of radiation responses in complex cell microenvironments such as human EpiDerm tissue constructs. Cellular responses to low and high doses of low LET radiation will be assessed from the view points of DNA damage signaling, DNA double strand break repair and cell cycle checkpoint regulation by analyzing the activities (i.e. post-translational modifications and kinetics of protein-protein interactions) of the key target proteins for PI-3 kinase like kinases both at the intra-cellular and molecular levels. The proteins chosen for this proposal are placed under three categories: (I) sensors/initiators include ATM ser1981, ATR, 53BP1, gamma-H2AX, MDC1, MRE11, Rad50 and Nbs1; (II) signal transducers include Chk1, Chk2, FANCD2 and SMC1; and (III) effectors include p53, CDC25A and CDC25C. The primary goal of this proposal is to elucidate the differences in cellular defense mechanisms between low and high doses of low LET radiation and to define the radiation doses where the cellular DNA damage signaling and repair mechanisms tend to shift. This information is critically important to address and advance some of the low dose research program objectives of DOE. The results of this proposed study will lead to a better understanding of the mechanisms for the cellular responses to low and high doses of low LET radiation. Further, systematic analysis of the role of PIKK signaling pathways as a function of radiation dose in tissue microenvironment will provide useful mechanistic information for improving the accuracy of radiation risk assessment for low doses. Knowledge of radiation responses in tissue microenvironment is important for the accurate prediction of ionizing radiation risks associated with cancer and tissue degeneration in humans.« less

Improved methods of DNA extraction from human spermatozoa that mitigate experimentally-induced oxidative DNA damage.

PubMed

Xavier, Miguel J; Nixon, Brett; Roman, Shaun D; Aitken, Robert John

2018-01-01

Current approaches for DNA extraction and fragmentation from mammalian spermatozoa provide several challenges for the investigation of the oxidative stress burden carried in the genome of male gametes. Indeed, the potential introduction of oxidative DNA damage induced by reactive oxygen species, reducing agents (dithiothreitol or beta-mercaptoethanol), and DNA shearing techniques used in the preparation of samples for chromatin immunoprecipitation and next-generation sequencing serve to cofound the reliability and accuracy of the results obtained. Here we report optimised methodology that minimises, or completely eliminates, exposure to DNA damaging compounds during extraction and fragmentation procedures. Specifically, we show that Micrococcal nuclease (MNase) digestion prior to cellular lysis generates a greater DNA yield with minimal collateral oxidation while randomly fragmenting the entire paternal genome. This modified methodology represents a significant improvement over traditional fragmentation achieved via sonication in the preparation of genomic DNA from human spermatozoa for downstream applications, such as next-generation sequencing. We also present a redesigned bioinformatic pipeline framework adjusted to correctly analyse this form of data and detect statistically relevant targets of oxidation.

Replication-mediated disassociation of replication protein A-XPA complex upon DNA damage: implications for RPA handing off.

PubMed

Jiang, Gaofeng; Zou, Yue; Wu, Xiaoming

2012-08-01

RPA (replication protein A), the eukaryotic ssDNA (single-stranded DNA)-binding protein, participates in most cellular processes in response to genotoxic insults, such as NER (nucleotide excision repair), DNA, DSB (double-strand break) repair and activation of cell cycle checkpoint signalling. RPA interacts with XPA (xeroderma pigmentosum A) and functions in early stage of NER. We have shown that in cells the RPA-XPA complex disassociated upon exposure of cells to high dose of UV irradiation. The dissociation required replication stress and was partially attributed to tRPA hyperphosphorylation. Treatment of cells with CPT (camptothecin) and HU (hydroxyurea), which cause DSB DNA damage and replication fork collapse respectively and also leads to the disruption of RPA-XPA complex. Purified RPA and XPA were unable to form complex in vitro in the presence of ssDNA. We propose that the competition-based RPA switch among different DNA metabolic pathways regulates the dissociation of RPA with XPA in cells after DNA damage. The biological significances of RPA-XPA complex disruption in relation with checkpoint activation, DSB repair and RPA hyperphosphorylation are discussed.

A Role for the Host DNA Damage Response in Hepatitis B Virus cccDNA Formation—and Beyond?

PubMed Central

Schreiner, Sabrina; Nassal, Michael

2017-01-01

Chronic hepatitis B virus (HBV) infection puts more than 250 million people at a greatly increased risk to develop end-stage liver disease. Like all hepadnaviruses, HBV replicates via protein-primed reverse transcription of a pregenomic (pg) RNA, yielding an unusually structured, viral polymerase-linked relaxed-circular (RC) DNA as genome in infectious particles. Upon infection, RC-DNA is converted into nuclear covalently closed circular (ccc) DNA. Associating with cellular proteins into an episomal minichromosome, cccDNA acts as template for new viral RNAs, ensuring formation of progeny virions. Hence, cccDNA represents the viral persistence reservoir that is not directly targeted by current anti-HBV therapeutics. Eliminating cccDNA will thus be at the heart of a cure for chronic hepatitis B. The low production of HBV cccDNA in most experimental models and the associated problems in reliable cccDNA quantitation have long hampered a deeper understanding of cccDNA molecular biology. Recent advancements including cccDNA-dependent cell culture systems have begun to identify select host DNA repair enzymes that HBV usurps for RC-DNA to cccDNA conversion. While this list is bound to grow, it may represent just one facet of a broader interaction with the cellular DNA damage response (DDR), a network of pathways that sense and repair aberrant DNA structures and in the process profoundly affect the cell cycle, up to inducing cell death if repair fails. Given the divergent interactions between other viruses and the DDR it will be intriguing to see how HBV copes with this multipronged host system. PMID:28531167

Effect of lycopene and {beta}-carotene on peroxynitrite-mediated cellular modifications

DOE Office of Scientific and Technical Information (OSTI.GOV)

Muzandu, Kaampwe; Ishizuka, Mayumi; Sakamoto, Kentaro Q.

2006-09-15

Peroxynitrite formed by the reaction of superoxide and nitric oxide is a highly reactive species with a role in various pathological processes such as cancer, chronic inflammation, and cardiovascular and neurological diseases. In the present study, the effect of the carotenoids, lycopene and {beta}-carotene, on peroxynitrite-mediated modifications in plasmid DNA as well as cellular DNA and proteins were investigated. In pUC18 plasmid DNA, these carotenoids strongly inhibited DNA strand breaks caused by peroxynitrite generated from 3-morpholinosydnonimine (SIN-1). SIN-1 was also used to determine effects on DNA damage and protein tyrosine nitration in Chinese hamster lung fibroblasts. SIN-1 dose-dependently increased nitrationmore » of proteins in cells above basal levels as determined by Western blotting. This nitration was inhibited in the presence of the uric acid as well as lycopene. Physiological concentrations (0.31-10 {mu}M) of lycopene and {beta}-carotene also had protective effects on DNA damage, as measured by the comet assay. Lycopene significantly reduced DNA damage particularly, in the median range of concentrations (2.5 {mu}M). The protective effects of lycopene and {beta}-carotene could be due to their scavenging of reactive oxygen (ROS) and/or nitrogen species (RNS) as they reduce the amount of intracellular ROS/RNS produced following treatment with SIN-1 by as much as 47.5% and 42.4%, respectively. The results obtained in this study suggest that carotenoids may alleviate some of the deleterious effects of peroxynitrite and possibly other reactive nitrogen species as well in vivo.« less

Effect of Treatment Media on the Agglomeration of Titanium Dioxide Nanoparticles: Impact on Genotoxicity, Cellular Interaction, and Cell Cycle

EPA Science Inventory

ABSTRACT The widespread use of titanium dioxide (TiO2) nanoparticles in consumer products increases the probability of exposure to humans and the environment. Although TiO2 nanoparticles have been shown to induce DNA damage (comet assay) and chromosome damage (micronucleus ass...

Serine is the major residue for ADP-ribosylation upon DNA damage

PubMed Central

Dauben, Helen

2018-01-01

Poly(ADP-ribose) polymerases (PARPs) are a family of enzymes that synthesise ADP-ribosylation (ADPr), a reversible modification of proteins that regulates many different cellular processes. Several mammalian PARPs are known to regulate the DNA damage response, but it is not clear which amino acids in proteins are the primary ADPr targets. Previously, we reported that ARH3 reverses the newly discovered type of ADPr (ADPr on serine residues; Ser-ADPr) and developed tools to analyse this modification (Fontana et al., 2017). Here, we show that Ser-ADPr represents the major fraction of ADPr synthesised after DNA damage in mammalian cells and that globally Ser-ADPr is dependent on HPF1, PARP1 and ARH3. In the absence of HPF1, glutamate/aspartate becomes the main target residues for ADPr. Furthermore, we describe a method for site-specific validation of serine ADP-ribosylated substrates in cells. Our study establishes serine as the primary form of ADPr in DNA damage signalling. PMID:29480802

Novel DNA lesions generated by the interaction between therapeutic thiopurines and UVA light.

PubMed

Zhang, Xiaohong; Jeffs, Graham; Ren, Xiaolin; O'Donovan, Peter; Montaner, Beatriz; Perrett, Conal M; Karran, Peter; Xu, Yao-Zhong

2007-03-01

The therapeutic effect of the thiopurines, 6-thioguanine (6-TG), 6-mercaptopurine, and its prodrug azathioprine, depends on the incorporation of 6-TG into cellular DNA. Unlike normal DNA bases, 6-TG absorbs UVA radiation, and UVA-mediated photochemical damage of DNA 6-TG has potentially harmful side effects. When free 6-TG is UVA irradiated in solution in the presence of molecular oxygen, reactive oxygen species are generated and 6-TG is oxidized to guanine-6-sulfonate (G(SO3)) and guanine-6-thioguanine in reactions involving singlet oxygen. This conversion is prevented by antioxidants, including the dietary vitamin ascorbate. DNA G(SO3) is also the major photoproduct of 6-TG in DNA and it can be selectively introduced into DNA or oligonucleotides in vitro by mild chemical oxidation. Thermal stability measurements indicate that G(SO3) does not form stable base pairs with any of the normal DNA bases in duplex oligonucleotides and is a powerful block for elongation by Klenow DNA polymerase in primer extension experiments. In cultured human cells, DNA damage produced by 6-TG and UVA treatment is associated with replication inhibition and provokes a p53-dependent DNA damage response.

DNA damage and repair after high LET radiation

NASA Astrophysics Data System (ADS)

O'Neill, Peter; Cucinotta, Francis; Anderson, Jennifer

Predictions from biophysical models of interactions of radiation tracks with cellular DNA indicate that clustered DNA damage sites, defined as two or more lesions formed within one or two helical turns of the DNA by passage of a single radiation track, are formed in mammalian cells. These complex DNA damage sites are regarded as a signature of ionizing radiation exposure particularly as the likelihood of clustered damage sites arising endogenously is low. For instance, it was predicted from biophysical modelling that 30-40% of low LET-induced double strand breaks (DSB), a form of clustered damage, are complex with the yield increasing to >90% for high LET radiation, consistent with the reduced reparability of DSB with increasing ionization density of the radiation. The question arises whether the increased biological effects such as mutagenesis, carcinogenesis and lethality is in part related to DNA damage complexity and/or spatial distribution of the damage sites, which may lead to small DNA fragments. With particle radiation it is also important to consider not only delta-rays which may cause clustered damaged sites and may be highly mutagenic but the non-random spatial distribution of DSB which may lead to deletions. In this overview I will concentrate on the molecular aspects of the variation of the complexity of DNA damage on radiation quality and the challenges this complexity presents the DNA damage repair pathways. I will draw on data from micro-irradiations which indicate that the repair of DSBs by non-homologous end joining is highly regulated with pathway choice and kinetics of repair dependent on the chemical complexity of the DSB. In summary the aim is to emphasis the link between the spatial distribution of energy deposition events related to the track, the molecular products formed and the consequence of damage complexity contributing to biological effects and to present some of the outstanding molecular challenges with particle radiation.

Disintegration of cruciform and G-quadruplex structures during the course of helicase-dependent amplification (HDA).

PubMed

Li, Dawei; Lv, Bei; Zhang, Hao; Lee, Jasmine Yiqin; Li, Tianhu

2015-04-15

Unlike chemical damages on DNA, physical alterations of B-form of DNA occur commonly in organisms that serve as signals for specified cellular events. Although the modes of action for repairing of chemically damaged DNA have been well studied nowadays, the repairing mechanisms for physically altered DNA structures have not yet been understood. Our current in vitro studies show that both breakdown of stable non-B DNA structures and resumption of canonical B-conformation of DNA can take place during the courses of isothermal helicase-dependent amplification (HDA). The pathway that makes the non-B DNA structures repairable is presumably the relieving of the accumulated torsional stress that was caused by the positive supercoiling. Our new findings suggest that living organisms might have evolved this distinct and economical pathway for repairing their physically altered DNA structures. Copyright © 2015 Elsevier Ltd. All rights reserved.

DNA oxidative damage and life expectancy in houseflies.

PubMed Central

Agarwal, S; Sohal, R S

1994-01-01

The objective of this study was to explore the relationship between oxidative molecular damage and the aging process by determining whether such damage is associated with the rate of aging, using the adult housefly as the experimental organism. Because the somatic tissues in the housefly consist of long-lived postmitotic cells, it provides an excellent model system for studying cumulative age-related cellular alterations. Rate of aging in the housefly was manipulated by varying the rate of metabolism (physical activity). The concentration of 8-hydroxydeoxyguanosine (80HdG) was used as an indicator of DNA oxidation. Exposure of live flies to x-rays and hyperoxia elevated the level of 8OHdG. The level of 8OHdG in mitochondrial as well as total DNA increased with the age of flies. Mitochondrial DNA was 3 times more susceptible to age-related oxidative damage than nuclear DNA. A decrease in the level of physical activity of the flies was found to prolong the life-span and corresponding reduce the level of 8OHdG in both mitochondrial and total DNA. Under all conditions examined, mitochondrial DNA exhibited a higher level of oxidative damage than total DNA. The 8OHdG levels were found to be inversely associated with the life expectancy of houseflies. The pattern of age-associated accrural of 8OHdG was virtually identical to that of protein carbonyl content. Altoghether, results of this study support the hypothesis that oxidative molecular damage is a causal factor in senescence. PMID:7991627

Roles of PCNA ubiquitination and TLS polymerases κ and η in the bypass of methyl methanesulfonate-induced DNA damage.

PubMed

Wit, Niek; Buoninfante, Olimpia Alessandra; van den Berk, Paul C M; Jansen, Jacob G; Hogenbirk, Marc A; de Wind, Niels; Jacobs, Heinz

2015-01-01

Translesion synthesis (TLS) provides a highly conserved mechanism that enables DNA synthesis on a damaged template. TLS is performed by specialized DNA polymerases of which polymerase (Pol) κ is important for the cellular response to DNA damage induced by benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE), ultraviolet (UV) light and the alkylating agent methyl methanesulfonate (MMS). As TLS polymerases are intrinsically error-prone, tight regulation of their activity is required. One level of control is provided by ubiquitination of the homotrimeric DNA clamp PCNA at lysine residue 164 (PCNA-Ub). We here show that Polκ can function independently of PCNA modification and that Polη can function as a backup during TLS of MMS-induced lesions. Compared to cell lines deficient for PCNA modification (Pcna(K164R)) or Polκ, double mutant cell lines display hypersensitivity to MMS but not to BPDE or UV-C. Double mutant cells also displayed delayed post-replicative TLS, accumulate higher levels of replication stress and delayed S-phase progression. Furthermore, we show that Polη and Polκ are redundant in the DNA damage bypass of MMS-induced DNA damage. Taken together, we provide evidence for PCNA-Ub-independent activation of Polκ and establish Polη as an important backup polymerase in the absence of Polκ in response to MMS-induced DNA damage. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

When theory and observation collide: Can non-ionizing radiation cause cancer?

PubMed

Havas, Magda

2017-02-01

This paper attempts to resolve the debate about whether non-ionizing radiation (NIR) can cause cancer-a debate that has been ongoing for decades. The rationale, put forward mostly by physicists and accepted by many health agencies, is that, "since NIR does not have enough energy to dislodge electrons, it is unable to cause cancer." This argument is based on a flawed assumption and uses the model of ionizing radiation (IR) to explain NIR, which is inappropriate. Evidence of free-radical damage has been repeatedly documented among humans, animals, plants and microorganisms for both extremely low frequency (ELF) electromagnetic fields (EMF) and for radio frequency (RF) radiation, neither of which is ionizing. While IR directly damages DNA, NIR interferes with the oxidative repair mechanisms resulting in oxidative stress, damage to cellular components including DNA, and damage to cellular processes leading to cancer. Furthermore, free-radical damage explains the increased cancer risks associated with mobile phone use, occupational exposure to NIR (ELF EMF and RFR), and residential exposure to power lines and RF transmitters including mobile phones, cell phone base stations, broadcast antennas, and radar installations. Copyright © 2016 Elsevier Ltd. All rights reserved.

Restriction Endonucleases from Invasive Neisseria gonorrhoeae Cause Double-Strand Breaks and Distort Mitosis in Epithelial Cells during Infection

PubMed Central

Weyler, Linda; Engelbrecht, Mattias; Mata Forsberg, Manuel; Brehwens, Karl; Vare, Daniel; Vielfort, Katarina; Wojcik, Andrzej; Aro, Helena

2014-01-01

The host epithelium is both a barrier against, and the target for microbial infections. Maintaining regulated cell growth ensures an intact protective layer towards microbial-induced cellular damage. Neisseria gonorrhoeae infections disrupt host cell cycle regulation machinery and the infection causes DNA double strand breaks that delay progression through the G2/M phase. We show that intracellular gonococci upregulate and release restriction endonucleases that enter the nucleus and damage human chromosomal DNA. Bacterial lysates containing restriction endonucleases were able to fragment genomic DNA as detected by PFGE. Lysates were also microinjected into the cytoplasm of cells in interphase and after 20 h, DNA double strand breaks were identified by 53BP1 staining. In addition, by using live-cell microscopy and NHS-ester stained live gonococci we visualized the subcellular location of the bacteria upon mitosis. Infected cells show dysregulation of the spindle assembly checkpoint proteins MAD1 and MAD2, impaired and prolonged M-phase, nuclear swelling, micronuclei formation and chromosomal instability. These data highlight basic molecular functions of how gonococcal infections affect host cell cycle regulation, cause DNA double strand breaks and predispose cellular malignancies. PMID:25460012

Restriction endonucleases from invasive Neisseria gonorrhoeae cause double-strand breaks and distort mitosis in epithelial cells during infection.

PubMed

Weyler, Linda; Engelbrecht, Mattias; Mata Forsberg, Manuel; Brehwens, Karl; Vare, Daniel; Vielfort, Katarina; Wojcik, Andrzej; Aro, Helena

2014-01-01

The host epithelium is both a barrier against, and the target for microbial infections. Maintaining regulated cell growth ensures an intact protective layer towards microbial-induced cellular damage. Neisseria gonorrhoeae infections disrupt host cell cycle regulation machinery and the infection causes DNA double strand breaks that delay progression through the G2/M phase. We show that intracellular gonococci upregulate and release restriction endonucleases that enter the nucleus and damage human chromosomal DNA. Bacterial lysates containing restriction endonucleases were able to fragment genomic DNA as detected by PFGE. Lysates were also microinjected into the cytoplasm of cells in interphase and after 20 h, DNA double strand breaks were identified by 53BP1 staining. In addition, by using live-cell microscopy and NHS-ester stained live gonococci we visualized the subcellular location of the bacteria upon mitosis. Infected cells show dysregulation of the spindle assembly checkpoint proteins MAD1 and MAD2, impaired and prolonged M-phase, nuclear swelling, micronuclei formation and chromosomal instability. These data highlight basic molecular functions of how gonococcal infections affect host cell cycle regulation, cause DNA double strand breaks and predispose cellular malignancies.

Modeling Space Radiation with Radiomimetic Agent Bleomycin

NASA Technical Reports Server (NTRS)

Lu, Tao

2017-01-01

Space radiation consists of proton and helium from solar particle events (SPE) and high energy heavy ions from galactic cosmic ray (GCR). This mixture of radiation with particles at different energy levels has different effects on biological systems. Currently, majority studies of radiation effects on human were based on single-source radiation due to the limitation of available method to model effects of space radiation on living organisms. While NASA Space Radiation Laboratory is working on advanced switches to make it possible to have a mixed field radiation with particles of different energies, the radiation source will be limited. Development of an easily available experimental model for studying effects of mixed field radiation could greatly speed up our progress in our understanding the molecular mechanisms of damage and responses from exposure to space radiation, and facilitate the discovery of protection and countermeasures against space radiation, which is critical for the mission to Mars. Bleomycin, a radiomimetic agent, has been widely used to study radiation induced DNA damage and cellular responses. Previously, bleomycin was often compared to low low Linear Energy Transfer (LET) gamma radiation without defined characteristics. Our recent work demonstrated that bleomycin could induce complex clustered DNA damage in human fibroblasts that is similar to DNA damage induced by high LET radiation. These type of DNA damage is difficult to repair and can be visualized by gamma-H2Ax staining weeks after the initial insult. The survival ratio between early and late plating of human fibroblasts after bleomycin treatment is between low LET and high LET radiation. Our results suggest that bleomycin induces DNA damage and other cellular stresses resembling those resulted from mixed field radiation with both low and high LET particles. We hypothesize that bleomycin could be used to mimic space radiation in biological systems. Potential advantages and limitations of using bleomycin to treat biological specimen as an easily available model to study effects of space radiation on biological systems and to develop countermeasures for space radiation associated risks will be discussed.

Activation of WIP1 Phosphatase by HTLV-1 Tax Mitigates the Cellular Response to DNA Damage

PubMed Central

Dayaram, Tajhal; Lemoine, Francene J.; Donehower, Lawrence A.; Marriott, Susan J.

2013-01-01

Genomic instability stemming from dysregulation of cell cycle checkpoints and DNA damage response (DDR) is a common feature of many cancers. The cancer adult T cell leukemia (ATL) can occur in individuals infected with human T cell leukemia virus type 1 (HTLV-1), and ATL cells contain extensive chromosomal abnormalities, suggesting that they have defects in the recognition or repair of DNA damage. Since Tax is the transforming protein encoded by HTLV-1, we asked whether Tax can affect cell cycle checkpoints and the DDR. Using a combination of flow cytometry and DNA repair assays we showed that Tax-expressing cells exit G1 phase and initiate DNA replication prematurely following damage. Reduced phosphorylation of H2AX (γH2AX) and RPA2, phosphoproteins that are essential to properly initiate the DDR, was also observed in Tax-expressing cells. To determine the cause of decreased DDR protein phosphorylation in Tax-expressing cells, we examined the cellular phosphatase, WIP1, which is known to dephosphorylate γH2AX. We found that Tax can interact with Wip1 in vivo and in vitro, and that Tax-expressing cells display elevated levels of Wip1 mRNA. In vitro phosphatase assays showed that Tax can enhance Wip1 activity on a γH2AX peptide target by 2-fold. Thus, loss of γH2AX in vivo could be due, in part, to increased expression and activity of WIP1 in the presence of Tax. siRNA knockdown of WIP1 in Tax-expressing cells rescued γH2AX in response to damage, confirming the role of WIP1 in the DDR. These studies demonstrate that Tax can disengage the G1/S checkpoint by enhancing WIP1 activity, resulting in reduced DDR. Premature G1 exit of Tax-expressing cells in the presence of DNA lesions creates an environment that tolerates incorporation of random mutations into the host genome. PMID:23405243

Nicotinamide Suppresses the DNA Damage Sensitivity of Saccharomyces cerevisiae Independently of Sirtuin Deacetylases.

PubMed

Rössl, Anthony; Bentley-DeSousa, Amanda; Tseng, Yi-Chieh; Nwosu, Christine; Downey, Michael

2016-10-01

Nicotinamide is both a reaction product and an inhibitor of the conserved sirtuin family of deacetylases, which have been implicated in a broad range of cellular functions in eukaryotes from yeast to humans. Phenotypes observed following treatment with nicotinamide are most often assumed to stem from inhibition of one or more of these enzymes. Here, we used this small molecule to inhibit multiple sirtuins at once during treatment with DNA damaging agents in the Saccharomyces cerevisiae model system. Since sirtuins have been previously implicated in the DNA damage response, we were surprised to observe that nicotinamide actually increased the survival of yeast cells exposed to the DNA damage agent MMS. Remarkably, we found that enhanced resistance to MMS in the presence of nicotinamide was independent of all five yeast sirtuins. Enhanced resistance was also independent of the nicotinamide salvage pathway, which uses nicotinamide as a substrate to generate NAD+, and of a DNA damage-induced increase in the salvage enzyme Pnc1 Our data suggest a novel and unexpected function for nicotinamide that has broad implications for its use in the study of sirtuin biology across model systems. Copyright © 2016 by the Genetics Society of America.

Nicotinamide Suppresses the DNA Damage Sensitivity of Saccharomyces cerevisiae Independently of Sirtuin Deacetylases

PubMed Central

Rössl, Anthony; Bentley-DeSousa, Amanda; Tseng, Yi-Chieh; Nwosu, Christine; Downey, Michael

2016-01-01

Nicotinamide is both a reaction product and an inhibitor of the conserved sirtuin family of deacetylases, which have been implicated in a broad range of cellular functions in eukaryotes from yeast to humans. Phenotypes observed following treatment with nicotinamide are most often assumed to stem from inhibition of one or more of these enzymes. Here, we used this small molecule to inhibit multiple sirtuins at once during treatment with DNA damaging agents in the Saccharomyces cerevisiae model system. Since sirtuins have been previously implicated in the DNA damage response, we were surprised to observe that nicotinamide actually increased the survival of yeast cells exposed to the DNA damage agent MMS. Remarkably, we found that enhanced resistance to MMS in the presence of nicotinamide was independent of all five yeast sirtuins. Enhanced resistance was also independent of the nicotinamide salvage pathway, which uses nicotinamide as a substrate to generate NAD+, and of a DNA damage-induced increase in the salvage enzyme Pnc1. Our data suggest a novel and unexpected function for nicotinamide that has broad implications for its use in the study of sirtuin biology across model systems. PMID:27527516

Red light improves spermatozoa motility and does not induce oxidative DNA damage

NASA Astrophysics Data System (ADS)

Preece, Daryl; Chow, Kay W.; Gomez-Godinez, Veronica; Gustafson, Kyle; Esener, Selin; Ravida, Nicole; Durrant, Barbara; Berns, Michael W.

2017-04-01

The ability to successfully fertilize ova relies upon the swimming ability of spermatozoa. Both in humans and in animals, sperm motility has been used as a metric for the viability of semen samples. Recently, several studies have examined the efficacy of low dosage red light exposure for cellular repair and increasing sperm motility. Of prime importance to the practical application of this technique is the absence of DNA damage caused by radiation exposure. In this study, we examine the effect of 633 nm coherent, red laser light on sperm motility using a novel wavelet-based algorithm that allows for direct measurement of curvilinear velocity under red light illumination. This new algorithm gives results comparable to the standard computer-assisted sperm analysis (CASA) system. We then assess the safety of red light treatment of sperm by analyzing, (1) the levels of double-strand breaks in the DNA, and (2) oxidative damage in the sperm DNA. The results demonstrate that for the parameters used there are insignificant differences in oxidative DNA damage as a result of irradiation.

Role of Mitochondrial Oxidative Stress in Spaceflight-Induced Tissue Degeneration

NASA Technical Reports Server (NTRS)

Torres, Samantha M.; Schreurs, Ann-Sofie; Truong, Tiffany A.; Tahimic, Candice; Globus, Ruth

2017-01-01

Microgravity and ionizing radiation in the spaceflight environment poses multiple challenges to homeostasis and may contribute to cellular stress. Effects may include increased generation of reactive oxygen species (ROS), DNA damage and repair error, cell cycle arrest, cell senescence or death. Our central hypothesis is that prolonged exposure to the spaceflight environment leads to the excess production of ROS and oxidative damage, culminating in accelerated tissue degeneration. The main goal of this project is to determine the importance of cellular redox defense for physiological adaptations and tissue degeneration in the space environment.

NEK8 regulates DNA damage-induced RAD51 foci formation and replication fork protection

PubMed Central

Abeyta, Antonio; Castella, Maria; Jacquemont, Celine; Taniguchi, Toshiyasu

2017-01-01

ABSTRACT Proteins essential for homologous recombination play a pivotal role in the repair of DNA double strand breaks, DNA inter-strand crosslinks and replication fork stability. Defects in homologous recombination also play a critical role in the development of cancer and the sensitivity of these cancers to chemotherapy. RAD51, an essential factor for homologous recombination and replication fork protection, accumulates and forms immunocytochemically detectable nuclear foci at sites of DNA damage. To identify kinases that may regulate RAD51 localization to sites of DNA damage, we performed a human kinome siRNA library screen, using DNA damage-induced RAD51 foci formation as readout. We found that NEK8, a NIMA family kinase member, is required for efficient DNA damage-induced RAD51 foci formation. Interestingly, knockout of Nek8 in murine embryonic fibroblasts led to cellular sensitivity to the replication inhibitor, hydroxyurea, and inhibition of the ATR kinase. Furthermore, NEK8 was required for proper replication fork protection following replication stall with hydroxyurea. Loading of RAD51 to chromatin was decreased in NEK8-depleted cells and Nek8-knockout cells. Single-molecule DNA fiber analyses revealed that nascent DNA tracts were degraded in the absence of NEK8 following treatment with hydroxyurea. Consistent with this, Nek8-knockout cells showed increased chromosome breaks following treatment with hydroxyurea. Thus, NEK8 plays a critical role in replication fork stability through its regulation of the DNA repair and replication fork protection protein RAD51. PMID:27892797

NEK8 regulates DNA damage-induced RAD51 foci formation and replication fork protection.

PubMed

Abeyta, Antonio; Castella, Maria; Jacquemont, Celine; Taniguchi, Toshiyasu

2017-02-16

Proteins essential for homologous recombination play a pivotal role in the repair of DNA double strand breaks, DNA inter-strand crosslinks and replication fork stability. Defects in homologous recombination also play a critical role in the development of cancer and the sensitivity of these cancers to chemotherapy. RAD51, an essential factor for homologous recombination and replication fork protection, accumulates and forms immunocytochemically detectable nuclear foci at sites of DNA damage. To identify kinases that may regulate RAD51 localization to sites of DNA damage, we performed a human kinome siRNA library screen, using DNA damage-induced RAD51 foci formation as readout. We found that NEK8, a NIMA family kinase member, is required for efficient DNA damage-induced RAD51 foci formation. Interestingly, knockout of Nek8 in murine embryonic fibroblasts led to cellular sensitivity to the replication inhibitor, hydroxyurea, and inhibition of the ATR kinase. Furthermore, NEK8 was required for proper replication fork protection following replication stall with hydroxyurea. Loading of RAD51 to chromatin was decreased in NEK8-depleted cells and Nek8-knockout cells. Single-molecule DNA fiber analyses revealed that nascent DNA tracts were degraded in the absence of NEK8 following treatment with hydroxyurea. Consistent with this, Nek8-knockout cells showed increased chromosome breaks following treatment with hydroxyurea. Thus, NEK8 plays a critical role in replication fork stability through its regulation of the DNA repair and replication fork protection protein RAD51.

Protoparvovirus Interactions with the Cellular DNA Damage Response

PubMed Central

Majumder, Kinjal; Etingov, Igor

2017-01-01

Protoparvoviruses are simple single-stranded DNA viruses that infect many animal species. The protoparvovirus minute virus of mice (MVM) infects murine and transformed human cells provoking a sustained DNA damage response (DDR). This DDR is dependent on signaling by the ATM kinase and leads to a prolonged pre-mitotic cell cycle block that features the inactivation of ATR-kinase mediated signaling, proteasome-targeted degradation of p21, and inhibition of cyclin B1 expression. This review explores how protoparvoviruses, and specifically MVM, co-opt the common mechanisms regulating the DDR and cell cycle progression in order to prepare the host nuclear environment for productive infection. PMID:29088070

Protoparvovirus Interactions with the Cellular DNA Damage Response.

PubMed

Majumder, Kinjal; Etingov, Igor; Pintel, David J

2017-10-31

Protoparvoviruses are simple single-stranded DNA viruses that infect many animal species. The protoparvovirus minute virus of mice (MVM) infects murine and transformed human cells provoking a sustained DNA damage response (DDR). This DDR is dependent on signaling by the ATM kinase and leads to a prolonged pre-mitotic cell cycle block that features the inactivation of ATR-kinase mediated signaling, proteasome-targeted degradation of p21, and inhibition of cyclin B1 expression. This review explores how protoparvoviruses, and specifically MVM, co-opt the common mechanisms regulating the DDR and cell cycle progression in order to prepare the host nuclear environment for productive infection.

Impact of Air Pollutants on Oxidative Stress in Common Autophagy-Mediated Aging Diseases

PubMed Central

Numan, Mohamed Saber; Brown, Jacques P.; Michou, Laëtitia

2015-01-01

Atmospheric pollution-induced cellular oxidative stress is probably one of the pathogenic mechanisms involved in most of the common autophagy-mediated aging diseases, including neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS), Alzheimer’s, disease, as well as Paget’s disease of bone with or without frontotemporal dementia and inclusion body myopathy. Oxidative stress has serious damaging effects on the cellular contents: DNA, RNA, cellular proteins, and cellular organelles. Autophagy has a pivotal role in recycling these damaged non-functional organelles and misfolded or unfolded proteins. In this paper, we highlight, through a narrative review of the literature, that when autophagy processes are impaired during aging, in presence of cumulative air pollution-induced cellular oxidative stress and due to a direct effect on air pollutant, autophagy-mediated aging diseases may occur. PMID:25690002

[Pulse-modulated Electromagnetic Radiation of Extremely High Frequencies Protects Cellular DNA against Damaging Effect of Physico-Chemical Factors in vitro].

PubMed

Gapeyev, A B; Lukyanova, N A

2015-01-01

Using a comet assay technique, we investigated protective effects of. extremely high frequency electromagnetic radiation in combination with the damaging effect of X-ray irradiation, the effect of damaging agents hydrogen peroxide and methyl methanesulfonate on DNA in mouse whole blood leukocytes. It was shown that the preliminary exposure of the cells to low intensity pulse-modulated electromagnetic radiation (42.2 GHz, 0.1 mW/cm2, 20-min exposure, modulation frequencies of 1 and 16 Hz) caused protective effects decreasing the DNA damage by 20-45%. The efficacy of pulse-modulated electromagnetic radiation depended on the type of genotoxic agent and increased in a row methyl methanesulfonate--X-rays--hydrogen peroxide. Continuous electromagnetic radiation was ineffective. The mechanisms of protective effects may be connected with an induction of the adaptive response by nanomolar concentrations of reactive oxygen species formed by pulse-modulated electromagnetic radiation.

Tumor Suppressor p53 Stimulates the Expression of Epstein-Barr Virus Latent Membrane Protein 1.

PubMed

Wang, Qianli; Lingel, Amy; Geiser, Vicki; Kwapnoski, Zachary; Zhang, Luwen

2017-10-15

Epstein-Barr virus (EBV) is associated with multiple human malignancies. EBV latent membrane protein 1 (LMP1) is required for the efficient transformation of primary B lymphocytes in vitro and possibly in vivo The tumor suppressor p53 plays a seminal role in cancer development. In some EBV-associated cancers, p53 tends to be wild type and overly expressed; however, the effects of p53 on LMP1 expression is not clear. We find LMP1 expression to be associated with p53 expression in EBV-transformed cells under physiological and DNA damaging conditions. DNA damage stimulates LMP1 expression, and p53 is required for the stimulation. Ectopic p53 stimulates endogenous LMP1 expression. Moreover, endogenous LMP1 blocks DNA damage-mediated apoptosis. Regarding the mechanism of p53-mediated LMP1 expression, we find that interferon regulatory factor 5 (IRF5), a direct target of p53, is associated with both p53 and LMP1. IRF5 binds to and activates a LMP1 promoter reporter construct. Ectopic IRF5 increases the expression of LMP1, while knockdown of IRF5 leads to reduction of LMP1. Furthermore, LMP1 blocks IRF5-mediated apoptosis in EBV-infected cells. All of the data suggest that cellular p53 stimulates viral LMP1 expression, and IRF5 may be one of the factors for p53-mediated LMP1 stimulation. LMP1 may subsequently block DNA damage- and IRF5-mediated apoptosis for the benefits of EBV. The mutual regulation between p53 and LMP1 may play an important role in EBV infection and latency and its related cancers. IMPORTANCE The tumor suppressor p53 is a critical cellular protein in response to various stresses and dictates cells for various responses, including apoptosis. This work suggests that an Epstein-Bar virus (EBV) principal viral oncogene is activated by cellular p53. The viral oncogene blocks p53-mediated adverse effects during viral infection and transformation. Therefore, the induction of the viral oncogene by p53 provides a means for the virus to cope with infection and DNA damage-mediated cellular stresses. This seems to be the first report that p53 activates a viral oncogene; therefore, the discovery would be interesting to a broad readership from the fields of oncology to virology. Copyright © 2017 American Society for Microbiology.

Identification of ATM Protein Kinase Phosphorylation Sites by Mass Spectrometry.

PubMed

Graham, Mark E; Lavin, Martin F; Kozlov, Sergei V

2017-01-01

ATM (ataxia-telangiectasia mutated) protein kinase is a key regulator of cellular responses to DNA damage and oxidative stress. DNA damage triggers complex cascade of signaling events leading to numerous posttranslational modification on multitude of proteins. Understanding the regulation of ATM kinase is therefore critical not only for understanding the human genetic disorder ataxia-telangiectasia and potential treatment strategies, but essential for deciphering physiological responses of cells to stress. These responses play an important role in carcinogenesis, neurodegeneration, and aging. We focus here on the identification of DNA damage inducible ATM phosphorylation sites to understand the importance of autophosphorylation in the mechanism of ATM kinase activation. We demonstrate the utility of using immunoprecipitated ATM in quantitative LC-MS/MS workflow with stable isotope dimethyl labeling of ATM peptides for identification of phosphorylation sites.

What Combined Measurements From Structures and Imaging Tell Us About DNA Damage Responses

PubMed Central

Brosey, Chris A.; Ahmed, Zamal; Lees-Miller, Susan P.; Tainer, John A.

2017-01-01

DNA damage outcomes depend upon the efficiency and fidelity of DNA damage responses (DDRs) for different cells and damage. As such, DDRs represent tightly regulated prototypical systems for linking nanoscale biomolecular structure and assembly to the biology of genomic regulation and cell signaling. However, the dynamic and multifunctional nature of DDR assemblies can render elusive the correlation between the structures of DDR factors and specific biological disruptions to the DDR when these structures are altered. In this chapter, we discuss concepts and strategies for combining structural, biophysical, and imaging techniques to investigate DDR recognition and regulation, and thus bridge sequence-level structural biochemistry to quantitative biological outcomes visualized in cells. We focus on representative DDR responses from PARP/PARG/AIF damage signaling in DNA single-strand break repair and nonhomologous end joining complexes in double-strand break repair. Methods with exemplary experimental results are considered with a focus on strategies for probing flexibility, conformational changes, and assembly processes that shape a predictive understanding of DDR mechanisms in a cellular context. Integration of structural and imaging measurements promises to provide foundational knowledge to rationally control and optimize DNA damage outcomes for synthetic lethality and for immune activation with resulting insights for biology and cancer interventions. PMID:28668129

Base excision repair: NMR backbone assignments of Escherichia coli formamidopyrimidine-DNA glycosylase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Buchko, Garry W.; Wallace, Susan S.; Kennedy, Michael A.

2002-03-01

Oxidative damage is emerging as one of the most important mechanisms responsible for mutagenesis, carcinogenesis, aging, and various diseases (Farr and Kogma, 1991). One of the potential targets for oxidation is cellular DNA. While exposure to exogenous agents, such as ionizing radiation and chemicals, contributes to damaging DNA, the most important oxidative agents are endogenous, such as the reactive free radicals produced during normal oxidative metabolism (Adelman et., 1988). To mitigate the potentially deleterious effects of oxidative DNA damage virtually all aerobic organisms have developed complex repair mechanisms (Petit and Sancar, 1999). One repair mechanism, base excision repair (BER), appearsmore » to be responsible for replacing most oxidative DNA damage (David and Williams, 1998). Formamidopyrimidine-DNA glycosylase (Fpg), a 269-residue metalloprotein with a molecular weight of 30.2 kDa, is a key BER enzyme in prokaryotes (Boiteaux et al., 1987). Substrates recognized and released by Fpg include 7,8-dihydro-8-oxoguanine (8-oxoG), 2,6 diamino-4-hydroxy-5-formamido pyrimidine (Fapy-G), the adenine equivalents 8-oxoA and Fapy-A, 5-hydroxycytosine, 5-hydroxyuracil, B ureidoisobutiric acid, and a-R-hydroxy-B-ureidoisobutiric acid (Freidberg et al., 1995). In vitro Fpg bind double-stranded DNA and performs three catalytic activities: (i) DNA glycosylase, (ii) AP lyase, and (iii) deoxyribophosphodiesterase.« less

Shedding Light on the Role of UV Exposure in Melanoma | Center for Cancer Research

Cancer.gov

When a cell is exposed to UV radiation, the chemical makeup of its DNA is changed in a specific manner, resulting in a recognizable modification that can be measured by scientists. These changes are normally detected and fixed by cellular mechanisms for DNA repair. However, if the damage is extensive or if a cell has defective DNA repair machinery, permanent mutations can be

Parvovirus Minute Virus of Mice Induces a DNA Damage Response That Facilitates Viral Replication

PubMed Central

Adeyemi, Richard O.; Landry, Sebastien; Davis, Meredith E.; Weitzman, Matthew D.; Pintel, David J.

2010-01-01

Infection by DNA viruses can elicit DNA damage responses (DDRs) in host cells. In some cases the DDR presents a block to viral replication that must be overcome, and in other cases the infecting agent exploits the DDR to facilitate replication. We find that low multiplicity infection with the autonomous parvovirus minute virus of mice (MVM) results in the activation of a DDR, characterized by the phosphorylation of H2AX, Nbs1, RPA32, Chk2 and p53. These proteins are recruited to MVM replication centers, where they co-localize with the main viral replication protein, NS1. The response is seen in both human and murine cell lines following infection with either the MVMp or MVMi strains. Replication of the virus is required for DNA damage signaling. Damage response proteins, including the ATM kinase, accumulate in viral-induced replication centers. Using mutant cell lines and specific kinase inhibitors, we show that ATM is the main transducer of the signaling events in the normal murine host. ATM inhibitors restrict MVM replication and ameliorate virus-induced cell cycle arrest, suggesting that DNA damage signaling facilitates virus replication, perhaps in part by promoting cell cycle arrest. Thus it appears that MVM exploits the cellular DNA damage response machinery early in infection to enhance its replication in host cells. PMID:20949077

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

PubMed Central

Kodaira, Satoshi; Konishi, Teruaki; Kobayashi, Alisa; Maeda, Takeshi; Ahmad, Tengku Ahbrizal Farizal Tengku; Yang, Gen; Akselrod, Mark S.; Furusawa, Yoshiya; Uchihori, Yukio

2015-01-01

Abstract 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. PMID:25324538

Protein Phosphatase Pph3 and Its Regulatory Subunit Psy2 Regulate Rad53 Dephosphorylation and Cell Morphogenesis during Recovery from DNA Damage in Candida albicans ▿

PubMed Central

Sun, Ling Ling; Li, Wan Jie; Wang, Hai Tao; Chen, Jie; Deng, Ping; Wang, Yue; Sang, Jian Li

2011-01-01

The ability of the pathogenic fungus Candida albicans to switch cellular morphologies is important for infection and virulence. Recent studies have revealed that C. albicans yeast cells can switch to filamentous growth under genotoxic stress in a manner dependent on the DNA replication/damage checkpoint. Here, we have investigated the functions of Pph3 (orf19.4378) and Psy2 (orf19.3685), whose orthologues in Saccharomyces cerevisiae mediate the dephosphorylation of the DNA damage checkpoint kinase Rad53 and the histone variant H2AX during recovery from DNA damage. Deleting PPH3 or PSY2 causes hypersensitivity to DNA-damaging agents, including cisplatin, methylmethane sulfonate (MMS), and UV light. In addition, pph3Δ and psy2Δ cells exhibit strong filamentous growth under genotoxic stress. Flow cytometry analysis shows that the mutant cells have lost the ability to adapt to genotoxic stress and remain arrested even after the stress is withdrawn. Furthermore, we show that Pph3 and Psy2 are required for the dephosphorylation of Rad53, but not H2AX, during DNA damage recovery. Taken together, these results show that C. albicans Pph3 and Psy2 have important roles in mediating genotoxin-induced filamentous growth and regulating Rad53 dephosphorylation. PMID:21890819

Quantitative Proteomics Reveals Dynamic Interactions of the Minichromosome Maintenance Complex (MCM) in the Cellular Response to Etoposide Induced DNA Damage.

PubMed

Drissi, Romain; Dubois, Marie-Line; Douziech, Mélanie; Boisvert, François-Michel

2015-07-01

The minichromosome maintenance complex (MCM) proteins are required for processive DNA replication and are a target of S-phase checkpoints. The eukaryotic MCM complex consists of six proteins (MCM2-7) that form a heterohexameric ring with DNA helicase activity, which is loaded on chromatin to form the pre-replication complex. Upon entry in S phase, the helicase is activated and opens the DNA duplex to recruit DNA polymerases at the replication fork. The MCM complex thus plays a crucial role during DNA replication, but recent work suggests that MCM proteins could also be involved in DNA repair. Here, we employed a combination of stable isotope labeling with amino acids in cell culture (SILAC)-based quantitative proteomics with immunoprecipitation of green fluorescent protein-tagged fusion proteins to identify proteins interacting with the MCM complex, and quantify changes in interactions in response to DNA damage. Interestingly, the MCM complex showed very dynamic changes in interaction with proteins such as Importin7, the histone chaperone ASF1, and the Chromodomain helicase DNA binding protein 3 (CHD3) following DNA damage. These changes in interactions were accompanied by an increase in phosphorylation and ubiquitination on specific sites on the MCM proteins and an increase in the co-localization of the MCM complex with γ-H2AX, confirming the recruitment of these proteins to sites of DNA damage. In summary, our data indicate that the MCM proteins is involved in chromatin remodeling in response to DNA damage. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Biological Clues to Potent DNA-Damaging Activities in Food and Flavoring

PubMed Central

Hossain, M. Zulfiquer; Gilbert, Samuel F.; Patel, Kalpesh; Ghosh, Soma; Bhunia, Anil K.; Kern, Scott E.

2013-01-01

Population differences in age-related diseases and cancer could stem from differences in diet. To characterize DNA strand-breaking activities in selected foods/beverages, flavorings, and some of their constituent chemicals, we used p53R cells, a cellular assay sensitive to such breaks. Substances testing positive included reference chemicals: quinacrine (peak response, 51X) and etoposide (33X); flavonoids: EGCG (19X), curcumin (12X), apigenin (9X), and quercetin (7X); beverages: chamomile (11X), green (21X), and black tea (26X) and coffee (3 to 29X); and liquid smoke (4 to 28X). Damage occurred at dietary concentrations: etoposide near 5 μg/ml produced responses similar to a 1:1000 dilution of liquid smoke, a 1:20 dilution of coffee, and a 1:5 dilution of tea. Pyrogallol-related chemicals and tannins are present in dietary sources and individually produced strong activity: pyrogallol (30X), 3-methoxycatechol (25X), gallic acid (21X), and 1,2,4-benzenetriol (21X). From structure-activity relationships, high activities depended on specific orientations of hydroxyls on the benzene ring. Responses accompanied cellular signals characteristic of DNA breaks such as H2AX phosphorylation. Breaks were also directly detected by comet assay. Cellular toxicological effects of foods and flavorings could guide epidemiologic and experimental studies of potential disease risks from DNA strand-breaking chemicals in diets. PMID:23402862

Formation and processing of DNA damage substrates for the hNEIL enzymes.

PubMed

Fleming, Aaron M; Burrows, Cynthia J

2017-06-01

Reactive oxygen species (ROS) are harnessed by the cell for signaling at the same time as being detrimental to cellular components such as DNA. The genome and transcriptome contain instructions that can alter cellular processes when oxidized. The guanine (G) heterocycle in the nucleotide pool, DNA, or RNA is the base most prone to oxidation. The oxidatively-derived products of G consistently observed in high yields from hydroxyl radical, carbonate radical, or singlet oxygen oxidations under conditions modeling the cellular reducing environment are discussed. The major G base oxidation products are 8-oxo-7,8-dihydroguanine (OG), 5-carboxamido-5-formamido-2-iminohydantoin (2Ih), spiroiminodihydantoin (Sp), and 5-guanidinohydantoin (Gh). The yields of these products show dependency on the oxidant and the reaction context that includes nucleoside, single-stranded DNA (ssDNA), double-stranded DNA (dsDNA), and G-quadruplex DNA (G4-DNA) structures. Upon formation of these products in cells, they are recognized by the DNA glycosylases in the base excision repair (BER) pathway. This review focuses on initiation of BER by the mammalian Nei-like1-3 (NEIL1-3) glycosylases for removal of 2Ih, Sp, and Gh. The unique ability of the human NEILs to initiate removal of the hydantoins in ssDNA, bulge-DNA, bubble-DNA, dsDNA, and G4-DNA is outlined. Additionally, when Gh exists in a G4 DNA found in a gene promoter, NEIL-mediated repair is modulated by the plasticity of the G4-DNA structure provided by additional G-runs flanking the sequence. On the basis of these observations and cellular studies from the literature, the interplay between DNA oxidation and BER to alter gene expression is discussed. Copyright © 2017 Elsevier Inc. All rights reserved.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Park, J.F.

Research in the biomedical sciences at PNL is described. Activities reported include: inhaled plutonium in dogs; national radiobiology archives; statistical analysis of data from animal studies; genotoxicity of inhaled energy effluents; molecular events during tumor initiation; biochemistry of free radical induced DNA damage; radon hazards in homes; mechanisms of radon injury; genetics of radon induced lung cancer; and in vivo/in vitro radon induced cellular damage.

2016 Arte Poster Competition First Place Winner: Circadian Rhythm and UV-Induced Skin Damage: An In Vivo Study.

PubMed

Guan, Linna; Suggs, Amanda; Ahsanuddin, Sayeeda; Tarrillion, Madeline; Selph, Jacqueline; Lam, Minh; Baron, Elma

2016-09-01

Exposure of the skin to ultraviolet (UV) irradiation causes many detrimental effects through mechanisms related to oxidative stress and DNA damage. Excessive oxidative stress can cause apoptosis and cellular dysfunction of epidermal cells leading to cellular senescence and connective tissue degradation. Direct and indirect damage to DNA predisposes the skin to cancer formation. Chronic UV exposure also leads to skin aging manifested as wrinkling, loss of skin tone, and decreased resilience. Fortunately, human skin has several natural mechanisms for combating UV-induced damage. The mechanisms operate on a diurnal rhythm, a cycle that repeats approximately every 24 hours. It is known that the circadian rhythm is involved in many skin physiologic processes, including water regulation and epidermal stem cell function. This study evaluated whether UV damage and the skin's natural mechanisms of inflammation and repair are also affected by circadian rhythm. We looked at UV-induced erythema on seven human subjects irradiated with simulated solar radiation in the morning (at 08:00 h) versus in the afternoon (at 16:00 h). Our data suggest that the same dose of UV radiation induces significantly more inflammation in the morning than in the afternoon. Changes in protein expression relevant to DNA damage, such as xeroderma pigmentosum, complementation group A (XPA), and cyclobutane pyrimidine dimers (CPD) from skin biopsies correlated with our clinical results. Both XPA and CPD levels were higher after the morning UV exposure compared with the afternoon exposure.

J Drugs Dermatol. 2016;15(9):1124-1130.

Plant Flavone Apigenin Binds to Nucleic Acid Bases and Reduces Oxidative DNA Damage in Prostate Epithelial Cells

PubMed Central

Bhaskaran, Natarajan; Gupta, Sanjay

2014-01-01

Oxidative stress has been linked to prostate carcinogenesis as human prostate tissue is vulnerable to oxidative DNA damage. Apigenin, a dietary plant flavone, possesses anti-proliferative and anticancer effects; however, its antioxidant properties have not been fully elucidated. We investigated sub-cellular distribution of apigenin, it’s binding to DNA and protective effects against H2O2-induced DNA damage using transformed human prostate epithelial RWPE-1 cells and prostate cancer LNCaP, PC-3 and DU145 cells. Exposure of cells to apigenin exhibited higher accumulation in RWPE-1 and LNCaP cells, compared to PC-3 and DU145 cells. The kinetics of apigenin uptake in LNCaP cells was estimated with a Km value of 5 µmole/L and Vmax of 190 pmoles/million cells/h. Sub-cellular fractionation demonstrated that nuclear matrix retains the highest concentration of apigenin (45.3%), followed by cytosol (23.9%), nuclear membranes (17.9%) and microsomes (12.9%), respectively. Spectroscopic analysis of apigenin with calf-thymus DNA exhibited intercalation as the dominant binding mode to DNA duplex. Apigenin exposure resulted in significant genoprotective effects in H2O2-stressed RWPE-1 cells by reduction in reactive oxygen species levels. In addition, apigenin exposure suppressed the formation of 8-hydroxy-2′ deoxyguanosine and protected exposed cells from apoptosis. Our studies demonstrate that apigenin is readily taken up by normal prostatic epithelial cells and prostate cancer cells, and is incorporated into their nuclei, where its intercalation with nucleic acid bases may account for its antioxidant and chemopreventive activities. PMID:24614817

Plant flavone apigenin binds to nucleic acid bases and reduces oxidative DNA damage in prostate epithelial cells.

PubMed

Sharma, Haripaul; Kanwal, Rajnee; Bhaskaran, Natarajan; Gupta, Sanjay

2014-01-01

Oxidative stress has been linked to prostate carcinogenesis as human prostate tissue is vulnerable to oxidative DNA damage. Apigenin, a dietary plant flavone, possesses anti-proliferative and anticancer effects; however, its antioxidant properties have not been fully elucidated. We investigated sub-cellular distribution of apigenin, it's binding to DNA and protective effects against H2O2-induced DNA damage using transformed human prostate epithelial RWPE-1 cells and prostate cancer LNCaP, PC-3 and DU145 cells. Exposure of cells to apigenin exhibited higher accumulation in RWPE-1 and LNCaP cells, compared to PC-3 and DU145 cells. The kinetics of apigenin uptake in LNCaP cells was estimated with a Km value of 5 µmole/L and Vmax of 190 pmoles/million cells/h. Sub-cellular fractionation demonstrated that nuclear matrix retains the highest concentration of apigenin (45.3%), followed by cytosol (23.9%), nuclear membranes (17.9%) and microsomes (12.9%), respectively. Spectroscopic analysis of apigenin with calf-thymus DNA exhibited intercalation as the dominant binding mode to DNA duplex. Apigenin exposure resulted in significant genoprotective effects in H2O2-stressed RWPE-1 cells by reduction in reactive oxygen species levels. In addition, apigenin exposure suppressed the formation of 8-hydroxy-2' deoxyguanosine and protected exposed cells from apoptosis. Our studies demonstrate that apigenin is readily taken up by normal prostatic epithelial cells and prostate cancer cells, and is incorporated into their nuclei, where its intercalation with nucleic acid bases may account for its antioxidant and chemopreventive activities.

Cellular metabolism and oxidative stress as a possible determinant for longevity in small breed and large breed dogs

PubMed Central

Winward, Josh; Beattie, Ursula; Cipolli, William

2018-01-01

Among species, larger animals tend to live longer than smaller ones, however, the opposite seems to be true for dogs—smaller dogs tend to live significantly longer than larger dogs across all breeds. We were interested in the mechanism that may allow for small breeds to age more slowly compared with large breeds in the context of cellular metabolism and oxidative stress. Primary dermal fibroblasts from small and large breed dogs were grown in culture. We measured basal oxygen consumption (OCR), proton leak, and glycolysis using a Seahorse XF96 oxygen flux analyzer. Additionally, we measured rates of reactive species (RS) production, reduced glutathione (GSH) content, mitochondrial content, lipid peroxidation (LPO) damage and DNA (8-OHdg) damage. Our data suggests that as dogs of both size classes age, proton leak is significantly higher in older dogs, regardless of size class. We found that all aspects of glycolysis were significantly higher in larger breeds compared with smaller breeds. We found significant differences between age classes in GSH concentration, and a negative correlation between DNA damage in puppies and mean breed lifespan. Interestingly, RS production showed no differences across size and age class. Thus, large breed dogs may have higher glycolytic rates, and DNA damage, suggesting a potential mechanism for their decreased lifespan compared with small breed dogs. PMID:29694441

Distinct mechanisms act in concert to mediate cell cycle arrest.

PubMed

Toettcher, Jared E; Loewer, Alexander; Ostheimer, Gerard J; Yaffe, Michael B; Tidor, Bruce; Lahav, Galit

2009-01-20

In response to DNA damage, cells arrest at specific stages in the cell cycle. This arrest must fulfill at least 3 requirements: it must be activated promptly; it must be sustained as long as damage is present to prevent loss of genomic information; and after the arrest, cells must re-enter into the appropriate cell cycle phase to ensure proper ploidy. Multiple molecular mechanisms capable of arresting the cell cycle have been identified in mammalian cells; however, it is unknown whether each mechanism meets all 3 requirements or whether they act together to confer specific functions to the arrest. To address this question, we integrated mathematical models describing the cell cycle and the DNA damage signaling networks and tested the contributions of each mechanism to cell cycle arrest and re-entry. Predictions from this model were then tested with quantitative experiments to identify the combined action of arrest mechanisms in irradiated cells. We find that different arrest mechanisms serve indispensable roles in the proper cellular response to DNA damage over time: p53-independent cyclin inactivation confers immediate arrest, whereas p53-dependent cyclin downregulation allows this arrest to be sustained. Additionally, p21-mediated inhibition of cyclin-dependent kinase activity is indispensable for preventing improper cell cycle re-entry and endoreduplication. This work shows that in a complex signaling network, seemingly redundant mechanisms, acting in a concerted fashion, can achieve a specific cellular outcome.

Inhibition of Transforming Growth Factor-Beta1 SignalingAttenuates Ataxia Telangiectasia Mutated Activity in Response toGenotoxic Stress

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kirshner, Julia; Jobling, Michael F.; Pajares, Maria Jose

2006-01-01

Ionizing radiation causes DNA damage that elicits a cellular program of damage control coordinated by the kinase activity of ataxia telangiectasia mutated protein (ATM). Transforming growth factor {beta} (TGF{beta})-1, which is activated by radiation, is a potent and pleiotropic mediator of physiologic and pathologic processes. Here we show that TGF{beta} inhibition impedes the canonical cellular DNA damage stress response. Irradiated Tgf{beta}I null murine epithelial cells or human epithelial cells treated with a small-molecule inhibitor of TGF{beta} type I receptor kinase exhibit decreased phosphorylation of Chk2, Rad17, and p53; reduced H2AX radiation-induced foci; and increased radiosensitivity compared with TGF{beta} competent cells.more » We determined that loss of TGF{beta} signaling in epithelial cells truncated ATM autophosphorylation and significantly reduced its kinase activity, without affecting protein abundance. Addition of TGF{beta} restored functional ATM and downstream DNA damage responses. These data reveal a heretofore undetected critical link between the microenvironment and ATM, which directs epithelial cell stress responses, cell fate, and tissue integrity. Thus, Tgf{beta}I, in addition to its role in homoeostatic growth control, plays a complex role in regulating responses to genotoxic stress, the failure of which would contribute to the development of cancer; conversely, inhibiting TGF{beta} may be used to advantage in cancer therapy.« less

Cytotoxicity, genotoxicity, and metal release in patients with fixed orthodontic appliances: a longitudinal in-vivo study.

PubMed

Hafez, Hend Salah; Selim, Essam Mohamed Nassef; Kamel Eid, Faten Hussein; Tawfik, Wael Attia; Al-Ashkar, Emad A; Mostafa, Yehya Ahmed

2011-09-01

Treatment with fixed orthodontic appliances in the corrosive environment of the oral cavity warrants in-vivo investigations of biocompatibility. Eighteen control and 28 treated subjects were evaluated longitudinally. Four combinations of brackets and archwires were tested. Buccal mucosa cell samples were collected before treatment, and 3 and 6 months after appliance placement. The cells were processed for cytotoxicity, genotoxicity, and nickel and chromium contents. In the treatment group, buccal mucosa cell viability values were 8.1% at pretreatment, and 6.4% and 4.5% at 3 and 6 months, respectively. The composite score, a calculated DNA damage value, decreased from 125.6 to 98.8 at 6 months. Nickel cellular content increased from 0.52 to 0.68 and 0.78 ng per milliliter, and chromium increased from 0.31 to 0.41 and 0.78 ng per milliliter at 3 and 6 months, respectively. Compared with the control group, the treated subjects showed significant differences for DNA damage and chromium content at 3 months only. Fixed orthodontic appliances decreased cellular viability, induced DNA damage, and increased the nickel and chromium contents of the buccal mucosa cells. Compared to the control group, these changes were not evident at 6 months, possibly indicating tolerance for or repair of the cells and the DNA. Copyright © 2011 American Association of Orthodontists. Published by Mosby, Inc. All rights reserved.

Effect of ionizing radiation on the physical biology of head and neck squamous cell carcinoma cells.

PubMed

Baker-Groberg, Sandra M; Bornstein, Sophia; Zilberman-Rudenko, Jevgenia; Schmidt, Mark; Tormoen, Garth W; Kernan, Casey; Thomas, Charles R; Wong, Melissa H; Phillips, Kevin G; McCarty, Owen J T

2015-09-01

Head and neck squamous cell carcinoma (HNSCC) is the sixth leading cause of cancer worldwide. Although there are numerous treatment options for HNSCC, such as surgery, cytotoxic chemotherapy, molecularly targeted systemic therapeutics, and radiotherapy, overall survival has not significantly improved in the last 50 years. This suggests a need for a better understanding of how these cancer cells respond to current treatments in order to improve treatment paradigms. Ionizing radiation (IR) promotes cancer cell death through the creation of cytotoxic DNA lesions, including single strand breaks, base damage, crosslinks, and double strand breaks (DSBs). As unrepaired DSBs are the most cytotoxic DNA lesion, defining the downstream cellular responses to DSBs are critical for understanding the mechanisms of tumor cell responses to IR. The effects of experimental IR on HNSCC cells beyond DNA damage in vitro are ill-defined. Here we combined label-free, quantitative phase and fluorescent microscopy to define the effects of IR on the dry mass and volume of the HNSCC cell line, UM-SCC-22A. We quantified nuclear and cytoplasmic subcellular density alterations resulting from 8 Gy X-ray IR and correlated these signatures with DNA and γ-H2AX expression patterns. This study utilizes a synergistic imaging approach to study both biophysical and biochemical alterations in cells following radiation damage and will aid in future understanding of cellular responses to radiation therapy.

Quantitative and Dynamic Imaging of ATM Kinase Activity by Bioluminescence Imaging.

PubMed

Nyati, Shyam; Young, Grant; Ross, Brian Dale; Rehemtulla, Alnawaz

2017-01-01

Ataxia telangiectasia mutated (ATM) is a serine/threonine kinase critical to the cellular DNA damage response, including DNA double strand breaks (DSBs). ATM activation results in the initiation of a complex cascade of events facilitating DNA damage repair, cell cycle checkpoint control, and survival. Traditionally, protein kinases have been analyzed in vitro using biochemical methods (kinase assays using purified proteins or immunological assays) requiring a large number of cells and cell lysis. Genetically encoded biosensors based on optical molecular imaging such as fluorescence or bioluminescence have been developed to enable interrogation of kinase activities in live cells with a high signal to background. We have genetically engineered a hybrid protein whose bioluminescent activity is dependent on the ATM-mediated phosphorylation of a substrate. The engineered protein consists of the split luciferase-based protein complementation pair with a CHK2 (a substrate for ATM kinase activity) target sequence and a phospho-serine/threonine-binding domain, FHA2, derived from yeast Rad53. Phosphorylation of the serine residue within the target sequence by ATM would lead to its interaction with the phospho-serine-binding domain, thereby preventing complementation of the split luciferase pair and loss of reporter activity. Bioluminescence imaging of reporter-expressing cells in cultured plates or as mouse xenografts provides a quantitative surrogate for ATM kinase activity and therefore the cellular DNA damage response in a noninvasive, dynamic fashion.

Quantitative and Dynamic Imaging of ATM Kinase Activity.

PubMed

Nyati, Shyam; Young, Grant; Ross, Brian Dale; Rehemtulla, Alnawaz

2017-01-01

Ataxia telangiectasia mutated (ATM) is a serine/threonine kinase critical to the cellular DNA-damage response, including DNA double-strand breaks (DSBs). ATM activation results in the initiation of a complex cascade of events facilitating DNA damage repair, cell cycle checkpoint control, and survival. Traditionally, protein kinases have been analyzed in vitro using biochemical methods (kinase assays using purified proteins or immunological assays) requiring a large number of cells and cell lysis. Genetically encoded biosensors based on optical molecular imaging such as fluorescence or bioluminescence have been developed to enable interrogation of kinase activities in live cells with a high signal to background. We have genetically engineered a hybrid protein whose bioluminescent activity is dependent on the ATM-mediated phosphorylation of a substrate. The engineered protein consists of the split luciferase-based protein complementation pair with a CHK2 (a substrate for ATM kinase activity) target sequence and a phospho-serine/threonine-binding domain, FHA2, derived from yeast Rad53. Phosphorylation of the serine residue within the target sequence by ATM would lead to its interaction with the phospho-serine-binding domain, thereby preventing complementation of the split luciferase pair and loss of reporter activity. Bioluminescence imaging of reporter expressing cells in cultured plates or as mouse xenografts provides a quantitative surrogate for ATM kinase activity and therefore the cellular DNA damage response in a noninvasive, dynamic fashion.

Menadione-Induced DNA Damage Leads to Mitochondrial Dysfunction and Fragmentation During Rosette Formation in Fuchs Endothelial Corneal Dystrophy

PubMed Central

Halilovic, Adna; Schmedt, Thore; Benischke, Anne-Sophie; Hamill, Cecily; Chen, Yuming; Santos, Janine Hertzog

2016-01-01

Abstract Aims: Fuchs endothelial corneal dystrophy (FECD), a leading cause of age-related corneal edema requiring transplantation, is characterized by rosette formation of corneal endothelium with ensuing apoptosis. We sought to determine whether excess of mitochondrial reactive oxygen species leads to chronic accumulation of oxidative DNA damage and mitochondrial dysfunction, instigating cell death. Results: We modeled the pathognomonic rosette formation of postmitotic corneal cells by increasing endogenous cellular oxidative stress with menadione (MN) and performed a temporal analysis of its effect in normal (HCEnC, HCECi) and FECD (FECDi) cells and ex vivo specimens. FECDi and FECD ex vivo specimens exhibited extensive mtDNA and nDNA damage as detected by quantitative PCR. Exposure to MN triggered an increase in mitochondrial superoxide levels and led to mtDNA and nDNA damage, while DNA amplification was restored with NAC pretreatment. Furthermore, MN exposure led to a decrease in ΔΨm and adenosine triphosphate levels in normal cells, while FECDi exhibited mitochondrial dysfunction at baseline. Mitochondrial fragmentation and cytochrome c release were detected in FECD tissue and after MN treatment of HCEnCs. Furthermore, cleavage of caspase-9 and caspase-3 followed MN-induced cytochrome c release in HCEnCs. Innovation: This study provides the first line of evidence that accumulation of oxidative DNA damage leads to rosette formation, loss of functionally intact mitochondria via fragmentation, and subsequent cell death during postmitotic cell degeneration of ocular tissue. Conclusion: MN induced rosette formation, along with mtDNA and nDNA damage, mitochondrial dysfunction, and fragmentation, leading to activation of the intrinsic apoptosis via caspase cleavage and cytochrome c release. Antioxid. Redox Signal. 24, 1072–1083. PMID:26935406

Menadione-Induced DNA Damage Leads to Mitochondrial Dysfunction and Fragmentation During Rosette Formation in Fuchs Endothelial Corneal Dystrophy.

PubMed

Halilovic, Adna; Schmedt, Thore; Benischke, Anne-Sophie; Hamill, Cecily; Chen, Yuming; Santos, Janine Hertzog; Jurkunas, Ula V

2016-06-20

Fuchs endothelial corneal dystrophy (FECD), a leading cause of age-related corneal edema requiring transplantation, is characterized by rosette formation of corneal endothelium with ensuing apoptosis. We sought to determine whether excess of mitochondrial reactive oxygen species leads to chronic accumulation of oxidative DNA damage and mitochondrial dysfunction, instigating cell death. We modeled the pathognomonic rosette formation of postmitotic corneal cells by increasing endogenous cellular oxidative stress with menadione (MN) and performed a temporal analysis of its effect in normal (HCEnC, HCECi) and FECD (FECDi) cells and ex vivo specimens. FECDi and FECD ex vivo specimens exhibited extensive mtDNA and nDNA damage as detected by quantitative PCR. Exposure to MN triggered an increase in mitochondrial superoxide levels and led to mtDNA and nDNA damage, while DNA amplification was restored with NAC pretreatment. Furthermore, MN exposure led to a decrease in ΔΨm and adenosine triphosphate levels in normal cells, while FECDi exhibited mitochondrial dysfunction at baseline. Mitochondrial fragmentation and cytochrome c release were detected in FECD tissue and after MN treatment of HCEnCs. Furthermore, cleavage of caspase-9 and caspase-3 followed MN-induced cytochrome c release in HCEnCs. This study provides the first line of evidence that accumulation of oxidative DNA damage leads to rosette formation, loss of functionally intact mitochondria via fragmentation, and subsequent cell death during postmitotic cell degeneration of ocular tissue. MN induced rosette formation, along with mtDNA and nDNA damage, mitochondrial dysfunction, and fragmentation, leading to activation of the intrinsic apoptosis via caspase cleavage and cytochrome c release. Antioxid. Redox Signal. 24, 1072-1083.

Microcystin-LR induced DNA damage in human peripheral blood lymphocytes.

PubMed

Zegura, B; Gajski, G; Straser, A; Garaj-Vrhovac, V; Filipič, M

2011-12-24

Human exposure to microcystins, which are produced by freshwater cyanobacterial species, is of growing concern due to increasing appearance of cyanobacterial blooms as a consequence of global warming and increasing water eutrophication. Although microcystins are considered to be liver-specific, there is evidence that they may also affect other tissues. These substances have been shown to induce DNA damage in vitro and in vivo, but the mechanisms of their genotoxic activity remain unclear. In human peripheral blood lymphocytes (HPBLs) exposure to non-cytotoxic concentrations (0, 0.1, 1 and 10μg/ml) of microcystin-LR (MCLR) induced a dose- and time-dependent increase in DNA damage, as measured with the comet assay. Digestion of DNA from MCLR-treated HPBLs with purified formamidopyrimidine-DNA glycosylase (Fpg) displayed a greater number of DNA strand-breaks than non-digested DNA, confirming the evidence that MCLR induces oxidative DNA damage. With the cytokinesis-block micronucleus assay no statistically significant induction of micronuclei, nucleoplasmic bridges and nuclear buds was observed after a 24-h exposure to MCLR. At the molecular level, no changes in the expression of selected genes involved in the cellular response to DNA damage and oxidative stress were observed after a 4-h exposure to MCLR (1μg/ml). After 24h, DNA damage-responsive genes (p53, mdm2, gadd45a, cdkn1a), a gene involved in apoptosis (bax) and oxidative stress-responsive genes (cat, gpx1, sod1, gsr, gclc) were up-regulated. These results provide strong support that MCLR is an indirectly genotoxic agent, acting via induction of oxidative stress, and that lymphocytes are also the target of microcystin-induced toxicity. Copyright © 2011 Elsevier B.V. All rights reserved.

Comet Assay: A Method to Evaluate Genotoxicity of Nano-Drug Delivery System

PubMed Central

Vandghanooni, Somayeh; Eskandani, Morteza

2011-01-01

Introduction Drug delivery systems could induce cellular toxicity as side effect of nanomaterials. The mechanism of toxicity usually involves DNA damage. The comet assay or single cell gel electrophoresis (SCGE) is a sensitive method for detecting strand damages in the DNA of a cell with applications in genotoxicity testing and molecular epidemiology as well as fundamental research in DNA damage and repair. Methods In the current study, we reviewed recent drug delivery researches related to SCGE. Results We found that one preference for choosing the assay is that comet images may result from apoptosis-mediated nuclear fragmentation. This method has been widely used over the last decade in several different areas. Overall cells, such as cultured cells are embedded in agarose on a microscope slide, lysed with detergent, and treated with high salt. Nucleoids are supercoiled DNA form. When the slide is faced to alkaline electrophoresis any breakages present in the DNA cause the supercoiling to relax locally and loops of DNA extend toward the anode as a ‘‘comet tail’’. Conclusion This article provides a relatively comprehensive review upon potentiality of the comet assay for assessment of DNA damage and accordingly it can be used as an informative platform in genotoxicity studies of drug delivery systems. PMID:23678412

Preferential mitochondrial DNA injury caused by glucose oxidase as a steady generator of hydrogen peroxide in human fibroblasts.

PubMed

Salazar, J J; Van Houten, B

1997-11-01

To test the hypothesis that mitochondrial DNA (mtDNA) is more prone to reactive oxygen species (ROS) damage than nuclear DNA, a continuous flux of hydrogen peroxide (H2O2) was produced with the glucose/glucose oxidase system. Using a horse radish peroxidase (HRPO)-based colorimetric assay to detect H2O2, glucose oxidase (GO; 12 mU/ml) produced 95 microM of H2O2 in 1 h, whereas only 46 microM of hydrogen peroxide accumulated in the presence of SV40-transformed human fibroblasts ( approximately 1 x 10(6). DNA damage was assessed in the mitochondira and three nuclear regions using a quantitative PCR assay. GO (12 mU/ml) resulted in more damage to the mitochondrial DNA (2.250 +/- 0.045 lesions/10 kb) than in any one of three nuclear targets, which included the non-expressed beta-globin locus (0.436 +/- 0.029 lesions/10 kb); and the active DNA polymerase b gene (0.442 +/- 0.037 lesions/10 kb); and the active hprt gene (0.310 +/- 0.025). Damage to the mtDNA occurred within 15 min of GO treatment, whereas nuclear damage did not appear until after 30 min, and reached a maximum after 60 min. Repair of mitochondrial damage after a 15 min GO (6 mU/ml) treatment was examined. Mitochondria repaired 50% of the damage after 1 h, and by 6 h all the damage was repaired. Higher doses of GO-generated H202, or more extended treatment periods, lead to mitochondrial DNA damage which was not repaired. Mitochondrial function was monitored using the MTT (3,(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide) assay. A 15 min treatment with 6 mU/ml of GO decreased mitochondrial activity to 80% of the control; the activity recovered completely within 1 h after damage. These data show that GO-generated H202 causes acute damage to mtDNA and function, and demonstrate that this organelle is an important site for the cellular toxicity of ROS.

Physical and in silico approaches identify DNA-PK in a Tax DNA-damage response interactome

PubMed Central

Ramadan, Emad; Ward, Michael; Guo, Xin; Durkin, Sarah S; Sawyer, Adam; Vilela, Marcelo; Osgood, Christopher; Pothen, Alex; Semmes, Oliver J

2008-01-01

Background We have initiated an effort to exhaustively map interactions between HTLV-1 Tax and host cellular proteins. The resulting Tax interactome will have significant utility toward defining new and understanding known activities of this important viral protein. In addition, the completion of a full Tax interactome will also help shed light upon the functional consequences of these myriad Tax activities. The physical mapping process involved the affinity isolation of Tax complexes followed by sequence identification using tandem mass spectrometry. To date we have mapped 250 cellular components within this interactome. Here we present our approach to prioritizing these interactions via an in silico culling process. Results We first constructed an in silico Tax interactome comprised of 46 literature-confirmed protein-protein interactions. This number was then reduced to four Tax-interactions suspected to play a role in DNA damage response (Rad51, TOP1, Chk2, 53BP1). The first-neighbor and second-neighbor interactions of these four proteins were assembled from available human protein interaction databases. Through an analysis of betweenness and closeness centrality measures, and numbers of interactions, we ranked proteins in the first neighborhood. When this rank list was compared to the list of physical Tax-binding proteins, DNA-PK was the highest ranked protein common to both lists. An overlapping clustering of the Tax-specific second-neighborhood protein network showed DNA-PK to be one of three bridge proteins that link multiple clusters in the DNA damage response network. Conclusion The interaction of Tax with DNA-PK represents an important biological paradigm as suggested via consensus findings in vivo and in silico. We present this methodology as an approach to discovery and as a means of validating components of a consensus Tax interactome. PMID:18922151

Targeting Protein for Xenopus Kinesin-like Protein 2 (TPX2) Regulates γ-Histone 2AX (γ-H2AX) Levels upon Ionizing Radiation*

PubMed Central

Neumayer, Gernot; Helfricht, Angela; Shim, Su Yeon; Le, Hoa Thi; Lundin, Cecilia; Belzil, Camille; Chansard, Mathieu; Yu, Yaping; Lees-Miller, Susan P.; Gruss, Oliver J.; van Attikum, Haico; Helleday, Thomas; Nguyen, Minh Dang

2012-01-01

The microtubule-associated protein targeting protein for Xenopus kinesin-like protein 2 (TPX2) plays a key role in spindle assembly and is required for mitosis in human cells. In interphase, TPX2 is actively imported into the nucleus to prevent its premature activity in microtubule organization. To date, no function has been assigned to nuclear TPX2. We now report that TPX2 plays a role in the cellular response to DNA double strand breaks induced by ionizing radiation. Loss of TPX2 leads to inordinately strong and transient accumulation of ionizing radiation-dependent Ser-139-phosphorylated Histone 2AX (γ-H2AX) at G0 and G1 phases of the cell cycle. This is accompanied by the formation of increased numbers of high intensity γ-H2AX ionizing radiation-induced foci. Conversely, cells overexpressing TPX2 have reduced levels of γ-H2AX after ionizing radiation. Consistent with a role for TPX2 in the DNA damage response, we found that the protein accumulates at DNA double strand breaks and associates with the mediator of DNA damage checkpoint 1 (MDC1) and the ataxia telangiectasia mutated (ATM) kinase, both key regulators of γ-H2AX amplification. Pharmacologic inhibition or depletion of ATM or MDC1, but not of DNA-dependent protein kinase (DNA-PK), antagonizes the γ-H2AX phenotype caused by TPX2 depletion. Importantly, the regulation of γ-H2AX signals by TPX2 is not associated with apoptosis or the mitotic functions of TPX2. In sum, our study identifies a novel and the first nuclear function for TPX2 in the cellular responses to DNA damage. PMID:23045526

EXO1 is critical for embryogenesis and the DNA damage response in mice with a hypomorphic Nbs1 allele

PubMed Central

Rein, Katrin; Yanez, Diana A.; Terré, Berta; Palenzuela, Lluís; Aivio, Suvi; Wei, Kaichun; Edelmann, Winfried; Stark, Jeremy M.; Stracker, Travis H.

2015-01-01

The maintenance of genome stability is critical for the suppression of diverse human pathologies that include developmental disorders, premature aging, infertility and predisposition to cancer. The DNA damage response (DDR) orchestrates the appropriate cellular responses following the detection of lesions to prevent genomic instability. The MRE11 complex is a sensor of DNA double strand breaks (DSBs) and plays key roles in multiple aspects of the DDR, including DNA end resection that is critical for signaling and DNA repair. The MRE11 complex has been shown to function both upstream and in concert with the 5′-3′ exonuclease EXO1 in DNA resection, but it remains unclear to what extent EXO1 influences DSB responses independently of the MRE11 complex. Here we examine the genetic relationship of the MRE11 complex and EXO1 during mammalian development and in response to DNA damage. Deletion of Exo1 in mice expressing a hypomorphic allele of Nbs1 leads to severe developmental impairment, embryonic death and chromosomal instability. While EXO1 plays a minimal role in normal cells, its loss strongly influences DNA replication, DNA repair, checkpoint signaling and damage sensitivity in NBS1 hypomorphic cells. Collectively, our results establish a key role for EXO1 in modulating the severity of hypomorphic MRE11 complex mutations. PMID:26160886

Disruption of nucleotide excision repair by the human T-cell leukemia virus type 1 Tax protein.

PubMed

Kao, S Y; Marriott, S J

1999-05-01

The Tax protein of human T-cell leukemia virus type 1 (HTLV-1) is a transcriptional transactivator and viral oncogene. Since cellular transformation has been frequently linked to alterations in genome stability, we investigated the effect of Tax on nucleotide excision repair (NER), a prominent cellular DNA repair pathway. Cells expressing Tax exhibited a reduced capacity for NER as measured by unscheduled DNA synthesis and host cell reactivation assays. The cellular proliferating cell nuclear antigen (PCNA) gene product regulates DNA replication and repair pathways, including NER. Since Tax activates transcription of the PCNA promoter, we investigated whether this activity contributes to the reduction of NER. Tax increased endogenous PCNA protein expression, and analysis of Tax mutant proteins demonstrated that the reduction in NER correlated with Tax transactivation of PCNA gene expression. Direct overexpression of PCNA also reduced NER. We propose that overexpression of PCNA, and disruption of NER induced by Tax, predisposes cells to accumulate DNA damage and contributes to HTLV-1 transformation.

DNA Damage Levels Determine Cyclobutyl Pyrimidine Dimer Repair Mechanisms in Alfalfa Seedlings.

PubMed Central

Quaite, F. E.; Takayanagi, S.; Ruffini, J.; Sutherland, J. C.; Sutherland, B. M.

1994-01-01

Ultraviolet radiation in sunlight damages DNA in plants, but little is understood about the types, lesion capacity, and coordination of repair pathways. We challenged intact alfalfa seedlings with UV doses that induced different initial levels of cyclobutyl pyrimidine dimers and measured repair by excision and photoreactivation. By using alkaline gel electrophoresis of nonradioactive DNAs treated with a cyclobutyl pyrimidine dimer-specific UV endonuclease, we quantitated ethidium-stained DNA by electronic imaging and calculated lesion frequencies from the number average molecular lengths. At low initial dimer frequencies (less than ~30 dimers per million bases), the seedlings used only photoreactivation to repair dimers; excision repair was not significant. At higher damage levels, both excision and photorepair contributed significantly. This strategy would allow plants with low damage levels to use error-free repair requiring only an external light energy source, whereas seedlings subjected to higher damage frequencies could call on additional repair processes requiring cellular energy. Characterization of repair in plants thus requires an investigation of a range of conditions, including the level of initial damage. PMID:12244228

E2F1 induces p19INK4d, a protein involved in the DNA damage response, following UV irradiation.

PubMed

Carcagno, Abel L; Giono, Luciana E; Marazita, Mariela C; Castillo, Daniela S; Pregi, Nicolás; Cánepa, Eduardo T

2012-07-01

Central to the maintenance of genomic integrity is the cellular DNA damage response. Depending on the type of genotoxic stress and through the activation of multiple signaling cascades, it can lead to cell cycle arrest, DNA repair, senescence, and apoptosis. p19INK4d, a member of the INK4 family of CDK inhibitors, plays a dual role in the DNA damage response, inhibiting cell proliferation and promoting DNA repair. Consistently, p19INK4d has been reported to become upregulated in response to UV irradiation and a great variety of genotoxic agents. Here, this induction is shown to result from a transcriptional stimulatory mechanism that can occur at every phase of the cell cycle except during mitosis. Moreover, evidence is presented that demonstrates that E2F1 is involved in the induction of p19INK4d following UV treatment, as it is prevented by E2F1 protein ablation and DNA-binding inhibition. Specific inhibition of this regulation using triplex-forming oligonucleotides that target the E2F response elements present in the p19INK4d promoter also block p19INK4d upregulation and sensitize cells to DNA damage. These results constitute the first description of a mechanism for the induction of p19INK4d in response to UV irradiation and demonstrate the physiological relevance of this regulation following DNA damage.

Highlighting the DNA damage response with ultrashort laser pulses in the near infrared and kinetic modeling

PubMed Central

Ferrando-May, Elisa; Tomas, Martin; Blumhardt, Philipp; Stöckl, Martin; Fuchs, Matthias; Leitenstorfer, Alfred

2013-01-01

Our understanding of the mechanisms governing the response to DNA damage in higher eucaryotes crucially depends on our ability to dissect the temporal and spatial organization of the cellular machinery responsible for maintaining genomic integrity. To achieve this goal, we need experimental tools to inflict DNA lesions with high spatial precision at pre-defined locations, and to visualize the ensuing reactions with adequate temporal resolution. Near-infrared femtosecond laser pulses focused through high-aperture objective lenses of advanced scanning microscopes offer the advantage of inducing DNA damage in a 3D-confined volume of subnuclear dimensions. This high spatial resolution results from the highly non-linear nature of the excitation process. Here we review recent progress based on the increasing availability of widely tunable and user-friendly technology of ultrafast lasers in the near infrared. We present a critical evaluation of this approach for DNA microdamage as compared to the currently prevalent use of UV or VIS laser irradiation, the latter in combination with photosensitizers. Current and future applications in the field of DNA repair and DNA-damage dependent chromatin dynamics are outlined. Finally, we discuss the requirement for proper simulation and quantitative modeling. We focus in particular on approaches to measure the effect of DNA damage on the mobility of nuclear proteins and consider the pros and cons of frequently used analysis models for FRAP and photoactivation and their applicability to non-linear photoperturbation experiments. PMID:23882280

Identification and characterization of a DnaJ gene from red alga Pyropia yezoensis (Bangiales, Rhodophyta)

NASA Astrophysics Data System (ADS)

Liu, Jiao; Li, Xianchao; Tang, Xuexi; Zhou, Bin

2016-03-01

Members of the DnaJ family are proteins that play a pivotal role in various cellular processes, such as protein folding, protein transport and cellular responses to stress. In the present study, we identified and characterized the full-length DnaJ cDNA sequence from expressed sequence tags of Pyropia yezoensis ( PyDnaJ) via rapid identification of cDNA ends. This cDNA encoded a protein of 429 amino acids, which shared high sequence similarity with other identified DnaJ proteins, such as a heat shock protein 40/DnaJ from Pyropia haitanensis. The relative mRNA expression level of PyDnaJ was investigated using real-time PCR to determine its specific expression during the algal life cycle and during desiccation. The relative mRNA expression level in sporophytes was higher than that in gametophytes and significantly increased during the whole desiccation process. These results indicate that PyDnaJ is an authentic member of the DnaJ family in plants and red algae and might play a pivotal role in mitigating damage to P. yezoensis during desiccation.

A high-throughput and quantitative method to assess the mutagenic potential of translesion DNA synthesis

PubMed Central

Taggart, David J.; Camerlengo, Terry L.; Harrison, Jason K.; Sherrer, Shanen M.; Kshetry, Ajay K.; Taylor, John-Stephen; Huang, Kun; Suo, Zucai

2013-01-01

Cellular genomes are constantly damaged by endogenous and exogenous agents that covalently and structurally modify DNA to produce DNA lesions. Although most lesions are mended by various DNA repair pathways in vivo, a significant number of damage sites persist during genomic replication. Our understanding of the mutagenic outcomes derived from these unrepaired DNA lesions has been hindered by the low throughput of existing sequencing methods. Therefore, we have developed a cost-effective high-throughput short oligonucleotide sequencing assay that uses next-generation DNA sequencing technology for the assessment of the mutagenic profiles of translesion DNA synthesis catalyzed by any error-prone DNA polymerase. The vast amount of sequencing data produced were aligned and quantified by using our novel software. As an example, the high-throughput short oligonucleotide sequencing assay was used to analyze the types and frequencies of mutations upstream, downstream and at a site-specifically placed cis–syn thymidine–thymidine dimer generated individually by three lesion-bypass human Y-family DNA polymerases. PMID:23470999

The Quinone Based Antitumor Agent Sepantronium Bromide (YM155) Causes Oxygen Independent Redox Activated Oxidative DNA Damage.

PubMed

Wani, Tasaduq Hussain; Surendran, Sreeraj; Jana, Anal; Chakrabarty, Anindita; Chowdhury, Goutam

2018-06-13

Sepantronium bromide (YM155) is a small molecule antitumor agent currently in phase II clinical trials. Although developed as survivin suppressor, YM155's primary mode of action has recently been found to be DNA damage. However, the mechanism of DNA damage by YM155 is still unknown. Knowing the mechanism of action of an anticancer drug is necessary to formulate a rational drug combination and select a cancer type for achieving maximum clinical efficacy. Using cell-based assays we showed that YM155 cause extensive DNA cleavage and reactive oxygen species generation. DNA cleavage by YM155 was found to be inhibited by radical scavengers and desferal. The reducing agent DTT and the cellular reducing system xanthine/xanthine oxidase were found to reductively activate YM155 and cause DNA cleavage. Unlike quinones, DNA cleavage by YM155 occurs in the presence of catalase and under hypoxic conditions indicating that hydrogen peroxide and oxygen is not necessary. Although YM155 is a quinone, it does not follow a typical quinone mechanism. Consistent with these observations a mechanism has been proposed that suggests that YM155 can cause oxidative DNA cleavage upon two electron reductive activation.

Miscoding and mutagenic properties of 8-oxoguanine and abasic sites: Ubiquitous lesions in damaged DNA

DOE Office of Scientific and Technical Information (OSTI.GOV)

Grollman, A.P.; Takeshita, Masaru

1995-12-31

More than twenty oxidatively-damaged bases, including 8-oxoguanine, have been found to occur in genomic DNA. Some of these lesions block DNA replication and are potentially lethal; others generate mutations which can initiate carcinogenesis and promote cellular aging. In this report, the authors focus attention on the mutagenicity and repair of 8-oxoguanine. Kasai and Nishimura`s discovery that hydroxyl radicals react with guanine residues in DNA to form 8-oxoguanine and the development of sensitive methods for the detection and quantitation of this modified base led to the observation that approximately 1 in 10{sup 5} guanine residues in mammalian DNA are oxidized atmore » the C-8 position. DNA containing 8-oxoguanine and synthetic analogs of the abasic site have been used to investigate the miscoding and mutagenic potential of these ubiquitous lesions. Studies in the laboratory were facilitated by the development of solid state synthetic methods by which these lesions could be introduced at defined positions in DNA. In this paper, the authors review studies in which 8-oxoguanine and abasic sites have been used in model systems to explore various early events in the replication of selectively damaged DNA.« less

Replication-mediated disassociation of replication protein A–XPA complex upon DNA damage: implications for RPA handing off

PubMed Central

Jiang, Gaofeng; Zou, Yue; Wu, Xiaoming

2013-01-01

RPA (replication protein A), the eukaryotic ssDNA (single-stranded DNA)-binding protein, participates in most cellular processes in response to genotoxic insults, such as NER (nucleotide excision repair), DNA, DSB (double-strand break) repair and activation of cell cycle checkpoint signalling. RPA interacts with XPA (xeroderma pigmentosum A) and functions in early stage of NER. We have shown that in cells the RPA–XPA complex disassociated upon exposure of cells to high dose of UV irradiation. The dissociation required replication stress and was partially attributed to tRPA hyperphosphorylation. Treatment of cells with CPT (camptothecin) and HU (hydroxyurea), which cause DSB DNA damage and replication fork collapse respectively and also leads to the disruption of RPA–XPA complex. Purified RPA and XPA were unable to form complex in vitro in the presence of ssDNA. We propose that the competition-based RPA switch among different DNA metabolic pathways regulates the dissociation of RPA with XPA in cells after DNA damage. The biological significances of RPA–XPA complex disruption in relation with checkpoint activation, DSB repair and RPA hyperphosphorylation are discussed. PMID:22578086

L- and D-lactate enhance DNA repair and modulate the resistance of cervical carcinoma cells to anticancer drugs via histone deacetylase inhibition and hydroxycarboxylic acid receptor 1 activation.

PubMed

Wagner, Waldemar; Ciszewski, Wojciech M; Kania, Katarzyna D

2015-07-25

The consideration of lactate as an active metabolite is a newly emerging and attractive concept. Recently, lactate has been reported to regulate gene transcription via the inhibition of histone deacetylases (HDACs) and survival of cancer cells via hydroxycarboxylic acid receptor 1 (HCAR1). This study examined the role of L- and D-lactate in the DNA damage response in cervical cancer cells. Three cervical cancer cell lines were examined: HeLa, Ca Ski and C33A. The inhibitory activity of lactate on HDACs was analysed using Western blot and biochemical methods. The lactate-mediated stimulation of DNA repair and cellular resistance to neocarzinostatin, doxorubicin and cisplatin were studied using γ-H2AX, comet and clonogenic assays. HCAR1 and DNA repair gene expression was quantified by real-time PCR. DNA-PKcs activity and HCAR1 protein expression were evaluated via immunocytochemistry and Western blot, respectively. HCAR1 activation was investigated by measuring intracellular cAMP accumulation and Erk phosphorylation. HCAR1 expression was silenced using shRNA. L- and D-lactate inhibited HDACs, induced histone H3 and H4 hyperacetylation, and decreased chromatin compactness in HeLa cells. Treating cells with lactate increased LIG4, NBS1, and APTX expression by nearly 2-fold and enhanced DNA-PKcs activity. Based on γ-H2AX and comet assays, incubation of cells in lactate-containing medium increased the DNA repair rate. Furthermore, clonogenic assays demonstrated that lactate mediates cellular resistance to clinically used chemotherapeutics. Western blot and immunocytochemistry showed that all studied cell lines express HCAR1 on the cellular surface. Inhibiting HCAR1 function via pertussis toxin pretreatment partially abolished the effects of lactate on DNA repair. Down-regulating HCAR1 decreased the efficiency of DNA repair, abolished the cellular response to L-lactate and decreased the effect of D-lactate. Moreover, HCAR1 shRNA-expressing cells produced significantly lower mRNA levels of monocarboxylate transporter 4. Finally, the enhancement of DNA repair and cell survival by lactate was suppressed by pharmacologically inhibiting monocarboxylate transporters using the inhibitor α-cyano-4-hydroxycinnamic acid (α-CHCA). Our data indicate that L- and D-lactate present in the uterine cervix may participate in the modulation of cellular DNA damage repair processes and in the resistance of cervical carcinoma cells to anticancer therapy.

DNA polymerase β variant Ile260Met generates global gene expression changes related to cellular transformation

PubMed Central

Sweasy, Joann B.

2012-01-01

Maintenance of genomic stability is essential for cellular survival. The base excision repair (BER) pathway is critical for resolution of abasic sites and damaged bases, estimated to occur 20,000 times in cells daily. DNA polymerase β (Pol β) participates in BER by filling DNA gaps that result from excision of damaged bases. Approximately 30% of human tumours express Pol β variants, many of which have altered fidelity and activity in vitro and when expressed, induce cellular transformation. The prostate tumour variant Ile260Met transforms cells and is a sequence-context-dependent mutator. To test the hypothesis that mutations induced in vivo by Ile260Met lead to cellular transformation, we characterized the genome-wide expression profile of a clone expressing Ile260Met as compared with its non-induced counterpart. Using a 1.5-fold minimum cut-off with a false discovery rate (FDR) of <0.05, 912 genes exhibit altered expression. Microarray results were confirmed by quantitative real-time polymerase chain reaction (qRT-PCR) and revealed unique expression profiles in other clones. Gene Ontology (GO) clusters were analyzed using Ingenuity Pathways Analysis to identify altered gene networks and associated nodes. We determined three nodes of interest that exhibited dysfunctional regulation of downstream gene products without themselves having altered expression. One node, peroxisome proliferator-activated protein γ (PPARG), was sequenced and found to contain a coding region mutation in PPARG2 only in transformed cells. Further analysis suggests that this mutation leads to dominant negative activity of PPARG2. PPARG is a transcription factor implicated to have tumour suppressor function. This suggests that the PPARG2 mutant may have played a role in driving cellular transformation. We conclude that PPARG induces cellular transformation by a mutational mechanism. PMID:22914675

Mycobacterium tuberculosis promotes genomic instability in macrophages.

PubMed

Castro-Garza, Jorge; Luévano-Martínez, Miriam Lorena; Villarreal-Treviño, Licet; Gosálvez, Jaime; Fernández, José Luis; Dávila-Rodríguez, Martha Imelda; García-Vielma, Catalina; González-Hernández, Silvia; Cortés-Gutiérrez, Elva Irene

2018-03-01

Mycobacterium tuberculosis is an intracellular pathogen, which may either block cellular defensive mechanisms and survive inside the host cell or induce cell death. Several studies are still exploring the mechanisms involved in these processes. To evaluate the genomic instability of M. tuberculosis-infected macrophages and compare it with that of uninfected macrophages. We analysed the possible variations in the genomic instability of Mycobacterium-infected macrophages using the DNA breakage detection fluorescence in situ hybridisation (DBD-FISH) technique with a whole human genome DNA probe. Quantitative image analyses showed a significant increase in DNA damage in infected macrophages as compared with uninfected cells. DNA breaks were localised in nuclear membrane blebs, as confirmed with DNA fragmentation assay. Furthermore, a significant increase in micronuclei and nuclear abnormalities were observed in infected macrophages versus uninfected cells. Genomic instability occurs during mycobacterial infection and these data may be seminal for future research on host cell DNA damage in M. tuberculosis infection.

Evidence for a Role of FEN1 in Maintaining Mitochondrial DNA Integrity

PubMed Central

Kalifa, Lidza; Beutner, Gisela; Phadnis, Naina; Sheu, Shey-Shing; Sia, Elaine A.

2009-01-01

Although the nuclear processes responsible for genomic DNA replication and repair are well characterized, the pathways involved in mitochondrial DNA (mtDNA) replication and repair remain unclear. DNA repair has been identified as being particularly important within the mitochondrial compartment due to the organelle’s high propensity to accumulate oxidative DNA damage. It has been postulated that continual accumulation of mtDNA damage and subsequent mutagenesis may function in cellular aging. Mitochondrial base excision repair (mtBER) plays a major role in combating mtDNA oxidative damage; however, the proteins involved in mtBER have yet to be fully characterized. It has been established that during nuclear long-patch (LP) BER, FEN1 is responsible for cleavage of 5′ flap structures generated during DNA synthesis. Furthermore, removal of 5′ flaps has been observed in mitochondrial extracts of mammalian cell lines; yet, the mitochondrial localization of FEN1 has not been clearly demonstrated. In this study, we analyzed the effects of deleting the yeast FEN1 homolog, RAD27, on mtDNA stability in Saccharomyces cerevisiae. Our findings demonstrate that Rad27p/FEN1 is localized in the mitochondrial compartment of both yeast and mice and that Rad27p has a significant role in maintaining mtDNA integrity. PMID:19699691

DNA damage checkpoint kinase ATM regulates germination and maintains genome stability in seeds

PubMed Central

Waterworth, Wanda M.; Footitt, Steven; Bray, Clifford M.; Finch-Savage, William E.; West, Christopher E.

2016-01-01

Genome integrity is crucial for cellular survival and the faithful transmission of genetic information. The eukaryotic cellular response to DNA damage is orchestrated by the DNA damage checkpoint kinases ATAXIA TELANGIECTASIA MUTATED (ATM) and ATM AND RAD3-RELATED (ATR). Here we identify important physiological roles for these sensor kinases in control of seed germination. We demonstrate that double-strand breaks (DSBs) are rate-limiting for germination. We identify that desiccation tolerant seeds exhibit a striking transcriptional DSB damage response during germination, indicative of high levels of genotoxic stress, which is induced following maturation drying and quiescence. Mutant atr and atm seeds are highly resistant to aging, establishing ATM and ATR as determinants of seed viability. In response to aging, ATM delays germination, whereas atm mutant seeds germinate with extensive chromosomal abnormalities. This identifies ATM as a major factor that controls germination in aged seeds, integrating progression through germination with surveillance of genome integrity. Mechanistically, ATM functions through control of DNA replication in imbibing seeds. ATM signaling is mediated by transcriptional control of the cell cycle inhibitor SIAMESE-RELATED 5, an essential factor required for the aging-induced delay to germination. In the soil seed bank, seeds exhibit increased transcript levels of ATM and ATR, with changes in dormancy and germination potential modulated by environmental signals, including temperature and soil moisture. Collectively, our findings reveal physiological functions for these sensor kinases in linking genome integrity to germination, thereby influencing seed quality, crucial for plant survival in the natural environment and sustainable crop production. PMID:27503884

DNA damage and repair in oncogenic transformation by heavy ion radiation

NASA Technical Reports Server (NTRS)

Yang, T. C.; Mei, M.; George, K. A.; Craise, L. M.

1996-01-01

Energetic heavy ions are present in galactic cosmic rays and solar particle events. One of the most important late effects in risk assessment is carcinogenesis. We have studied the carcinogenic effects of heavy ions at the cellular and molecular levels and have obtained quantitative data on dose-response curves and on the repair of oncogenic lesions for heavy particles with various charges and energies. Studies with repair inhibitors and restriction endonucleases indicated that for oncogenic transformation DNA is the primary target. Results from heavy ion experiments showed that the cross section increased with LET and reached a maximum value of about 0.02 micrometer2 at about 500 keV/micrometer. This limited size of cross section suggests that only a fraction of cellular genomic DNA is important in radiogenic transformation. Free radical scavengers, such as DMSO, do not give any effect on induction of oncogenic transformation by 600 MeV/u iron particles, suggesting most oncogenic damage induced by high-LET heavy ions is through direct action. Repair studies with stationary phase cells showed that the amount of reparable oncogenic lesions decreased with an increase of LET and that heavy ions with LET greater than 200 keV/micrometer produced only irreparable oncogenic damage. An enhancement effect for oncogenic transformation was observed in cells irradiated by low-dose-rate argon ions (400 MeV/u; 120 keV/micrometer). Chromosomal aberrations, such as translocation and deletion, but not sister chromatid exchange, are essential for heavy-ion-induced oncogenic transformation. The basic mechanism(s) of misrepair of DNA damage, which form oncogenic lesions, is unknown.

DNA damage checkpoint kinase ATM regulates germination and maintains genome stability in seeds.

PubMed

Waterworth, Wanda M; Footitt, Steven; Bray, Clifford M; Finch-Savage, William E; West, Christopher E

2016-08-23

Genome integrity is crucial for cellular survival and the faithful transmission of genetic information. The eukaryotic cellular response to DNA damage is orchestrated by the DNA damage checkpoint kinases ATAXIA TELANGIECTASIA MUTATED (ATM) and ATM AND RAD3-RELATED (ATR). Here we identify important physiological roles for these sensor kinases in control of seed germination. We demonstrate that double-strand breaks (DSBs) are rate-limiting for germination. We identify that desiccation tolerant seeds exhibit a striking transcriptional DSB damage response during germination, indicative of high levels of genotoxic stress, which is induced following maturation drying and quiescence. Mutant atr and atm seeds are highly resistant to aging, establishing ATM and ATR as determinants of seed viability. In response to aging, ATM delays germination, whereas atm mutant seeds germinate with extensive chromosomal abnormalities. This identifies ATM as a major factor that controls germination in aged seeds, integrating progression through germination with surveillance of genome integrity. Mechanistically, ATM functions through control of DNA replication in imbibing seeds. ATM signaling is mediated by transcriptional control of the cell cycle inhibitor SIAMESE-RELATED 5, an essential factor required for the aging-induced delay to germination. In the soil seed bank, seeds exhibit increased transcript levels of ATM and ATR, with changes in dormancy and germination potential modulated by environmental signals, including temperature and soil moisture. Collectively, our findings reveal physiological functions for these sensor kinases in linking genome integrity to germination, thereby influencing seed quality, crucial for plant survival in the natural environment and sustainable crop production.

Oxidative Stress, Bone Marrow Failure, and Genome Instability in Hematopoietic Stem Cells

PubMed Central

Richardson, Christine; Yan, Shan; Vestal, C. Greer

2015-01-01

Reactive oxygen species (ROS) can be generated by defective endogenous reduction of oxygen by cellular enzymes or in the mitochondrial respiratory pathway, as well as by exogenous exposure to UV or environmental damaging agents. Regulation of intracellular ROS levels is critical since increases above normal concentrations lead to oxidative stress and DNA damage. A growing body of evidence indicates that the inability to regulate high levels of ROS leading to alteration of cellular homeostasis or defective repair of ROS-induced damage lies at the root of diseases characterized by both neurodegeneration and bone marrow failure as well as cancer. That these diseases may be reflective of the dynamic ability of cells to respond to ROS through developmental stages and aging lies in the similarities between phenotypes at the cellular level. This review summarizes work linking the ability to regulate intracellular ROS to the hematopoietic stem cell phenotype, aging, and disease. PMID:25622253

Cellular functions of TIP60.

PubMed

Sapountzi, Vasileia; Logan, Ian R; Robson, Craig N

2006-01-01

TIP60 was originally identified as a cellular acetyltransferase protein that interacts with HIV-1 Tat. As a consequence, the role of TIP60 in transcriptional regulation has been investigated intensively. Recent data suggest that TIP60 has more divergent functions than originally thought and roles for TIP60 in many processes, such as cellular signalling, DNA damage repair, cell cycle and checkpoint control and apoptosis are emerging. TIP60 is a tightly regulated transcriptional coregulator, acting in a large multiprotein complex for a range of transcription factors including androgen receptor, Myc, STAT3, NF-kappaB, E2F1 and p53. This usually involves recruitment of TIP60 acetyltransferase activities to chromatin. Additionally, in response to DNA double strand breaks, TIP60 is recruited to DNA lesions where it participates both in the initial as well as the final stages of repair. Here, we describe how TIP60 is a multifunctional enzyme involved in multiple nuclear transactions.

Aqueous extract of Crataegus azarolus protects against DNA damage in human lymphoblast Cell K562 and enhances antioxidant activity.

PubMed

Mustapha, Nadia; Bouhlel, Inès; Chaabane, Fadwa; Bzéouich, Imèn Mokdad; Ghedira, Kamel; Hennebelle, Thierry; Chekir-Ghedira, Leila

2014-02-01

The present study was carried out to characterize the cellular antioxidant effect of the aqueous extract of Crataegus azarolus and its antigenotoxic potential using human myelogenous cells, K562. The antioxidant capacity of this extract was evaluated by determining its cellular antioxidant activity (CAA) in K562 cells. Also, preceding antigenotoxicity assessment, its eventual genotoxicity property was investigated by evaluating its capacity to induce the DNA degradation of treated cell nuclei. As no genotoxicity was detected at different exposure times, its ability to protect cell DNA against H2O2 oxidative effect was investigated, using the "comet assay." It appears that 800 μg/mL of extract inhibited the genotoxicity induced by H2O2 with a rate of 41.30 %, after 4 h of incubation. In addition, this extract revealed a significant cellular antioxidant capacity against the reactive oxygen species in K562 cells.

Selective Cytotoxicity of Rhodium Metalloinsertors in Mismatch Repair-Deficient Cells†

PubMed Central

Ernst, Russell J.; Komor, Alexis C.; Barton, Jacqueline K.

2011-01-01

Mismatches in DNA occur naturally during replication and as a result of endogenous DNA damaging agents, but the mismatch repair (MMR) pathway acts to correct mismatches before subsequent rounds of replication. Rhodium metalloinsertors bind to DNA mismatches with high affinity and specificity and represent a promising strategy to target mismatches in cells. Here we examine the biological fate of rhodium metalloinsertors bearing dipyridylamine ancillary ligands in cells deficient in MMR versus those that are MMR-proficient. These complexes are shown to exhibit accelerated cellular uptake which permits the observation of various cellular responses, including disruption of the cell cycle, monitored by flow cytometry assays, and induction of necrosis, monitored by dye exclusion and caspase inhibition assays, that occur preferentially in the MMR-deficient cell line. These cellular responses provide insight into the mechanisms underlying the selective activity of this novel class of targeted anti-cancer agents. PMID:22103240

Persistent damaged bases in DNA allow mutagenic break repair in Escherichia coli.

PubMed

Moore, Jessica M; Correa, Raul; Rosenberg, Susan M; Hastings, P J

2017-07-01

Bacteria, yeast and human cancer cells possess mechanisms of mutagenesis upregulated by stress responses. Stress-inducible mutagenesis potentially accelerates adaptation, and may provide important models for mutagenesis that drives cancers, host pathogen interactions, antibiotic resistance and possibly much of evolution generally. In Escherichia coli repair of double-strand breaks (DSBs) becomes mutagenic, using low-fidelity DNA polymerases under the control of the SOS DNA-damage response and RpoS general stress response, which upregulate and allow the action of error-prone DNA polymerases IV (DinB), II and V to make mutations during repair. Pol IV is implied to compete with and replace high-fidelity DNA polymerases at the DSB-repair replisome, causing mutagenesis. We report that up-regulated Pol IV is not sufficient for mutagenic break repair (MBR); damaged bases in the DNA are also required, and that in starvation-stressed cells, these are caused by reactive-oxygen species (ROS). First, MBR is reduced by either ROS-scavenging agents or constitutive activation of oxidative-damage responses, both of which reduce cellular ROS levels. The ROS promote MBR other than by causing DSBs, saturating mismatch repair, oxidizing proteins, or inducing the SOS response or the general stress response. We find that ROS drive MBR through oxidized guanines (8-oxo-dG) in DNA, in that overproduction of a glycosylase that removes 8-oxo-dG from DNA prevents MBR. Further, other damaged DNA bases can substitute for 8-oxo-dG because ROS-scavenged cells resume MBR if either DNA pyrimidine dimers or alkylated bases are induced. We hypothesize that damaged bases in DNA pause the replisome and allow the critical switch from high fidelity to error-prone DNA polymerases in the DSB-repair replisome, thus allowing MBR. The data imply that in addition to the indirect stress-response controlled switch to MBR, a direct cis-acting switch to MBR occurs independently of DNA breakage, caused by ROS oxidation of DNA potentially regulated by ROS regulators.

Persistent damaged bases in DNA allow mutagenic break repair in Escherichia coli

PubMed Central

Moore, Jessica M.; Correa, Raul; Rosenberg, Susan M.

2017-01-01

Bacteria, yeast and human cancer cells possess mechanisms of mutagenesis upregulated by stress responses. Stress-inducible mutagenesis potentially accelerates adaptation, and may provide important models for mutagenesis that drives cancers, host pathogen interactions, antibiotic resistance and possibly much of evolution generally. In Escherichia coli repair of double-strand breaks (DSBs) becomes mutagenic, using low-fidelity DNA polymerases under the control of the SOS DNA-damage response and RpoS general stress response, which upregulate and allow the action of error-prone DNA polymerases IV (DinB), II and V to make mutations during repair. Pol IV is implied to compete with and replace high-fidelity DNA polymerases at the DSB-repair replisome, causing mutagenesis. We report that up-regulated Pol IV is not sufficient for mutagenic break repair (MBR); damaged bases in the DNA are also required, and that in starvation-stressed cells, these are caused by reactive-oxygen species (ROS). First, MBR is reduced by either ROS-scavenging agents or constitutive activation of oxidative-damage responses, both of which reduce cellular ROS levels. The ROS promote MBR other than by causing DSBs, saturating mismatch repair, oxidizing proteins, or inducing the SOS response or the general stress response. We find that ROS drive MBR through oxidized guanines (8-oxo-dG) in DNA, in that overproduction of a glycosylase that removes 8-oxo-dG from DNA prevents MBR. Further, other damaged DNA bases can substitute for 8-oxo-dG because ROS-scavenged cells resume MBR if either DNA pyrimidine dimers or alkylated bases are induced. We hypothesize that damaged bases in DNA pause the replisome and allow the critical switch from high fidelity to error-prone DNA polymerases in the DSB-repair replisome, thus allowing MBR. The data imply that in addition to the indirect stress-response controlled switch to MBR, a direct cis-acting switch to MBR occurs independently of DNA breakage, caused by ROS oxidation of DNA potentially regulated by ROS regulators. PMID:28727736

Sex as a response to oxidative stress: a twofold increase in cellular reactive oxygen species activates sex genes.

PubMed

Nedelcu, Aurora M; Marcu, Oana; Michod, Richard E

2004-08-07

Organisms are constantly subjected to factors that can alter the cellular redox balance and result in the formation of a series of highly reactive molecules known as reactive oxygen species (ROS). As ROS can be damaging to biological structures, cells evolved a series of mechanisms (e.g. cell-cycle arrest, programmed cell death) to respond to high levels of ROS (i.e. oxidative stress). Recently, we presented evidence that in a facultatively sexual lineage--the multicellular green alga Volvox carteri--sex is an additional response to increased levels of stress, and probably ROS and DNA damage. Here we show that, in V. carteri, (i) sex is triggered by an approximately twofold increase in the level of cellular ROS (induced either by the natural sex-inducing stress, namely heat, or by blocking the mitochondrial electron transport chain with antimycin A), and (ii) ROS are responsible for the activation of sex genes. As most types of stress result in the overproduction of ROS, we believe that our findings will prove to extend to other facultatively sexual lineages, which could be indicative of the ancestral role of sex as an adaptive response to stress and ROS-induced DNA damage. Copyright 2004 The Royal Society

DNA Damage Response Genes and the Development of Cancer Metastasis

PubMed Central

Broustas, Constantinos G.; Lieberman, Howard B.

2014-01-01

DNA damage response genes play vital roles in the maintenance of a healthy genome. Defects in cell cycle checkpoint and DNA repair genes, especially mutation or aberrant downregulation, are associated with a wide spectrum of human disease, including a predisposition to the development of neurodegenerative conditions and cancer. On the other hand, upregulation of DNA damage response and repair genes can also cause cancer, as well as increase resistance of cancer cells to DNA damaging therapy. In recent years, it has become evident that many of the genes involved in DNA damage repair have additional roles in tumorigenesis, most prominently by acting as transcriptional (co-) factors. Although defects in these genes are causally connected to tumor initiation, their role in tumor progression is more controversial and it seems to depend on tumor type. In some tumors like melanoma, cell cycle checkpoint/DNA repair gene upregulation is associated with tumor metastasis, whereas in a number of other cancers the opposite has been observed. Several genes that participate in the DNA damage response, such as RAD9, PARP1, BRCA1, ATM and TP53 have been associated with metastasis by a number of in vitro biochemical and cellular assays, by examining human tumor specimens by immunohistochemistry or by DNA genomewide gene expression profiling. Many of these genes act as transcriptional effectors to regulate other genes implicated in the pathogenesis of cancer. Furthermore, they are aberrantly expressed in numerous human tumors and are causally related to tumorigenesis. However, whether the DNA damage repair function of these genes is required to promote metastasis or another activity is responsible (e.g., transcription control) has not been determined. Importantly, despite some compelling in vitro evidence, investigations are still needed to demonstrate the role of cell cycle checkpoint and DNA repair genes in regulating metastatic phenotypes in vivo. PMID:24397478

DNA damage in the oligodendrocyte lineage and its role in brain aging.

PubMed

Tse, Kai-Hei; Herrup, Karl

2017-01-01

Myelination is a recent evolutionary addition that significantly enhances the speed of transmission in the neural network. Even slight defects in myelin integrity impair performance and enhance the risk of neurological disorders. Indeed, myelin degeneration is an early and well-recognized neuropathology that is age associated, but appears before cognitive decline. Myelin is only formed by fully differentiated oligodendrocytes, but the entire oligodendrocyte lineage are clear targets of the altered chemistry of the aging brain. As in neurons, unrepaired DNA damage accumulates in the postmitotic oligodendrocyte genome during normal aging, and indeed may be one of the upstream causes of cellular aging - a fact well illustrated by myelin co-morbidity in premature aging syndromes arising from deficits in DNA repair enzymes. The clinical and experimental evidence from Alzheimer's disease, progeroid syndromes, ataxia-telangiectasia and other conditions strongly suggest that oligodendrocytes may in fact be uniquely vulnerable to oxidative DNA damage. If this damage remains unrepaired, as is increasingly true in the aging brain, myelin gene transcription and oligodendrocyte differentiation is impaired. Delineating the relationships between early myelin loss and DNA damage in brain aging will offer an additional dimension outside the neurocentric view of neurodegenerative disease. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Squalene Inhibits ATM-Dependent Signaling in γIR-Induced DNA Damage Response through Induction of Wip1 Phosphatase.

PubMed

Tatewaki, Naoto; Konishi, Tetsuya; Nakajima, Yuki; Nishida, Miyako; Saito, Masafumi; Eitsuka, Takahiro; Sakamaki, Toshiyuki; Ikekawa, Nobuo; Nishida, Hiroshi

2016-01-01

Ataxia telangiectasia mutated (ATM) kinase plays a crucial role as a master controller in the cellular DNA damage response. Inhibition of ATM leads to inhibition of the checkpoint signaling pathway. Hence, addition of checkpoint inhibitors to anticancer therapies may be an effective targeting strategy. A recent study reported that Wip1, a protein phosphatase, de-phosphorylates serine 1981 of ATM during the DNA damage response. Squalene has been proposed to complement anticancer therapies such as chemotherapy and radiotherapy; however, there is little mechanistic information supporting this idea. Here, we report the inhibitory effect of squalene on ATM-dependent DNA damage signals. Squalene itself did not affect cell viability and the cell cycle of A549 cells, but it enhanced the cytotoxicity of gamma-irradiation (γIR). The in vitro kinase activity of ATM was not altered by squalene. However, squalene increased Wip1 expression in cells and suppressed ATM activation in γIR-treated cells. Consistent with the potential inhibition of ATM by squalene, IR-induced phosphorylation of ATM effectors such as p53 (Ser15) and Chk1 (Ser317) was inhibited by cell treatment with squalene. Thus, squalene inhibits the ATM-dependent signaling pathway following DNA damage through intracellular induction of Wip1 expression.

DNA repair efficiency in germ cells and early mouse embryos and consequences for radiation-induced transgenerational genomic damage

DOE Office of Scientific and Technical Information (OSTI.GOV)

Marchetti, Francesco; Wyrobek, Andrew J.

Exposure to ionizing radiation and other environmental agents can affect the genomic integrity of germ cells and induce adverse health effects in the progeny. Efficient DNA repair during gametogenesis and the early embryonic cycles after fertilization is critical for preventing transmission of DNA damage to the progeny and relies on maternal factors stored in the egg before fertilization. The ability of the maternal repair machinery to repair DNA damage in both parental genomes in the fertilizing egg is especially crucial for the fertilizing male genome that has not experienced a DNA repair-competent cellular environment for several weeks prior to fertilization.more » During the DNA repair-deficient period of spermatogenesis, DNA lesions may accumulate in sperm and be carried into the egg where, if not properly repaired, could result in the formation of heritable chromosomal aberrations or mutations and associated birth defects. Studies with female mice deficient in specific DNA repair genes have shown that: (i) cell cycle checkpoints are activated in the fertilized egg by DNA damage carried by the sperm; and (ii) the maternal genotype plays a major role in determining the efficiency of repairing genomic lesions in the fertilizing sperm and directly affect the risk for abnormal reproductive outcomes. There is also growing evidence that implicates DNA damage carried by the fertilizing gamete as a mediator of postfertilization processes that contribute to genomic instability in subsequent generations. Transgenerational genomic instability most likely involves epigenetic mechanisms or error-prone DNA repair processes in the early embryo. Maternal and embryonic DNA repair processes during the early phases of mammalian embryonic development can have far reaching consequences for the genomic integrity and health of subsequent generations.« less

Transcriptional activation of short interspersed elements by DNA-damaging agents.

PubMed

Rudin, C M; Thompson, C B

2001-01-01

Short interspersed elements (SINEs), typified by the human Alu repeat, are RNA polymerase III (pol III)-transcribed sequences that replicate within the genome through an RNA intermediate. Replication of SINEs has been extensive in mammalian evolution: an estimated 5% of the human genome consists of Alu repeats. The mechanisms regulating transcription, reverse transcription, and reinsertion of SINE elements in genomic DNA are poorly understood. Here we report that expression of murine SINE transcripts of both the B1 and B2 classes is strongly upregulated after prolonged exposure to cisplatin, etoposide, or gamma radiation. A similar induction of Alu transcripts in human cells occurs under these conditions. This induction is not due to a general upregulation of pol III activity in either species. Genotoxic treatment of murine cells containing an exogenous human Alu element induced Alu transcription. Concomitant with the increased expression of SINEs, an increase in cellular reverse transcriptase was observed after exposure to these same DNA-damaging agents. These findings suggest that genomic damage may be an important activator of SINEs, and that SINE mobility may contribute to secondary malignancy after exposure to DNA-damaging chemotherapy.

RAD9-dependent G1 arrest defines a second checkpoint for damaged DNA in the cell cycle of Saccharomyces cerevisiae.

PubMed

Siede, W; Friedberg, A S; Friedberg, E C

1993-09-01

Exposure of the yeast Saccharomyces cerevisiae to ultraviolet (UV) light, the UV-mimetic chemical 4-nitroquinoline-1-oxide (4NQO), or gamma radiation after release from G1 arrest induced by alpha factor results in delayed resumption of the cell cycle. As is the case with G2 arrest following ionizing radiation damage [Weinert, T. A. & Hartwell, L. H. (1988) Science 241, 317-322], the normal execution of DNA damage-induced G1 arrest depends on a functional yeast RAD9 gene. We suggest that the RAD9 gene product may interact with cellular components common to the G1/S and G2/M transition points in the cell cycle of this yeast. These observations define a checkpoint in the eukaryotic cell cycle that may facilitate the repair of lesions that are otherwise processed to lethal and/or mutagenic damage during DNA replication. This checkpoint apparently operates after the mating pheromone-induced G1 arrest point but prior to replicative DNA synthesis, S phase-associated maximal induction of histone H2A mRNA, and bud emergence.

Tip60 degradation by adenovirus relieves transcriptional repression of viral transcriptional activator EIA.

PubMed

Gupta, A; Jha, S; Engel, D A; Ornelles, D A; Dutta, A

2013-10-17

Adenoviruses are linear double-stranded DNA viruses that infect human and rodent cell lines, occasionally transform them and cause tumors in animal models. The host cell challenges the virus in multifaceted ways to restrain viral gene expression and DNA replication, and sometimes even eliminates the infected cells by programmed cell death. To combat these challenges, adenoviruses abrogate the cellular DNA damage response pathway. Tip60 is a lysine acetyltransferase that acetylates histones and other proteins to regulate gene expression, DNA damage response, apoptosis and cell cycle regulation. Tip60 is a bona fide tumor suppressor as mice that are haploid for Tip60 are predisposed to tumors. We have discovered that Tip60 is degraded by adenovirus oncoproteins EIB55K and E4orf6 by a proteasome-mediated pathway. Tip60 binds to the immediate early adenovirus promoter and suppresses adenovirus EIA gene expression, which is a master regulator of adenovirus transcription, at least partly through retention of the virally encoded repressor pVII on this promoter. Thus, degradation of Tip60 by the adenoviral early proteins is important for efficient viral early gene transcription and for changes in expression of cellular genes.

Ku protein as a potential human T-cell leukemia virus type 1 (HTLV-1) Tax target in clastogenic chromosomal instability of mammalian cells

PubMed Central

Majone, Franca; Luisetto, Roberto; Zamboni, Daniela; Iwanaga, Yoichi; Jeang, Kuan-Teh

2005-01-01

The HTLV-1 Tax oncoprotein rapidly induces cytogenetic damage which can be measured by a significant increase in the number of micronuclei (MN) in cells. Tax is thought to have both aneuploidogenic and clastogenic effects. To examine the cellular target for Tax which might mechanistically explain the clastogenic phenomenon, we tested the ability of Tax to induce MN in rodents cells genetically defective for either the Ku80 protein or the catalytic subunit of DNA protein kinase (DNAPKcs). We found that cells genetically mutated in Ku80 were refractory to Tax's induction of MN while cells knocked-out for DNAPKcs showed increased number of Tax-induced MN. Using a cytogenetic method termed FISHI (Fluorescent In Situ Hybridization and Incorporation) which measures the number of DNA-breaks in cells that contained unprotected 3'-OH ends, we observed that Tax increased the prevalence of unprotected DNA breaks in Ku80-intact cells, but not in Ku80-mutated cells. Taken together, our findings suggest Ku80 as a cellular factor targeted by Tax in engendering clastogenic DNA damage. PMID:16014171

Autophagy regulates DNA repair through SQSTM1/p62.

PubMed

Feng, Yuchen; Klionsky, Daniel J

2017-06-03

Macroautophagy/autophagy is primarily a degradative pathway that clears malfunctioning cellular components in response to various types of stress. Recent studies have indicated that autophagy also plays an important role in maintaining genome stability. Loss of autophagy is associated with increased damage to DNA, inappropriate amplification of genomic regions and abnormal chromosome number. In a recent paper by Wang et al. the authors uncover a mechanism through which autophagy regulates the ubiquitination of chromatin. In particular, the autophagy receptor and substrate SQSTM1/p62 inhibits the E3 ligase RNF168-dependent ubiquitination of histone in response to DNA double-strand breaks. Dysregulation of this process leads to a reduced ability to repair DNA and a corresponding increase in the sensitivity of cells to radiation-induced damage.

In Vitro Expansion of Bone Marrow Derived Mesenchymal Stem Cells Alters DNA Double Strand Break Repair of Etoposide Induced DNA Damage.

PubMed

Hare, Ian; Gencheva, Marieta; Evans, Rebecca; Fortney, James; Piktel, Debbie; Vos, Jeffrey A; Howell, David; Gibson, Laura F

2016-01-01

Mesenchymal stem cells (MSCs) are of interest for use in diverse cellular therapies. Ex vivo expansion of MSCs intended for transplantation must result in generation of cells that maintain fidelity of critical functions. Previous investigations have identified genetic and phenotypic alterations of MSCs with in vitro passage, but little is known regarding how culturing influences the ability of MSCs to repair double strand DNA breaks (DSBs), the most severe of DNA lesions. To investigate the response to DSB stress with passage in vitro, primary human MSCs were exposed to etoposide (VP16) at various passages with subsequent evaluation of cellular damage responses and DNA repair. Passage number did not affect susceptibility to VP16 or the incidence and repair kinetics of DSBs. Nonhomologous end joining (NHEJ) transcripts showed little alteration with VP16 exposure or passage; however, homologous recombination (HR) transcripts were reduced following VP16 exposure with this decrease amplified as MSCs were passaged in vitro. Functional evaluations of NHEJ and HR showed that MSCs were unable to activate NHEJ repair following VP16 stress in cells after successive passage. These results indicate that ex vivo expansion of MSCs alters their ability to perform DSB repair, a necessary function for cells intended for transplantation.

Suppression of E. multilocularis Hydatid Cysts after Ionizing Radiation Exposure

PubMed Central

Zhou, Rong; Zhang, Hong

2013-01-01

Background Heavy-ion therapy has an advantage over conventional radiotherapy due to its superb biological effectiveness and dose conformity in cancer therapy. It could be a potential alternate approach for hydatid cyst treatment. However, there is no information currently available on the cellular and molecular basis for heavy-ion irradiation induced cell death in cystic echinococcosis. Methododology/Principal Findings LD50 was scored by protoscolex death. Cellular and ultrastructural changes within the parasite were studied by light and electron microscopy, mitochondrial DNA (mtDNA) damage and copy number were measured by QPCR, and apoptosis was determined by caspase 3 expression and caspase 3 activity. Ionizing radiation induced sparse cytoplasm, disorganized and clumped organelles, large vacuoles and devoid of villi. The initial mtDNA damage caused by ionizing radiation increased in a dose-dependent manner. The kinetic of DNA repair was slower after carbon-ion radiation than that after X-rays radiation. High dose carbon-ion radiation caused irreversible mtDNA degradation. Cysts apoptosis was pronounced after radiation. Carbon-ion radiation was more effective to suppress hydatid cysts than X-rays. Conclusions These studies provide a framework to the evaluation of attenuation effect of heavy-ion radiation on cystic echinococcosis in vitro. Carbon-ion radiation is more effective to suppress E. multilocularis than X-rays. PMID:24205427

The FA pathway counteracts oxidative stress through selective protection of antioxidant defense gene promoters.

PubMed

Du, Wei; Rani, Reena; Sipple, Jared; Schick, Jonathan; Myers, Kasiani C; Mehta, Parinda; Andreassen, Paul R; Davies, Stella M; Pang, Qishen

2012-05-03

Oxidative stress has been implicated in the pathogenesis of many human diseases including Fanconi anemia (FA), a genetic disorder associated with BM failure and cancer. Here we show that major antioxidant defense genes are down-regulated in FA patients, and that gene down-regulation is selectively associated with increased oxidative DNA damage in the promoters of the antioxidant defense genes. Assessment of promoter activity and DNA damage repair kinetics shows that increased initial damage, rather than a reduced repair rate, contributes to the augmented oxidative DNA damage. Mechanistically, FA proteins act in concert with the chromatin-remodeling factor BRG1 to protect the promoters of antioxidant defense genes from oxidative damage. Specifically, BRG1 binds to the promoters of the antioxidant defense genes at steady state. On challenge with oxidative stress, FA proteins are recruited to promoter DNA, which correlates with significant increase in the binding of BRG1 within promoter regions. In addition, oxidative stress-induced FANCD2 ubiquitination is required for the formation of a FA-BRG1-promoter complex. Taken together, these data identify a role for the FA pathway in cellular antioxidant defense.

Oxidative Damage to RPA Limits the Nucleotide Excision Repair Capacity of Human Cells.

PubMed

Guven, Melisa; Brem, Reto; Macpherson, Peter; Peacock, Matthew; Karran, Peter

2015-11-01

Nucleotide excision repair (NER) protects against sunlight-induced skin cancer. Defective NER is associated with photosensitivity and a high skin cancer incidence. Some clinical treatments that cause photosensitivity can also increase skin cancer risk. Among these, the immunosuppressant azathioprine and the fluoroquinolone antibiotics ciprofloxacin and ofloxacin interact with UVA radiation to generate reactive oxygen species that diminish NER capacity by causing protein damage. The replication protein A (RPA) DNA-binding protein has a pivotal role in DNA metabolism and is an essential component of NER. The relationship between protein oxidation and NER inhibition was investigated in cultured human cells expressing different levels of RPA. We show here that RPA is limiting for NER and that oxidative damage to RPA compromises NER capability. Our findings reveal that cellular RPA is surprisingly vulnerable to oxidation, and we identify oxidized forms of RPA that are associated with impaired NER. The vulnerability of NER to inhibition by oxidation provides a connection between cutaneous photosensitivity, protein damage, and increased skin cancer risk. Our findings emphasize that damage to DNA repair proteins, as well as to DNA itself, is likely to be an important contributor to skin cancer risk.

The repair of low dose UV light-induced damage to human skin DNA in condition of trace amount Mg 2+

NASA Astrophysics Data System (ADS)

Gao, Fang; Guo, Zhouyi; Zheng, Changchun; Wang, Rui; Liu, Zhiming; Meng, Pei; Zhai, Juan

2008-12-01

Ultraviolet light-induced damage to human skin DNA was widely investigated. The primary mechanism of this damage contributed to form cyclobutane pyrimidine dimmers (CPDs). Although the distribution of UV light-induced CPDs within a defined sequence is similar, the damage in cellular environment which shields the nuclear DNA was higher than that in organism in apparent dose. So we use low UVB light as main study agent. Low dose UV-irradiated HDF-a cells (Human Dermal Fibroblasts-adult cells) which is weaker than epidermic cells were cultured with DMEM at different trace amount of Mg2+ (0mmol/L , 0.1mmol/L , 0.2mmol/L, 0.4mmol/L, 0.8mmol/L, 1.2mmol/L) free-serum DMEM and the repair of DNA strands injured were observed. Treat these cells with DNA strand breaks detection, photoproducts detection and the repair of photoproducts detection. Then quantitate the role of trace amount Mg2+ in repair of UV light-induced damage to human skin. The experiment results indicated that epidermic cells have capability of resistance to UV-radiation at a certain extent. And Mg2+ can regulate the UV-induced damage repair and relative vitality. It can offer a rationale and experiment data to relieve UV light-induced skin disease.

Impact of Age and Insulin-Like Growth Factor-1 on DNA Damage Responses in UV-Irradiated Human Skin.

PubMed

Kemp, Michael G; Spandau, Dan F; Travers, Jeffrey B

2017-02-26

The growing incidence of non-melanoma skin cancer (NMSC) necessitates a thorough understanding of its primary risk factors, which include exposure to ultraviolet (UV) wavelengths of sunlight and age. Whereas UV radiation (UVR) has long been known to generate photoproducts in genomic DNA that promote genetic mutations that drive skin carcinogenesis, the mechanism by which age contributes to disease pathogenesis is less understood and has not been sufficiently studied. In this review, we highlight studies that have considered age as a variable in examining DNA damage responses in UV-irradiated skin and then discuss emerging evidence that the reduced production of insulin-like growth factor-1 (IGF-1) by senescent fibroblasts in the dermis of geriatric skin creates an environment that negatively impacts how epidermal keratinocytes respond to UVR-induced DNA damage. In particular, recent data suggest that two principle components of the cellular response to DNA damage, including nucleotide excision repair and DNA damage checkpoint signaling, are both partially defective in keratinocytes with inactive IGF-1 receptors. Overcoming these tumor-promoting conditions in aged skin may therefore provide a way to lower aging-associated skin cancer risk, and thus we will consider how dermal wounding and related clinical interventions may work to rejuvenate the skin, re-activate IGF-1 signaling, and prevent the initiation of NMSC.

Impact of Age and Insulin-Like Growth Factor-1 on DNA Damage Responses in UV-Irradiated Human Skin

PubMed Central

Kemp, Michael G.; Spandau, Dan F; Travers, Jeffrey B.

2017-01-01

The growing incidence of non-melanoma skin cancer (NMSC) necessitates a thorough understanding of its primary risk factors, which include exposure to ultraviolet (UV) wavelengths of sunlight and age. Whereas UV radiation (UVR) has long been known to generate photoproducts in genomic DNA that promote genetic mutations that drive skin carcinogenesis, the mechanism by which age contributes to disease pathogenesis is less understood and has not been sufficiently studied. In this review, we highlight studies that have considered age as a variable in examining DNA damage responses in UV-irradiated skin and then discuss emerging evidence that the reduced production of insulin-like growth factor-1 (IGF-1) by senescent fibroblasts in the dermis of geriatric skin creates an environment that negatively impacts how epidermal keratinocytes respond to UVR-induced DNA damage. In particular, recent data suggest that two principle components of the cellular response to DNA damage, including nucleotide excision repair and DNA damage checkpoint signaling, are both partially defective in keratinocytes with inactive IGF-1 receptors. Overcoming these tumor-promoting conditions in aged skin may therefore provide a way to lower aging-associated skin cancer risk, and thus we will consider how dermal wounding and related clinical interventions may work to rejuvenate the skin, re-activate IGF-1 signaling, and prevent the initiation of NMSC. PMID:28245638

The effect of 2-[(aminopropyl)amino] ethanethiol on fission-neutron-induced DNA damage and repair.

PubMed Central

Grdina, D. J.; Sigdestad, C. P.; Dale, P. J.; Perrin, J. M.

1989-01-01

The effect(s) of the radioprotector 2-[(aminopropyl)amino] ethanethiol (WR 1065) on fission-neutron-induced DNA damage and repair in V79 Chinese hamster cells was determined by using a neutral filter elution procedure (pH 7.2). When required, WR1065, at a final working concentration of 4 mM, was added to the culture medium, either 30 min before and during irradiation with fission spectrum neutrons (beam energy of 0.85 MeV) from the JANUS research reactor, or for selected intervals of time following exposure. The frequency of neutron-induced DNA strand breaks as measured by neutral elution as a function of dose equalled that observed for 60Co gamma-ray-induced damage (relative biological effectiveness of one). In contrast to the protective effect exhibited by WR1065 in reducing 60Co-induced DNA damage, WR1065 was ineffective in reducing or protecting against induction of DNA strand breaks by JANUS neutrons. The kinetics of DNA double-strand rejoining were measured following neutron irradiation. In the absence of WR1065, considerable DNA degradation by cellular enzymes was observed. This process was inhibited when WR1065 was present. These results indicate that, under the conditions used, the quality (i.e. nature), rather than quantity, of DNA lesions (measured by neutral elution) formed by neutrons was significantly different from that formed by gamma-rays. PMID:2667608

Structure of transcribed chromatin is a sensor of DNA damage

PubMed Central

Pestov, Nikolay A.; Gerasimova, Nadezhda S.; Kulaeva, Olga I.; Studitsky, Vasily M.

2015-01-01

Early detection and repair of damaged DNA is essential for cell functioning and survival. Although multiple cellular systems are involved in the repair of single-strand DNA breaks (SSBs), it remains unknown how SSBs present in the nontemplate strand (NT-SSBs) of DNA organized in chromatin are detected. The effect of NT-SSBs on transcription through chromatin by RNA polymerase II was studied. NT-SSBs localized in the promoter-proximal region of nucleosomal DNA and hidden in the nucleosome structure can induce a nearly quantitative arrest of RNA polymerase downstream of the break, whereas more promoter-distal SSBs moderately facilitate transcription. The location of the arrest sites on nucleosomal DNA suggests that formation of small intranucleosomal DNA loops causes the arrest. This mechanism likely involves relief of unconstrained DNA supercoiling accumulated during transcription through chromatin by NT-SSBs. These data suggest the existence of a novel chromatin-specific mechanism that allows the detection of NT-SSBs by the transcribing enzyme. PMID:26601207

DNA Damage Repair and the Emerging Role of Poly(ADP-ribose) Polymerase Inhibition in Cancer Therapeutics.

PubMed

Rabenau, Karen; Hofstatter, Erin

2016-07-01

As a result of improved understanding of DNA repair mechanisms, poly(ADP-ribose) polymerase inhibitors (PARPi) are increasingly recognized to play an important therapeutic role in the treatment of cancer. The aim of this article is to provide a review of PARPi function in DNA damage repair and synthetic lethality and to demonstrate how these mechanisms can be exploited to provide new PARPi-based therapies to patients with solid tumors. Literature from a range of sources, including PubMed and MEDLINE, were searched to identify recent reports regarding DNA damage repair and PARPi. DNA damage repair is central to cellular viability. The family of poly(ADP-ribose) polymerase proteins play multiple intracellular roles in DNA repair, but function primarily in the resolution of repair of single-strand DNA breaks. Insights through the discovery of germline BRCA1/2 mutations led to the understanding of synthetic lethality and the potential therapeutic role of PARPi in the treatment of cancer. Further understanding of DNA damage repair and the concept of BRCA-like tumors have catalyzed PARPi clinical investigation in multiple oncologic settings. PARPi hold great promise in the treatment of solid tumors, both as monotherapy and in combination with other cancer therapeutics. Multiple PARPi clinical trials are currently underway. Further understanding of aberrant DNA repair mechanisms in the germline and in the tumor genome will allow clinicians and researchers to apply PARPi most strategically in the era of personalized medicine. Copyright © 2016 Elsevier HS Journals, Inc. All rights reserved.

p21 Activated kinase 1: Nuclear activity and its role during DNA damage repair.

PubMed

Pérez-Yépez, Eloy Andrés; Saldívar-Cerón, Héctor Iván; Villamar-Cruz, Olga; Pérez-Plasencia, Carlos; Arias-Romero, Luis Enrique

2018-05-01

p21-activated kinase 1 (PAK1) is a serine/threonine kinase activated by the small GTPases Rac1 and Cdc42. It is located in the chromosome 11q13 and is amplified and/or overexpressed in several human cancer types including 25-30% of breast tumors. This enzyme plays a pivotal role in the control of a number of fundamental cellular processes by phosphorylating its downstream substrates. In addition to its role in the cytoplasm, it is well documented that PAK1 also plays crucial roles in the nucleus participating in mitotic events and gene expression through its association and/or phosphorylation of several transcription factors, transcriptional co-regulators and cell cycle-related proteins, including Aurora kinase A (AURKA), polo-like kinase 1 (PLK1), the forkhead transcription factor (FKHR), estrogen receptor α (ERα), and Snail. More recently, PAK signaling has emerged as a component of the DNA damage response (DDR) as PAK1 activity influences the cellular sensitivity to ionizing radiation and promotes the expression of several genes involved in the Fanconi Anemia/BRCA pathway. This review will focus on the nuclear functions of PAK1 and its role in the regulation of DNA damage repair. Copyright © 2018 Elsevier B.V. All rights reserved.

Constitutive role of the Fanconi anemia D2 gene in the replication stress response.

PubMed

Tian, Yanyan; Shen, Xi; Wang, Rui; Klages-Mundt, Naeh L; Lynn, Erica J; Martin, Sara K; Ye, Yin; Gao, Min; Chen, Junjie; Schlacher, Katharina; Li, Lei

2017-12-08

In response to DNA cross-linking damage, the Fanconi anemia (FA) core complex activates the FA pathway by monoubiquitinating Fanconi anemia complementation group D2 (FANCD2) for the initiation of the nucleolytic processing of the DNA cross-links and stabilization of stalled replication forks. Given that all the classic FA proteins coordinately monoubiquitinate FANCD2, it is unclear why losses of individual classic FA genes yield varying cellular sensitivities to cross-linking damage. To address this question, we generated cellular knock-out models of FA core complex components and FANCD2 and found that FANCD2-null mutants display higher levels of spontaneous chromosomal damage and hypersensitivity to replication-blocking lesions than Fanconi anemia complementation group L (FANCL)-null mutants, suggesting that FANCD2 provides a basal level of DNA protection countering endogenous lesions in the absence of monoubiquitination. FANCD2's ubiquitination-independent function is likely involved in optimized recruitment of nucleolytic activities for the processing and protection of stressed replication forks. Our results reveal that FANCD2 has a ubiquitination-independent role in countering endogenous levels of replication stress, a function that is critical for the maintenance of genomic stability. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Oxidative Stress Resistance in Deinococcus radiodurans†

PubMed Central

Slade, Dea; Radman, Miroslav

2011-01-01

Summary: Deinococcus radiodurans is a robust bacterium best known for its capacity to repair massive DNA damage efficiently and accurately. It is extremely resistant to many DNA-damaging agents, including ionizing radiation and UV radiation (100 to 295 nm), desiccation, and mitomycin C, which induce oxidative damage not only to DNA but also to all cellular macromolecules via the production of reactive oxygen species. The extreme resilience of D. radiodurans to oxidative stress is imparted synergistically by an efficient protection of proteins against oxidative stress and an efficient DNA repair mechanism, enhanced by functional redundancies in both systems. D. radiodurans assets for the prevention of and recovery from oxidative stress are extensively reviewed here. Radiation- and desiccation-resistant bacteria such as D. radiodurans have substantially lower protein oxidation levels than do sensitive bacteria but have similar yields of DNA double-strand breaks. These findings challenge the concept of DNA as the primary target of radiation toxicity while advancing protein damage, and the protection of proteins against oxidative damage, as a new paradigm of radiation toxicity and survival. The protection of DNA repair and other proteins against oxidative damage is imparted by enzymatic and nonenzymatic antioxidant defense systems dominated by divalent manganese complexes. Given that oxidative stress caused by the accumulation of reactive oxygen species is associated with aging and cancer, a comprehensive outlook on D. radiodurans strategies of combating oxidative stress may open new avenues for antiaging and anticancer treatments. The study of the antioxidation protection in D. radiodurans is therefore of considerable potential interest for medicine and public health. PMID:21372322

Chromatin Collapse during Caspase-dependent Apoptotic Cell Death Requires DNA Fragmentation Factor, 40-kDa Subunit-/Caspase-activated Deoxyribonuclease-mediated 3′-OH Single-strand DNA Breaks*

PubMed Central

Iglesias-Guimarais, Victoria; Gil-Guiñon, Estel; Sánchez-Osuna, María; Casanelles, Elisenda; García-Belinchón, Mercè; Comella, Joan X.; Yuste, Victor J.

2013-01-01

Apoptotic nuclear morphology and oligonucleosomal double-strand DNA fragments (also known as DNA ladder) are considered the hallmarks of apoptotic cell death. From a classic point of view, these two processes occur concomitantly. Once activated, DNA fragmentation factor, 40-kDa subunit (DFF40)/caspase-activated DNase (CAD) endonuclease hydrolyzes the DNA into oligonucleosomal-size pieces, facilitating the chromatin package. However, the dogma that the apoptotic nuclear morphology depends on DNA fragmentation has been questioned. Here, we use different cellular models, including MEF CAD−/− cells, to unravel the mechanism by which DFF40/CAD influences chromatin condensation and nuclear collapse during apoptosis. Upon apoptotic insult, SK-N-AS cells display caspase-dependent apoptotic nuclear alterations in the absence of internucleosomal DNA degradation. The overexpression of a wild-type form of DFF40/CAD endonuclease, but not of different catalytic-null mutants, restores the cellular ability to degrade the chromatin into oligonucleosomal-length fragments. We show that apoptotic nuclear collapse requires a 3′-OH endonucleolytic activity even though the internucleosomal DNA degradation is impaired. Moreover, alkaline unwinding electrophoresis and In Situ End-Labeling (ISEL)/In Situ Nick Translation (ISNT) assays reveal that the apoptotic DNA damage observed in the DNA ladder-deficient SK-N-AS cells is characterized by the presence of single-strand nicks/breaks. Apoptotic single-strand breaks can be impaired by DFF40/CAD knockdown, abrogating nuclear collapse and disassembly. In conclusion, the highest order of chromatin compaction observed in the later steps of caspase-dependent apoptosis relies on DFF40/CAD-mediated DNA damage by generating 3′-OH ends in single-strand rather than double-strand DNA nicks/breaks. PMID:23430749

Strand-specific Recognition of DNA Damages by XPD Provides Insights into Nucleotide Excision Repair Substrate Versatility*

PubMed Central

Buechner, Claudia N.; Heil, Korbinian; Michels, Gudrun; Carell, Thomas; Kisker, Caroline; Tessmer, Ingrid

2014-01-01

Recognition and removal of DNA damages is essential for cellular and organismal viability. Nucleotide excision repair (NER) is the sole mechanism in humans for the repair of carcinogenic UV irradiation-induced photoproducts in the DNA, such as cyclobutane pyrimidine dimers. The broad substrate versatility of NER further includes, among others, various bulky DNA adducts. It has been proposed that the 5′-3′ helicase XPD (xeroderma pigmentosum group D) protein plays a decisive role in damage verification. However, despite recent advances such as the identification of a DNA-binding channel and central pore in the protein, through which the DNA is threaded, as well as a dedicated lesion recognition pocket near the pore, the exact process of target site recognition and verification in eukaryotic NER still remained elusive. Our single molecule analysis by atomic force microscopy reveals for the first time that XPD utilizes different recognition strategies to verify structurally diverse lesions. Bulky fluorescein damage is preferentially detected on the translocated strand, whereas the opposite strand preference is observed for a cyclobutane pyrimidine dimer lesion. Both states, however, lead to similar conformational changes in the resulting specific complexes, indicating a merge to a “final” verification state, which may then trigger the recruitment of further NER proteins. PMID:24338567

Inactivation of the budding yeast cohesin loader Scc2 alters gene expression both globally and in response to a single DNA double strand break

PubMed Central

Lindgren, Emma; Hägg, Sara; Giordano, Fosco; Björkegren, Johan; Ström, Lena

2014-01-01

Genome integrity is fundamental for cell survival and cell cycle progression. Important mechanisms for keeping the genome intact are proper sister chromatid segregation, correct gene regulation and efficient repair of damaged DNA. Cohesin and its DNA loader, the Scc2/4 complex have been implicated in all these cellular actions. The gene regulation role has been described in several organisms. In yeast it has been suggested that the proteins in the cohesin network would effect transcription based on its role as insulator. More recently, data are emerging indicating direct roles for gene regulation also in yeast. Here we extend these studies by investigating whether the cohesin loader Scc2 is involved in regulation of gene expression. We performed global gene expression profiling in the absence and presence of DNA damage, in wild type and Scc2 deficient G2/M arrested cells, when it is known that Scc2 is important for DNA double strand break repair and formation of damage induced cohesion. We found that not only the DNA damage specific transcriptional response is distorted after inactivation of Scc2 but also the overall transcription profile. Interestingly, these alterations did not correlate with changes in cohesin binding. PMID:25483075

Co-expression of antioxidant enzymes with expression of p53, DNA repair, and heat shock protein genes in the gamma ray-irradiated hermaphroditic fish Kryptolebias marmoratus larvae.

PubMed

Rhee, Jae-Sung; Kim, Bo-Mi; Kim, Ryeo-Ok; Seo, Jung Soo; Kim, Il-Chan; Lee, Young-Mi; Lee, Jae-Seong

2013-09-15

To investigate effects of gamma ray irradiation in the hermaphroditic fish, Kryptolebias marmoratus larvae, we checked expression of p53, DNA repair, and heat shock protein genes with several antioxidant enzyme activities by quantitative real-time RT-PCR and biochemical methods in response to different doses of gamma radiation. As a result, the level of gamma radiation-induced DNA damage was initiated after 4Gy of radiation, and biochemical and molecular damage became substantial from 8Gy. In particular, several DNA repair mechanism-related genes were significantly modulated in the 6Gy gamma radiation-exposed fish larvae, suggesting that upregulation of such DNA repair genes was closely associated with cell survival after gamma irradiation. The mRNA expression of p53 and most hsps was also significantly upregulated at high doses of gamma radiation related to cellular damage. This finding indicates that gamma radiation can induce oxidative stress with associated antioxidant enzyme activities, and linked to modulation of the expression of DNA repair-related genes as one of the defense mechanisms against radiation damage. This study provides a better understanding of the molecular mode of action of defense mechanisms upon gamma radiation in fish larvae. Copyright © 2013 Elsevier B.V. All rights reserved.

DNA repair and aging: the impact of the p53 family.

PubMed

Nicolai, Sara; Rossi, Antonello; Di Daniele, Nicola; Melino, Gerry; Annicchiarico-Petruzzelli, Margherita; Raschellà, Giuseppe

2015-12-01

Cells are constantly exposed to endogenous and exogenous factors that threaten the integrity of their DNA. The maintenance of genome stability is of paramount importance in the prevention of both cancer and aging processes. To deal with DNA damage, cells put into operation a sophisticated and coordinated mechanism, collectively known as DNA damage response (DDR). The DDR orchestrates different cellular processes, such as DNA repair, senescence and apoptosis. Among the key factors of the DDR, the related proteins p53, p63 and p73, all belonging to the same family of transcription factors, play multiple relevant roles. Indeed, the members of this family are directly involved in the induction of cell cycle arrest that is necessary to allow the cells to repair. Alternatively, they can promote cell death in case of prolonged or irreparable DNA damage. They also take part in a more direct task by modulating the expression of core factors involved in the process of DNA repair or by directly interacting with them. In this review we will analyze the fundamental roles of the p53 family in the aging process through their multifaceted function in DDR.

DNA repair and aging: the impact of the p53 family

PubMed Central

Nicolai, Sara; Rossi, Antonello; Di Daniele, Nicola; Melino, Gerry; Annicchiarico-Petruzzelli, Margherita; Raschellà, Giuseppe

2015-01-01

Cells are constantly exposed to endogenous and exogenous factors that threaten the integrity of their DNA. The maintenance of genome stability is of paramount importance in the prevention of both cancer and aging processes. To deal with DNA damage, cells put into operation a sophisticated and coordinated mechanism, collectively known as DNA damage response (DDR). The DDR orchestrates different cellular processes, such as DNA repair, senescence and apoptosis. Among the key factors of the DDR, the related proteins p53, p63 and p73, all belonging to the same family of transcription factors, play multiple relevant roles. Indeed, the members of this family are directly involved in the induction of cell cycle arrest that is necessary to allow the cells to repair. Alternatively, they can promote cell death in case of prolonged or irreparable DNA damage. They also take part in a more direct task by modulating the expression of core factors involved in the process of DNA repair or by directly interacting with them. In this review we will analyze the fundamental roles of the p53 family in the aging process through their multifaceted function in DDR. PMID:26668111

Sunlight damage to cellular DNA: Focus on oxidatively generated lesions.

PubMed

Schuch, André Passaglia; Moreno, Natália Cestari; Schuch, Natielen Jacques; Menck, Carlos Frederico Martins; Garcia, Camila Carrião Machado

2017-06-01

The routine and often unavoidable exposure to solar ultraviolet (UV) radiation makes it one of the most significant environmental DNA-damaging agents to which humans are exposed. Sunlight, specifically UVB and UVA, triggers various types of DNA damage. Although sunlight, mainly UVB, is necessary for the production of vitamin D, which is necessary for human health, DNA damage may have several deleterious consequences, such as cell death, mutagenesis, photoaging and cancer. UVA and UVB photons can be directly absorbed not only by DNA, which results in lesions, but also by the chromophores that are present in skin cells. This process leads to the formation of reactive oxygen species, which may indirectly cause DNA damage. Despite many decades of investigation, the discrimination among the consequences of these different types of lesions is not clear. However, human cells have complex systems to avoid the deleterious effects of the reactive species produced by sunlight. These systems include antioxidants, that protect DNA, and mechanisms of DNA damage repair and tolerance. Genetic defects in these mechanisms that have clear harmful effects in the exposed skin are found in several human syndromes. The best known of these is xeroderma pigmentosum (XP), whose patients are defective in the nucleotide excision repair (NER) and translesion synthesis (TLS) pathways. These patients are mainly affected due to UV-induced pyrimidine dimers, but there is growing evidence that XP cells are also defective in the protection against other types of lesions, including oxidized DNA bases. This raises a question regarding the relative roles of the various forms of sunlight-induced DNA damage on skin carcinogenesis and photoaging. Therefore, knowledge of what occurs in XP patients may still bring important contributions to the understanding of the biological impact of sunlight-induced deleterious effects on the skin cells. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Gamma and Ion-Beam Irradiation of DNA: Free Radical Mechanisms, Electron Effects, and Radiation Chemical Track Structure

PubMed Central

Sevilla, Michael D.; Becker, David; Kumar, Anil; Adhikary, Amitava

2016-01-01

The focus of our laboratory’s investigation is to study the direct-type DNA damage mechanisms resulting from γ-ray and ion-beam radiation-induced free radical processes in DNA which lead to molecular damage important to cellular survival. This work compares the results of low LET (γ−) and high LET (ion-beam) radiation to develop a chemical track structure model for ion-beam radiation damage to DNA. Recent studies on protonation states of cytosine cation radicals in the N1-substituted cytosine derivatives in their ground state and 5-methylcytosine cation radicals in ground as well as in excited state are described. Our results exhibit a radical signature of excitations in 5-methylcytosine cation radical. Moreover, our recent theoretical studies elucidate the role of electron-induced reactions (low energy electrons (LEE), presolvated electrons (epre−), and aqueous (or, solvated) electrons (eaq−)). Finally DFT calculations of the ionization potentials of various sugar radicals show the relative reactivity of these species. PMID:27695205

Gamma and ion-beam irradiation of DNA: Free radical mechanisms, electron effects, and radiation chemical track structure

NASA Astrophysics Data System (ADS)

Sevilla, Michael D.; Becker, David; Kumar, Anil; Adhikary, Amitava

2016-11-01

The focus of our laboratory's investigation is to study the direct-type DNA damage mechanisms resulting from γ-ray and ion-beam radiation-induced free radical processes in DNA which lead to molecular damage important to cellular survival. This work compares the results of low LET (γ-) and high LET (ion-beam) radiation to develop a chemical track structure model for ion-beam radiation damage to DNA. Recent studies on protonation states of cytosine cation radicals in the N1-substituted cytosine derivatives in their ground state and 5-methylcytosine cation radicals in ground as well as in excited state are described. Our results exhibit a radical signature of excitations in 5-methylcytosine cation radical. Moreover, our recent theoretical studies elucidate the role of electron-induced reactions (low energy electrons (LEE), presolvated electrons (epre-), and aqueous (or, solvated) electrons (eaq-)). Finally DFT calculations of the ionization potentials of various sugar radicals show the relative reactivity of these species.

Ionization Cross Sections and Dissociation Channels of the DNA Sugar-Phosphate Backbone by Electron Collisions

NASA Technical Reports Server (NTRS)

Dateo, Christopher; Huo, Winifred M.; Fletcher, Graham D.

2004-01-01

It has been suggested that the genotoxic effects of ionizing radiation in living cells are not caused by the highly energetic incident radiation, but rather are induced by less energetic secondary species generated, the most abundant of which are free electrons.' The secondary electrons will further react to cause DNA damage via indirect and direct mechanisms. Detailed knowledge of these mechanisms is ultimately important for the development of global models of cellular radiation damage. We are studying one possible mechanism for the formation cf DNA strand breaks involving dissociative ionization of the DNA sugar-phosphate backbone induced by secondary electron co!lisions. We will present ionization cross sections at electron collision energies between threshold and 10 KeV using the improved binary encounter dipole (iBED) formulation' Preliminary results of the possible dissociative ionization pathways will be presented. It is speculated that radical fragments produced from the dissociative ionization can further react, providing a possible mechanism for double strand breaks and base damage.

Multifunctional Role of ATM/Tel1 Kinase in Genome Stability: From the DNA Damage Response to Telomere Maintenance

PubMed Central

2014-01-01

The mammalian protein kinase ataxia telangiectasia mutated (ATM) is a key regulator of the DNA double-strand-break response and belongs to the evolutionary conserved phosphatidylinositol-3-kinase-related protein kinases. ATM deficiency causes ataxia telangiectasia (AT), a genetic disorder that is characterized by premature aging, cerebellar neuropathy, immunodeficiency, and predisposition to cancer. AT cells show defects in the DNA damage-response pathway, cell-cycle control, and telomere maintenance and length regulation. Likewise, in Saccharomyces cerevisiae, haploid strains defective in the TEL1 gene, the ATM ortholog, show chromosomal aberrations and short telomeres. In this review, we outline the complex role of ATM/Tel1 in maintaining genomic stability through its control of numerous aspects of cellular survival. In particular, we describe how ATM/Tel1 participates in the signal transduction pathways elicited by DNA damage and in telomere homeostasis and its importance as a barrier to cancer development. PMID:25247188

Cytosolic NADP(+)-dependent isocitrate dehydrogenase status modulates oxidative damage to cells.

PubMed

Lee, Su Min; Koh, Ho-Jin; Park, Dong-Chan; Song, Byoung J; Huh, Tae-Lin; Park, Jeen-Woo

2002-06-01

NADPH is an important cofactor in many biosynthesis pathways and the regeneration of reduced glutathione, critically important in cellular defense against oxidative damage. It is mainly produced by glucose 6-phosphate dehydrogenase (G6PD), malic enzyme, and the cytosolic form of NADP(+)-dependent isocitrate dehydrogenase (IDPc). Little information is available about the role of IDPc in antioxidant defense. In this study we investigated the role of IDPc against cytotoxicity induced by oxidative stress by comparing the relative degree of cellular responses in three different NIH3T3 cells with stable transfection with the cDNA for mouse IDPc in sense and antisense orientations, where IDPc activities were 3-4-fold higher and 35% lower, respectively, than that in the parental cells carrying the vector alone. Although the activities of other antioxidant enzymes, such as superoxide dismutase, catalase, glutathione reductase, glutathione peroxidase, and G6PD, were comparable in all transformed cells, the ratio of GSSG to total glutathione was significantly higher in the cells expressing the lower level of IDPc. This finding indicates that IDPc is essential for the efficient glutathione recycling. Upon transient exposure to increasing concentrations of H(2)O(2) or menadione, an intracellular source of free radicals and reactive oxygen species, the cells with low levels of IDPc became more sensitive to oxidative damage by H(2)O(2) or menadione. Lipid peroxidation, oxidative DNA damage, and intracellular peroxide generation were higher in the cell-line expressing the lower level of IDPc. However, the cells with the highly over-expressed IDPc exhibited enhanced resistance against oxidative stress, compared to the control cells. This study provides direct evidence correlating the activities of IDPc and the maintenance of the cellular redox state, suggesting that IDPc plays an important role in cellular defense against oxidative stress.

Radiation induced pulmonary fibrosis as a model of progressive fibrosis: Contributions of DNA damage, inflammatory response and cellular senescence genes.

PubMed

Beach, Tyler A; Johnston, Carl J; Groves, Angela M; Williams, Jacqueline P; Finkelstein, Jacob N

2017-04-01

Purpose/Aim of Study: Studies of pulmonary fibrosis (PF) have resulted in DNA damage, inflammatory response, and cellular senescence being widely hypothesized to play a role in the progression of the disease. Utilizing these aforementioned terms, genomics databases were interrogated along with the term, "pulmonary fibrosis," to identify genes common among all 4 search terms. Findings were compared to data derived from a model of radiation-induced progressive pulmonary fibrosis (RIPF) to verify that these genes are similarly expressed, supporting the use of radiation as a model for diseases involving PF, such as human idiopathic pulmonary fibrosis (IPF). In an established model of RIPF, C57BL/6J mice were exposed to 12.5 Gy thorax irradiation and sacrificed at 24 hours, 1, 4, 12, and 32 weeks following exposure, and lung tissue was compared to age-matched controls by RNA sequencing. Of 176 PF associated gene transcripts identified by database interrogation, 146 (>82%) were present in our experimental model, throughout the progression of RIPF. Analysis revealed that nearly 85% of PF gene transcripts were associated with at least 1 other search term. Furthermore, of 22 genes common to all four terms, 16 were present experimentally in RIPF. This illustrates the validity of RIPF as a model of progressive PF/IPF based on the numbers of transcripts reported in both literature and observed experimentally. Well characterized genes and proteins are implicated in this model, supporting the hypotheses that DNA damage, inflammatory response and cellular senescence are associated with the pathogenesis of PF.

Biomonitoring of DNA damage in peripheral blood lymphocytes of subjects with dental restorative fillings.

PubMed

Di Pietro, Angela; Visalli, Giuseppa; La Maestra, Sebastiano; Micale, Rosanna; Baluce, Barbara; Matarese, Giovanni; Cingano, Luciano; Scoglio, Maria Elena

2008-02-29

Dental fillings provide a major iatrogenic exposure to xenobiotic compounds due to the high prevalence of surface restorations in developed countries. Experimental data suggest that both amalgams, which contain mercury, and resin-based dental materials cause an impairment of the cellular pro- and anti-oxidant redox balance. The aim of this study was to assess the potential genotoxicity of dental restorative compounds in peripheral blood lymphocytes of young exposed subjects compared with controls. The study examined, by use of the comet assay, 68 carefully selected subjects taking into account the major known confounding factors. In the 44 exposed subjects, the mean numbers of restored surfaces was 3.0 and 3.8 in males and females, respectively. Tail length, percentage of DNA in the tail, tail moment or Olive tail moment were twofold higher in the exposed group than in unexposed controls, with significant differences. No significant difference was observed between amalgam and composite fillings. Furthermore, as shown by multivariate analysis, the association between dental fillings and DNA damage was enhanced by the number of fillings and by the exposure time. Among the lifestyle variables, a moderate physical activity showed a protective effect, being inversely correlated to the DNA damage parameters evaluated. On the whole, the use of DNA-migration allowed us to detect for the first time the potential adverse impact on human health of both kinds of dental filling constituents, the amalgams and the methacrylates. The main mechanism underlying the genotoxicity of dental restorative materials of various nature may be ascribed to the ability of both amalgams and methacrylates to trigger the generation of cellular reactive oxygen species, able to cause oxidative DNA lesions.

Quantitation of Radiation Induced Deletion and Recombination Events Associated with Repeated DNA Sequences

NASA Technical Reports Server (NTRS)

Sinden, Richard R.

1999-01-01

Manned exploration of space exposes the explorers to a complex and novel radiation environment. The galactic cosmic ray and trapped belt radiation (predominantly proton) components of this environment are relatively constant, and the variations with the solar cycle are well understood and predictable. The level of radiation encountered in low earth orbits is determined by several factors, including altitude, inclination of orbit with respect to the equator, and spacecraft shielding. At higher altitudes, and on a Mars mission, the level of radiation exposure will increase significantly. A significant fraction of the dose may be delivered by solar particle events which vary dramatically in dose rate and incident particle spectrum. High-LET radiation is of particular concern. High-LET radiation, a component of galactic cosmic rays (GCR), is comprised of a variety of charged particles of various energies (10 MeV/n to 10 GeV/n), including about 87% photons, 12% helium ions, and heavy ions (including iron). These high energy particles can cause significant damage to target cells. The different particle types and energies result in different patterns of energy deposition at the molecular and cellular level in a primary target cell. They can also cause significant damage to other, nearby cells as a result of secondary particles. Protons, for instance produce secondaries that include photons, neutrons, pions, heavy particles, as well as gamma rays. Heavy ions deposit energy in a "track" in which the magnitude of the damage varies as the particle loses energy. Heavy ions produce secondary delta rays, or electrons. The distribution of damage through tissue is described by a Bragg curve which will be characteristic for different energies. Needless to say there are differences in the RBE of protons and a particles. High-LET heavy ions are particularly damaging to cells as they do continual damage throughout their track. Differences in these energy deposition patterns can significantly influence the nature of DNA damage and the ability of cellular systems to repair such damage. It has been suspected that these differences also affect the spatial distribution of damage within the DNA of the interphase cell nucleus and produce corresponding differences in endpoints related to health effects. The interaction of a single high-LET particle with chromatin has been suggested to cause multiple double strand breaks within a relatively short distance. In part this is due to the organization of DNA into chromatin fibers in which distant regions of the DNA helix can be physically juxtaposed by the various levels of coiling of the DNA. This prediction was confirmed by the detection of the generation of double strand DNA fragments of 100-2000 bp following exposure to high-LET ions (including iron).

DNA damage and repair capacity in workers exposed to low concentrations of benzene.

PubMed

Lovreglio, Piero; Doria, Denise; Fracasso, Maria Enrica; Barbieri, Anna; Sabatini, Laura; Drago, Ignazio; Violante, Francesco S; Soleo, Leonardo

2016-03-01

DNA damage and cellular repair capacity were studied in 18 male fuel tanker drivers and 13 male filling-station attendants exposed to low and very low concentrations of benzene, respectively, and compared to 20 males with no occupational exposure (controls). Exposure to airborne benzene was measured using passive personal samplers, and internal doses were assayed through the biomarkers t,t-muconic acid, S-phenylmercapturic acid and urinary benzene. DNA damage was evaluated using tail intensity (TI) determined by the comet assay in peripheral lymphocytes. Urinary 7-hydro-8-oxo-2'-deoxyguanosine (8-oxodG) was measured as a biomarker of oxidative damage. DNA repair kinetics were assessed using the comet assay in lymphocytes sampled 20 and 60 min post H2O2 exposure. Benzene exposure differed significantly between the drivers (median 246.3 µg/m(3)), attendants (median 13.8 µg/m(3)), and controls (median 4.1 µg/m(3)). There were no differences in TI and 8-oxodG among the three groups, or between smokers and non-smokers. DNA repair kinetics were similar among the drivers, attendants and controls, although the comet assay on H2 O2 -damaged lymphocytes after 60 min revealed significantly lower levels of TI only in drivers. The DNA repair process in smokers was similar to that observed in drivers. In conclusion, this study found no relationship between low levels of benzene exposure and DNA damage, although there was evidence that exposure interferes with DNA repair kinetics. The biological impact of this finding on the onset of genotoxic effects in exposed workers has still to be ascertained. © 2015 Wiley Periodicals, Inc.

Increased plasma levels of circulating cell-free mitochondrial DNA in suicide attempters: associations with HPA-axis hyperactivity

PubMed Central

Lindqvist, D; Fernström, J; Grudet, C; Ljunggren, L; Träskman-Bendz, L; Ohlsson, L; Westrin, Å

2016-01-01

Preclinical data suggest that chronic stress may cause cellular damage and mitochondrial dysfunction, potentially leading to the release of mitochondrial DNA (mtDNA) into the bloodstream. Major depressive disorder has been associated with an increased amount of mtDNA in leukocytes from saliva samples and blood; however, no previous studies have measured plasma levels of free-circulating mtDNA in a clinical psychiatric sample. In this study, free circulating mtDNA was quantified in plasma samples from 37 suicide attempters, who had undergone a dexamethasone suppression test (DST), and 37 healthy controls. We hypothesized that free circulating mtDNA would be elevated in the suicide attempters and would be associated with hypothalamic–pituitary–adrenal (HPA)-axis hyperactivity. Suicide attempters had significantly higher plasma levels of free-circulating mtDNA compared with healthy controls at different time points (pre- and post-DST; all P-values<2.98E−12, Cohen's d ranging from 2.55 to 4.01). Pre-DST plasma levels of mtDNA were positively correlated with post-DST cortisol levels (rho=0.49, P<0.003). Suicide attempters may have elevated plasma levels of free-circulating mtDNA, which are related to impaired HPA-axis negative feedback. This peripheral index is consistent with an increased cellular or mitochondrial damage. The specific cells and tissues contributing to plasma levels of free-circulating mtDNA are not known, as is the specificity of this finding for suicide attempters. Future studies are needed in order to better understand the relevance of increased free-circulating mtDNA in relation to the pathophysiology underlying suicidal behavior and depression. PMID:27922635

Epstein-Barr Virus Hijacks DNA Damage Response Transducers to Orchestrate Its Life Cycle.

PubMed

Hau, Pok Man; Tsao, Sai Wah

2017-11-16

The Epstein-Barr virus (EBV) is a ubiquitous virus that infects most of the human population. EBV infection is associated with multiple human cancers, including Burkitt's lymphoma, Hodgkin's lymphoma, a subset of gastric carcinomas, and almost all undifferentiated non-keratinizing nasopharyngeal carcinoma. Intensive research has shown that EBV triggers a DNA damage response (DDR) during primary infection and lytic reactivation. The EBV-encoded viral proteins have been implicated in deregulating the DDR signaling pathways. The consequences of DDR inactivation lead to genomic instability and promote cellular transformation. This review summarizes the current understanding of the relationship between EBV infection and the DDR transducers, including ATM (ataxia telangiectasia mutated), ATR (ATM and Rad3-related), and DNA-PK (DNA-dependent protein kinase), and discusses how EBV manipulates the DDR signaling pathways to complete the replication process of viral DNA during lytic reactivation.

Fe65 is required for Tip60-directed histone H4 acetylation at DNA strand breaks

PubMed Central

Stante, Maria; Minopoli, Giuseppina; Passaro, Fabiana; Raia, Maddalena; Vecchio, Luigi Del; Russo, Tommaso

2009-01-01

Fe65 is a binding partner of the Alzheimer's β-amyloid precursor protein APP. The possible involvement of this protein in the cellular response to DNA damage was suggested by the observation that Fe65 null mice are more sensitive to genotoxic stress than WT counterpart. Fe65 associated with chromatin under basal conditions and its involvement in DNA damage repair requires this association. A known partner of Fe65 is the histone acetyltransferase Tip60. Considering the crucial role of Tip60 in DNA repair, we explored the hypothesis that the phenotype of Fe65 null cells depended on its interaction with Tip60. We demonstrated that Fe65 knockdown impaired recruitment of Tip60-TRRAP complex to DNA double strand breaks and decreased histone H4 acetylation. Accordingly, the efficiency of DNA repair was decreased upon Fe65 suppression. To explore whether APP has a role in this mechanism, we analyzed a Fe65 mutant unable to bind to APP. This mutant failed to rescue the phenotypes of Fe65 null cells; furthermore, APP/APLP2 suppression results in the impairment of recruitment of Tip60-TRRAP complex to DNA double strand breaks, decreased histone H4 acetylation and repair efficiency. On these bases, we propose that Fe65 and its interaction with APP play an important role in the response to DNA damage by assisting the recruitment of Tip60-TRRAP to DNA damage sites. PMID:19282473

EXO1 is critical for embryogenesis and the DNA damage response in mice with a hypomorphic Nbs1 allele.

PubMed

Rein, Katrin; Yanez, Diana A; Terré, Berta; Palenzuela, Lluís; Aivio, Suvi; Wei, Kaichun; Edelmann, Winfried; Stark, Jeremy M; Stracker, Travis H

2015-09-03

The maintenance of genome stability is critical for the suppression of diverse human pathologies that include developmental disorders, premature aging, infertility and predisposition to cancer. The DNA damage response (DDR) orchestrates the appropriate cellular responses following the detection of lesions to prevent genomic instability. The MRE11 complex is a sensor of DNA double strand breaks (DSBs) and plays key roles in multiple aspects of the DDR, including DNA end resection that is critical for signaling and DNA repair. The MRE11 complex has been shown to function both upstream and in concert with the 5'-3' exonuclease EXO1 in DNA resection, but it remains unclear to what extent EXO1 influences DSB responses independently of the MRE11 complex. Here we examine the genetic relationship of the MRE11 complex and EXO1 during mammalian development and in response to DNA damage. Deletion of Exo1 in mice expressing a hypomorphic allele of Nbs1 leads to severe developmental impairment, embryonic death and chromosomal instability. While EXO1 plays a minimal role in normal cells, its loss strongly influences DNA replication, DNA repair, checkpoint signaling and damage sensitivity in NBS1 hypomorphic cells. Collectively, our results establish a key role for EXO1 in modulating the severity of hypomorphic MRE11 complex mutations. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

Regulation and Modulation of Human DNA Polymerase δ Activity and Function

PubMed Central

Wang, Xiaoxiao; Zhang, Sufang; Zhang, Zhongtao; Lee, Ernest Y. C.

2017-01-01

This review focuses on the regulation and modulation of human DNA polymerase δ (Pol δ). The emphasis is on the mechanisms that regulate the activity and properties of Pol δ in DNA repair and replication. The areas covered are the degradation of the p12 subunit of Pol δ, which converts it from a heterotetramer (Pol δ4) to a heterotrimer (Pol δ3), in response to DNA damage and also during the cell cycle. The biochemical mechanisms that lead to degradation of p12 are reviewed, as well as the properties of Pol δ4 and Pol δ3 that provide insights into their functions in DNA replication and repair. The second focus of the review involves the functions of two Pol δ binding proteins, polymerase delta interaction protein 46 (PDIP46) and polymerase delta interaction protein 38 (PDIP38), both of which are multi-functional proteins. PDIP46 is a novel activator of Pol δ4, and the impact of this function is discussed in relation to its potential roles in DNA replication. Several new models for the roles of Pol δ3 and Pol δ4 in leading and lagging strand DNA synthesis that integrate a role for PDIP46 are presented. PDIP38 has multiple cellular localizations including the mitochondria, the spliceosomes and the nucleus. It has been implicated in a number of cellular functions, including the regulation of specialized DNA polymerases, mitosis, the DNA damage response, mouse double minute 2 homolog (Mdm2) alternative splicing and the regulation of the NADPH oxidase 4 (Nox4). PMID:28737709

Non-equilibrium repressor binding kinetics link DNA damage dose to transcriptional timing within the SOS gene network.

PubMed

Culyba, Matthew J; Kubiak, Jeffrey M; Mo, Charlie Y; Goulian, Mark; Kohli, Rahul M

2018-06-01

Biochemical pathways are often genetically encoded as simple transcription regulation networks, where one transcription factor regulates the expression of multiple genes in a pathway. The relative timing of each promoter's activation and shut-off within the network can impact physiology. In the DNA damage repair pathway (known as the SOS response) of Escherichia coli, approximately 40 genes are regulated by the LexA repressor. After a DNA damaging event, LexA degradation triggers SOS gene transcription, which is temporally separated into subsets of 'early', 'middle', and 'late' genes. Although this feature plays an important role in regulating the SOS response, both the range of this separation and its underlying mechanism are not experimentally defined. Here we show that, at low doses of DNA damage, the timing of promoter activities is not separated. Instead, timing differences only emerge at higher levels of DNA damage and increase as a function of DNA damage dose. To understand mechanism, we derived a series of synthetic SOS gene promoters which vary in LexA-operator binding kinetics, but are otherwise identical, and then studied their activity over a large dose-range of DNA damage. In distinction to established models based on rapid equilibrium assumptions, the data best fit a kinetic model of repressor occupancy at promoters, where the drop in cellular LexA levels associated with higher doses of DNA damage leads to non-equilibrium binding kinetics of LexA at operators. Operators with slow LexA binding kinetics achieve their minimal occupancy state at later times than operators with fast binding kinetics, resulting in a time separation of peak promoter activity between genes. These data provide insight into this remarkable feature of the SOS pathway by demonstrating how a single transcription factor can be employed to control the relative timing of each gene's transcription as a function of stimulus dose.

Toxicity of nano- and micro-sized silver particles in human hepatocyte cell line L02

NASA Astrophysics Data System (ADS)

Liu, Pengpeng; Guan, Rongfa; Ye, Xingqian; Jiang, Jiaxin; Liu, Mingqi; Huang, Guangrong; Chen, Xiaoting

2011-07-01

Silver nanoparticles (Ag NPs) previously classified as antimicrobial agents have been widely used in consumers and industrial products, especially food storage material. Ag NPs used as antimicrobial agents may be found in liver. Thus, examination of the ability of Ag NPs to penetrate the liver is warranted. The aim of the study was to determine the optimal viability assay for using with Ag NPs in order to assess their toxicity to liver cells. For toxicity evaluations, cellular morphology, mitochondrial function (3-(4, 5-dimethylazol-2-yl)-2, 5-diphenyl-tetrazolium bromide, MTT assay), membrane leakage of lactate dehydrogenase (lactate dehydrogenase, LDH release assay), Oxidative stress markers (malonaldehyde (MDA), glutathione (GSH) and superoxide dismutase (SOD)), DNA damage (single cell gel eletrophoresis, SCGE assay), and protein damage were assessed under control and exposed conditions (24 h of exposure). The results showed that mitochondrial function decreased significantly in cells exposed to Ag NPs at 25 μg·mL-1. LDH leakage significantly increased in cells exposed to Ag NPs (>= 25 μg mL-1) while micro-sized silver particles tested displayed LDH leakage only at higher doses (100 μg·mL-1). The microscopic studies demonstrated that nanoparticle-exposed cells at higher doses became abnormal in size, displaying cellular shrinkage, and an acquisition of an irregular shape. Due to toxicity of silver, further study conducted with reference to its oxidative stress. The results exhibited significant depletion of GSH level, increase in SOD levels and lead to lipid peroxidation, which suggested that cytotoxicity of Ag NPs in liver cells might be mediated through oxidative stress. The results demonstrates that Ag NPs lead to cellular morphological modifications, LDH leakage, mitochondrial dysfunction, and cause increased generation of ROS, depletion of GSH, lipid peroxidation, oxidative DNA damage and protein damage. Though the exact mechanism behind Ag NPs toxicity is suggested oxidative stress and lipid peroxidation playing an important role in Ag NPs elicited cell membrane disruption, DNA damage, protein damage and subsequent cell death. Our preliminary data suggest that oxidative stress might contribute to Ag NPs cytotoxicity. To reveal whether apoptosis involved in Ag NPs toxicity, further studies are underway.

Sulforaphane inhibits damage-induced poly (ADP-ribosyl)ation via direct interaction of its cellular metabolites with PARP-1.

PubMed

Piberger, Ann Liza; Keil, Claudia; Platz, Stefanie; Rohn, Sascha; Hartwig, Andrea

2015-11-01

The isothiocyanate sulforaphane, a major breakdown product of the broccoli glucosinolate glucoraphanin, has frequently been proposed to exert anticarcinogenic properties. Potential underlying mechanisms include a zinc release from Kelch-like ECH-associated protein 1 followed by the induction of detoxifying enzymes. This suggests that sulforaphane may also interfere with other zinc-binding proteins, e.g. those essential for DNA repair. Therefore, we explored the impact of sulforaphane on poly (ADP-ribose)polymerase-1 (PARP-1), poly (ADP-ribosyl)ation (PARylation), and DNA single-strand break repair (SSBR) in cell culture. Immunofluorescence analyses showed that sulforaphane diminished H2 O2 -induced PARylation in HeLa S3 cells starting from 15 μM despite increased lesion induction under these conditions. Subcellular experiments quantifying the damage-induced incorporation of (32) P-ADP-ribose by PARP-1 displayed no direct impact of sulforaphane itself, but cellular metabolites, namely the glutathione conjugates of sulforaphane and its interconversion product erucin, reduced PARP-1 activity concentration dependently. Interestingly, this sulforaphane metabolite-induced PARP-1 inhibition was prevented by thiol compounds. PARP-1 is a stimulating factor for DNA SSBR-rate and we further demonstrated that 25 μM sulforaphane also delayed the rejoining of H2 O2 -induced DNA strand breaks, although this might be partly due to increased lesion frequencies. Sulforaphane interferes with damage-induced PARylation and SSBR, which implies a sulforaphane-dependent impairment of genomic stability. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Characterization of a DNA damage-recognition protein mammalian cells that binds specifically to intrastrand d(GpG) and d(ApG) DNA adducts of the anticancer drug cisplatin

DOE Office of Scientific and Technical Information (OSTI.GOV)

Donahue, B.A.; Augot, M.; Bellon, S.F.

1990-06-19

A factor has been identified in extracts from human HeLa and hamster V79 cells that retards the electrophoretic mobility of several DNA restriction fragments modified with the antitumor drug cis-diamminedichloroplatinum(II) (cisplatin). Binding of the factor to cisplatin-modified DNA was sensitive to pretreatment with proteinase K, establishing that the factor is a protein. Gel mobility shifts were observed with probes containing as few as seven Pt atoms per kilobase of duplex DNA. By competition experiments the dissociation constant, K{sub d}, of the protein from cisplatin-modified DNA was estimated to be (1-20) {times} 10{sup {minus}10} M. Protein binding is selective for DNAmore » modified with cisplatin, (Pt(en)Cl{sub 2}) (en, ethylenediamine), and (Pt(dach)Cl{sub 2}) (dach, 1,2-diaminocyclohexane) but not with chemotherapeutically inactive trans-diamminedichloroplatinum(II) or monofunctionally coordinating (Pt(dien)Cl)Cl (dien, diethylenetriamine) complexes. The protein binds specifically to 1,2-intrastrand d(GpG) and d(ApG) cross-links formed by cisplatin. The apparent molecular weight of the protein is 91,000, as determined by sucrose gradient centrifugation of a preparation partially purified by ammonium sulfate fractionation. Binding of the protein to platinum-modified DNA does not require cofactors but is sensitive to treatment with 5 mM MnCl{sub 2}, CdCl{sub 2}, CoCl{sub 2}, or ZnCl{sub 2} and with 1 mM HgCl{sub 2}. This protein, alone or in conjunction with other cellular constituents, could be of general importance in the initial stages of processing of mammalian DNA damaged by cisplatin or other genotoxic agents and may belong to a wider class of such cellular damage-recognition proteins (DRPs).« less

Cellular defense against UVB-induced phototoxicity by cytosolic NADP(+)-dependent isocitrate dehydrogenase.

PubMed

Jo, Seung-Hee; Lee, So-Hyun; Chun, Hang Suk; Lee, Su Min; Koh, Ho-Jin; Lee, Sung-Eun; Chun, Jang-Soo; Park, Jeen-Woo; Huh, Tae-Lin

2002-03-29

Ultraviolet (UV) radiation is known as a major cause of skin photoaging and photocarcinogenesis. Many harmful effects of UV radiation are associated with the generation of reactive oxygen species. Recently, we have shown that NADP(+)-dependent isocitrate dehydrogenase is involved in the supply of NADPH needed for GSH production against cellular oxidative damage. In this study we investigated the role of cytosolic form of NADP(+)-dependent isocitrate dehydrogenase (IDPc) against UV radiation-induced cytotoxicity by comparing the relative degree of cellular responses in three different NIH3T3 cells with stable transfection with the cDNA for mouse IDPc in sense and antisense orientations, where IDPc activities were 2.3-fold higher and 39% lower, respectively, than that in the parental cells carrying the vector alone. Upon exposure to UVB (312 nm), the cells with low levels of IDPc became more sensitive to cell killing. Lipid peroxidation, protein oxidation, oxidative DNA damage, and intracellular peroxide generation were higher in the cell-line expressing the lower level of IDPc. However, the cells with the highly overexpressed IDPc exhibited enhanced resistance against UV radiation, compared to the control cells. The data indicate that IDPc plays an important role in cellular defense against UV radiation-induced oxidative injury. (c)2002 Elsevier Science (USA).

[Fanconi anemia: cellular and molecular features].

PubMed

Macé, G; Briot, D; Guervilly, J-H; Rosselli, F

2007-02-01

Fanconi anemia (FA) is a recessive human cancer prone syndrome featuring bone marrow failure, developmental abnormalities and hypersensitivity to DNA crosslinking agents exposure. 11 among 12 FA gene have been isolated. The biochemical functions of the FANC proteins remain poorly understood. Anyhow, to cope with DNA crosslinks a cell needs a functional FANC pathway. Moreover, the FANC proteins appear to be involved in cell protection against oxidative damage and in the control of TNF-alpha activity. In this review, we describe the current understanding of the FANC pathway and we present how it may be integrated in the complex networks of proteins involved in maintaining the cellular homeostasis.

Regulatory mechanisms of RNA function: emerging roles of DNA repair enzymes.

PubMed

Jobert, Laure; Nilsen, Hilde

2014-07-01

The acquisition of an appropriate set of chemical modifications is required in order to establish correct structure of RNA molecules, and essential for their function. Modification of RNA bases affects RNA maturation, RNA processing, RNA quality control, and protein translation. Some RNA modifications are directly involved in the regulation of these processes. RNA epigenetics is emerging as a mechanism to achieve dynamic regulation of RNA function. Other modifications may prevent or be a signal for degradation. All types of RNA species are subject to processing or degradation, and numerous cellular mechanisms are involved. Unexpectedly, several studies during the last decade have established a connection between DNA and RNA surveillance mechanisms in eukaryotes. Several proteins that respond to DNA damage, either to process or to signal the presence of damaged DNA, have been shown to participate in RNA quality control, turnover or processing. Some enzymes that repair DNA damage may also process modified RNA substrates. In this review, we give an overview of the DNA repair proteins that function in RNA metabolism. We also discuss the roles of two base excision repair enzymes, SMUG1 and APE1, in RNA quality control.

Contribution of indirect effects to clustered damage in DNA irradiated with protons.

PubMed

Pachnerová Brabcová, K; Štěpán, V; Karamitros, M; Karabín, M; Dostálek, P; Incerti, S; Davídková, M; Sihver, L

2015-09-01

Protons are the dominant particles both in galactic cosmic rays and in solar particle events and, furthermore, proton irradiation becomes increasingly used in tumour treatment. It is believed that complex DNA damage is the determining factor for the consequent cellular response to radiation. DNA plasmid pBR322 was irradiated at U120-M cyclotron with 30 MeV protons and treated with two Escherichia coli base excision repair enzymes. The yields of SSBs and DSBs were analysed using agarose gel electrophoresis. DNA has been irradiated in the presence of hydroxyl radical scavenger (coumarin-3-carboxylic acid) in order to distinguish between direct and indirect damage of the biological target. Pure scavenger solution was used as a probe for measurement of induced OH· radical yields. Experimental OH· radical yield kinetics was compared with predictions computed by two theoretical models-RADAMOL and Geant4-DNA. Both approaches use Geant4-DNA for description of physical stages of radiation action, and then each of them applies a distinct model for description of the pre-chemical and chemical stage. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Co-opting the Fanconi Anemia Genomic Stability Pathway Enables Herpesvirus DNA Synthesis and Productive Growth

PubMed Central

Karttunen, Heidi; Savas, Jeffrey N.; McKinney, Caleb; Chen, Yu-Hung; Yates, John R.; Hukkanen, Veijo; Huang, Tony T.; Mohr, Ian

2015-01-01

SUMMARY DNA damage associated with viral DNA synthesis can result in double strand breaks that threaten genome integrity and must be repaired. Here, we establish that the cellular Fanconi Anemia (FA) genomic stability pathway is exploited by HSV1 to promote viral DNA synthesis and enable its productive growth. Potent FA pathway activation in HSV1-infected cells resulted in monoubiquitination of FA effector proteins, FANCI and FANCD2 (FANCI-D2) and required the viral DNA polymerase. FANCD2 relocalized to viral replication compartments and FANCI-D2 interacted with a multi-subunit complex containing the virus-encoded single-stranded DNA-binding protein ICP8. Significantly, while HSV1 productive growth was impaired in monoubiquitination-defective FA patient cells, this restriction was partially surmounted by antagonizing the DNA-dependent protein kinase (DNA-PK), a critical enzyme required for non-homologous end-joining (NHEJ). This identifies the FA-pathway as a new cellular factor required for herpesvirus productive growth and suggests that FA-mediated suppression of NHEJ is a fundamental step in the viral lifecycle. PMID:24954902

ATM activation and its recruitment to damaged DNA require binding to the C terminus of Nbs1.

PubMed

You, Zhongsheng; Chahwan, Charly; Bailis, Julie; Hunter, Tony; Russell, Paul

2005-07-01

ATM has a central role in controlling the cellular responses to DNA damage. It and other phosphoinositide 3-kinase-related kinases (PIKKs) have giant helical HEAT repeat domains in their amino-terminal regions. The functions of these domains in PIKKs are not well understood. ATM activation in response to DNA damage appears to be regulated by the Mre11-Rad50-Nbs1 (MRN) complex, although the exact functional relationship between the MRN complex and ATM is uncertain. Here we show that two pairs of HEAT repeats in fission yeast ATM (Tel1) interact with an FXF/Y motif at the C terminus of Nbs1. This interaction resembles nucleoporin FXFG motif binding to HEAT repeats in importin-beta. Budding yeast Nbs1 (Xrs2) appears to have two FXF/Y motifs that interact with Tel1 (ATM). In Xenopus egg extracts, the C terminus of Nbs1 recruits ATM to damaged DNA, where it is subsequently autophosphorylated. This interaction is essential for ATM activation. A C-terminal 147-amino-acid fragment of Nbs1 that has the Mre11- and ATM-binding domains can restore ATM activation in an Nbs1-depleted extract. We conclude that an interaction between specific HEAT repeats in ATM and the C-terminal FXF/Y domain of Nbs1 is essential for ATM activation. We propose that conformational changes in the MRN complex that occur upon binding to damaged DNA are transmitted through the FXF/Y-HEAT interface to activate ATM. This interaction also retains active ATM at sites of DNA damage.

Replicative stress and alterations in cell cycle checkpoint controls following acetaminophen hepatotoxicity restrict liver regeneration.

PubMed

Viswanathan, Preeti; Sharma, Yogeshwar; Gupta, Priya; Gupta, Sanjeev

2018-03-05

Acetaminophen hepatotoxicity is a leading cause of hepatic failure with impairments in liver regeneration producing significant mortality. Multiple intracellular events, including oxidative stress, mitochondrial damage, inflammation, etc., signify acetaminophen toxicity, although how these may alter cell cycle controls has been unknown and was studied for its significance in liver regeneration. Assays were performed in HuH-7 human hepatocellular carcinoma cells, primary human hepatocytes and tissue samples from people with acetaminophen-induced acute liver failure. Cellular oxidative stress, DNA damage and cell proliferation events were investigated by mitochondrial membrane potential assays, flow cytometry, fluorescence staining, comet assays and spotted arrays for protein expression after acetaminophen exposures. In experimental groups with acetaminophen toxicity, impaired mitochondrial viability and substantial DNA damage were observed with rapid loss of cells in S and G2/M and cell cycle restrictions or even exit in the remainder. This resulted from altered expression of the DNA damage regulator, ATM and downstream transducers, which imposed G1/S checkpoint arrest, delayed entry into S and restricted G2 transit. Tissues from people with acute liver failure confirmed hepatic DNA damage and cell cycle-related lesions, including restrictions of hepatocytes in aneuploid states. Remarkably, treatment of cells with a cytoprotective cytokine reversed acetaminophen-induced restrictions to restore cycling. Cell cycle lesions following mitochondrial and DNA damage led to failure of hepatic regeneration in acetaminophen toxicity but their reversibility offers molecular targets for treating acute liver failure. © 2018 John Wiley & Sons Ltd.

Retinoic acid-related orphan receptor-α is induced in the setting of DNA damage and promotes pulmonary emphysema.

PubMed

Shi, Ying; Cao, Jiaofei; Gao, Jane; Zheng, Liang; Goodwin, Andrew; An, Chang Hyoek; Patel, Avignat; Lee, Janet S; Duncan, Steven R; Kaminski, Naftali; Pandit, Kusum V; Rosas, Ivan O; Choi, Augustine M K; Morse, Danielle

2012-09-01

The discovery that retinoic acid-related orphan receptor (Rora)-α is highly expressed in lungs of patients with COPD led us to hypothesize that Rora may contribute to the pathogenesis of emphysema. To determine the role of Rora in smoke-induced emphysema. Cigarette smoke extract in vitro and elastase or cigarette smoke exposure in vivo were used to model smoke-related cell stress and airspace enlargement. Lung tissue from patients undergoing lung transplantation was examined for markers of DNA damage and Rora expression. Rora expression was induced by cigarette smoke in mice and in cell culture. Gene expression profiling of Rora-null mice exposed to cigarette smoke demonstrated enrichment for genes involved in DNA repair. Rora expression increased and Rora translocated to the nucleus after DNA damage. Inhibition of ataxia telangiectasia mutated decreased the induction of Rora. Gene silencing of Rora attenuated apoptotic cell death in response to cigarette smoke extract, whereas overexpression of Rora enhanced apoptosis. Rora-deficient mice were protected from elastase and cigarette smoke induced airspace enlargement. Finally, lungs of patients with COPD showed evidence of increased DNA damage even in the absence of active smoking. Taken together, these findings suggest that DNA damage may contribute to the pathogenesis of emphysema, and that Rora has a previously unrecognized role in cellular responses to genotoxicity. These findings provide a potential link between emphysema and features of premature ageing, including enhanced susceptibility to lung cancer.

Influence of anoxia on the induction of mutations by phenylalanine radicals during gamma-irradiation of plasmid DNA in aqueous solution.

PubMed

Kuipers, Gitta K; Slotman, Ben J; Reitsma-Wijker, Carola A; van Andel, Rob J; Poldervaart, Hester A; Lafleur, M Vincent M

2004-12-21

When DNA is irradiated in aqueous solution, most of the damage is inflicted by water-derived radicals. This is called the indirect effect of ionizing radiation. However in whole cells not only the primary formed water radicals play a role, because some cellular compounds form secondary radicals which can also damage DNA. It is known that the amino acid phenylalanine is able to react with water radicals, resulting in the production of secondary phenylalanine radicals which can damage and inactivate DNA. In a previous study the influence of the presence of phenylalanine during gamma-irradiation of DNA in aqueous solution under oxic conditions was studied. Under anoxic irradiation conditions different amounts and types of reactive water-derived radicals are formed compared to oxic conditions and also different phenylalanine radicals are formed. Therefore, this study examines the influence of the presence of phenylalanine under anoxic conditions on the gamma-radiation-induced mutation spectrum. The results indicate that phenylalanine radicals are damaging to DNA, but less effective compared to primary water radicals. On the mutational level, in the presence of phenylalanine radicals under anoxic conditions, the amount of mutations on G:C base pairs was significantly decreased as compared to oxic conditions. Furthermore, the results of this study indicate that nucleotide excision repair is involved in repair of both inactivating and mutagenic damage induced by phenylalanine radicals under anoxic conditions.

Enhancement of UVB radiation-mediated apoptosis by knockdown of cytosolic NADP+-dependent isocitrate dehydrogenase in HaCaT cells.

PubMed

Lee, Su Jeong; Park, Jeen-Woo

2014-04-01

Ultraviolet B (UVB) radiation induces the production of reactive oxygen species (ROS) that promote apoptotic cell death. We showed that cytosolic NADP+-dependent isocitrate dehydrogenase (IDPc) plays an essential role in the control of cellular redox balance and defense against oxidative damage, by supplying NADPH for antioxidant systems. In this study, we demonstrated that knockdown of IDPc expression by RNA interference enhances UVB-induced apoptosis of immortalized human HaCaT keratinocytes. This effect manifested as DNA fragmentation, changes in cellular redox status, mitochondrial dysfunction, and modulation of apoptotic marker expression. Based on our findings, we suggest that attenuation of IDPc expression may protect skin from UVB-mediated damage, by inducing the apoptosis of UV-damaged cells.

Mitochondria and mitochondrial DNA as relevant targets for environmental contaminants.

PubMed

Roubicek, Deborah A; Souza-Pinto, Nadja C de

2017-11-01

The mitochondrial DNA (mtDNA) is a closed circular molecule that encodes, in humans, 13 polypeptides components of the oxidative phosphorylation complexes. Integrity of the mitochondrial genome is essential for mitochondrial function and cellular homeostasis, and mutations and deletions in the mtDNA lead to oxidative stress, mitochondrial dysfunction and cell death. In vitro and in situ studies suggest that when exposed to certain genotoxins, mtDNA accumulates more damage than nuclear DNA, likely owing to its organization and localization in the mitochondrial matrix, which tends to accumulate lipophilic, positively charged molecules. In that regard, several relevant environmental and occupational contaminants have physical-chemical characteristics that indicate that they might accumulate in mitochondria and target mtDNA. Nonetheless, very little is known so far about mtDNA damage and mitochondrial dysfunction due to environmental exposure, either in model organisms or in humans. In this article, we discuss some of the characteristics of mtDNA which render it a potentially relevant target for damage by environmental contaminants, as well as possible functional consequences of damage/mutation accumulation. In addition, we review the data available in the literature focusing on mitochondrial effects of the most common classes of environmental pollutants. From that, we conclude that several lines of experimental evidence support the idea that mitochondria and mtDNA are susceptible and biologically relevant targets for pollutants, and more studies, including mechanistic ones, are needed to shed more light into the contribution of mitochondrial dysfunction to the environmental and human health effects of chemical exposure. Copyright © 2017 Elsevier B.V. All rights reserved.

Structure of human DNA polymerase iota and the mechanism of DNA synthesis.

PubMed

Makarova, A V; Kulbachinskiy, A V

2012-06-01

Cellular DNA polymerases belong to several families and carry out different functions. Highly accurate replicative DNA polymerases play the major role in cell genome replication. A number of new specialized DNA polymerases were discovered at the turn of XX-XXI centuries and have been intensively studied during the last decade. Due to the special structure of the active site, these enzymes efficiently perform synthesis on damaged DNA but are characterized by low fidelity. Human DNA polymerase iota (Pol ι) belongs to the Y-family of specialized DNA polymerases and is one of the most error-prone enzymes involved in DNA synthesis. In contrast to other DNA polymerases, Pol ι is able to use noncanonical Hoogsteen interactions for nucleotide base pairing. This allows it to incorporate nucleotides opposite various lesions in the DNA template that impair Watson-Crick interactions. Based on the data of X-ray structural analysis of Pol ι in complexes with various DNA templates and dNTP substrates, we consider the structural peculiarities of the Pol ι active site and discuss possible mechanisms that ensure the unique behavior of the enzyme on damaged and undamaged DNA.

Gamma rays induce DNA damage and oxidative stress associated with impaired growth and reproduction in the copepod Tigriopus japonicus.

PubMed

Han, Jeonghoon; Won, Eun-Ji; Lee, Bo-Young; Hwang, Un-Ki; Kim, Il-Chan; Yim, Joung Han; Leung, Kenneth Mei Yee; Lee, Yong Sung; Lee, Jae-Seong

2014-07-01

Nuclear radioisotope accidents are potentially ecologically devastating due to their impact on marine organisms. To examine the effects of exposure of a marine organism to radioisotopes, we irradiated the intertidal copepod Tigriopus japonicus with several doses of gamma radiation and analyzed the effects on mortality, fecundity, and molting by assessing antioxidant enzyme activities and gene expression patterns. No mortality was observed at 96h, even in response to exposure to a high dose (800Gy) of radiation, but mortality rate was significantly increased 120h (5 days) after exposure to 600 or 800Gy gamma ray radiation. We observed a dose-dependent reduction in fecundity of ovigerous females; even the group irradiated with 50Gy showed a significant reduction in fecundity, suggesting that gamma rays are likely to have a population level effect. In addition, we observed growth retardation, particularly at the nauplius stage, in individuals after gamma irradiation. In fact, nauplii irradiated with more than 200Gy, though able to molt to copepodite stage 1, did not develop into adults. Upon gamma radiation, T. japonicus showed a dose-dependent increase in reactive oxygen species (ROS) levels, the activities of several antioxidant enzymes, and expression of double-stranded DNA break damage genes (e.g. DNA-PK, Ku70, Ku80). At a low level (sub-lethal dose) of gamma irradiation, we found dose-dependent upregulation of p53, implying cellular damage in T. japonicus in response to sub-lethal doses of gamma irradiation, suggesting that T. japonicus is not susceptible to sub-lethal doses of gamma irradiation. Additionally, antioxidant genes, phase II enzyme (e.g. GSTs), and cellular chaperone genes (e.g. Hsps) that are involved in cellular defense mechanisms also showed the same expression patterns for sublethal doses of gamma irradiation (50-200Gy). These findings indicate that sublethal doses of gamma radiation can induce oxidative stress-mediated DNA damage and increase the expression of antioxidant enzymes and proteins with chaperone-related functions, thereby significantly affecting life history parameters such as fecundity and molting in the copepod T. japonicus. Copyright © 2014 Elsevier B.V. All rights reserved.

Selective antitumor activity of roscovitine in head and neck cancer

PubMed Central

Biktasova, Asel; Bellinger, Gary; Yarbrough, Wendell G.; Issaeva, Natalia

2016-01-01

Radiation and chemotherapy that are commonly used to treat human cancers damage cellular DNA. DNA damage appears to be more toxic to cancer cells than normal cells, most likely due to deregulated checkpoint activation and/or deficiency in DNA repair pathways that are characteristics of many tumors. However, unwanted side effects arise as a result of DNA damage to normal cells during the treatment. Here, we show that roscovitine, a cyclin-dependent kinase (CDK) inhibitor that inhibits CDK-1, CDK-2, CDK-5, CDK-7, and CDK-9 due to competitive binding to the ATP site on the kinases, causes significant DNA damage followed by p53-dependent cell death in human papilloma virus (HPV)-positive, but not in HPV-negative, head and neck cancer cells. Since HPV positivity was a molecular marker for increased sensitivity of cells to roscovitine, we reasoned that systemic roscovitine administration would not be toxic to healthy HPV-negative tissue. Indeed, low roscovitine doses significantly inhibited the growth of HPV-associated xenografted tumors in mice without causing any detectable side effects. Given that inhibition of CDKs has been shown to inhibit replication of several viruses, we suggest that roscovitine treatment may represent a selective and safe targeted therapeutic option against HPV-positive head and neck cancer. PMID:27233076

Genomic instability in human cancer: Molecular insights and opportunities for therapeutic attack and prevention through diet and nutrition

PubMed Central

Ferguson, Lynnette R.; Chen, Helen; Collins, Andrew R.; Connell, Marisa; Damia, Giovanna; Dasgupta, Santanu; Malhotra, Meenakshi; Meeker, Alan K.; Amedei, Amedeo; Amin, Amr; Ashraf, S. Salman; Aquilano, Katia; Azmi, Asfar S.; Bhakta, Dipita; Bilsland, Alan; Boosani, Chandra S.; Chen, Sophie; Ciriolo, Maria Rosa; Fujii, Hiromasa; Guha, Gunjan; Halicka, Dorota; Helferich, William G.; Keith, W. Nicol; Mohammed, Sulma I.; Niccolai, Elena; Yang, Xujuan; Honoki, Kanya; Parslow, Virginia R.; Prakash, Satya; Rezazadeh, Sarallah; Shackelford, Rodney E.; Sidransky, David; Tran, Phuoc T.; Yang, Eddy S.; Maxwell, Christopher A.

2015-01-01

Genomic instability can initiate cancer, augment progression, and influence the overall prognosis of the affected patient. Genomic instability arises from many different pathways, such as telomere damage, centrosome amplification, epigenetic modifications, and DNA damage from endogenous and exogenous sources, and can be perpetuating, or limiting, through the induction of mutations or aneuploidy, both enabling and catastrophic. Many cancer treatments induce DNA damage to impair cell division on a global scale but it is accepted that personalized treatments, those that are tailored to the particular patient and type of cancer, must also be developed. In this review, we detail the mechanisms from which genomic instability arises and can lead to cancer, as well as treatments and measures that prevent genomic instability or take advantage of the cellular defects caused by genomic instability. In particular, we identify and discuss five priority targets against genomic instability: (1) prevention of DNA damage; (2) enhancement of DNA repair; (3) targeting deficient DNA repair; (4) impairing centrosome clustering; and, (5) inhibition of telomerase activity. Moreover, we highlight vitamin D and B, selenium, carotenoids, PARP inhibitors, resveratrol, and isothiocyanates as priority approaches against genomic instability. The prioritized target sites and approaches were cross validated to identify potential synergistic effects on a number of important areas of cancer biology. PMID:25869442

Mycobacterium tuberculosis promotes genomic instability in macrophages

PubMed Central

Castro-Garza, Jorge; Luévano-Martínez, Miriam Lorena; Villarreal-Treviño, Licet; Gosálvez, Jaime; Fernández, José Luis; Dávila-Rodríguez, Martha Imelda; García-Vielma, Catalina; González-Hernández, Silvia; Cortés-Gutiérrez, Elva Irene

2018-01-01

BACKGROUND Mycobacterium tuberculosis is an intracellular pathogen, which may either block cellular defensive mechanisms and survive inside the host cell or induce cell death. Several studies are still exploring the mechanisms involved in these processes. OBJECTIVES To evaluate the genomic instability of M. tuberculosis-infected macrophages and compare it with that of uninfected macrophages. METHODS We analysed the possible variations in the genomic instability of Mycobacterium-infected macrophages using the DNA breakage detection fluorescence in situ hybridisation (DBD-FISH) technique with a whole human genome DNA probe. FINDINGS Quantitative image analyses showed a significant increase in DNA damage in infected macrophages as compared with uninfected cells. DNA breaks were localised in nuclear membrane blebs, as confirmed with DNA fragmentation assay. Furthermore, a significant increase in micronuclei and nuclear abnormalities were observed in infected macrophages versus uninfected cells. MAIN CONCLUSIONS Genomic instability occurs during mycobacterial infection and these data may be seminal for future research on host cell DNA damage in M. tuberculosis infection. PMID:29412354

AKT phosphorylates H3-threonine 45 to facilitate termination of gene transcription in response to DNA damage.

PubMed

Lee, Jong-Hyuk; Kang, Byung-Hee; Jang, Hyonchol; Kim, Tae Wan; Choi, Jinmi; Kwak, Sojung; Han, Jungwon; Cho, Eun-Jung; Youn, Hong-Duk

2015-05-19

Post-translational modifications of core histones affect various cellular processes, primarily through transcription. However, their relationship with the termination of transcription has remained largely unknown. In this study, we show that DNA damage-activated AKT phosphorylates threonine 45 of core histone H3 (H3-T45). By genome-wide chromatin immunoprecipitation sequencing (ChIP-seq) analysis, H3-T45 phosphorylation was distributed throughout DNA damage-responsive gene loci, particularly immediately after the transcription termination site. H3-T45 phosphorylation pattern showed close-resemblance to that of RNA polymerase II C-terminal domain (CTD) serine 2 phosphorylation, which establishes the transcription termination signal. AKT1 was more effective than AKT2 in phosphorylating H3-T45. Blocking H3-T45 phosphorylation by inhibiting AKT or through amino acid substitution limited RNA decay downstream of mRNA cleavage sites and decreased RNA polymerase II release from chromatin. Our findings suggest that AKT-mediated phosphorylation of H3-T45 regulates the processing of the 3' end of DNA damage-activated genes to facilitate transcriptional termination. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

Nucleotide Excision Repair and Vitamin D--Relevance for Skin Cancer Therapy.

PubMed

Pawlowska, Elzbieta; Wysokinski, Daniel; Blasiak, Janusz

2016-04-06

Ultraviolet (UV) radiation is involved in almost all skin cancer cases, but on the other hand, it stimulates the production of pre-vitamin D3, whose active metabolite, 1,25-dihydroxyvitamin D3 (1,25VD3), plays important physiological functions on binding with its receptor (vitamin D receptor, VDR). UV-induced DNA damages in the form of cyclobutane pyrimidine dimers or (6-4)-pyrimidine-pyrimidone photoproducts are frequently found in skin cancer and its precursors. Therefore, removing these lesions is essential for the prevention of skin cancer. As UV-induced DNA damages are repaired by nucleotide excision repair (NER), the interaction of 1,25VD3 with NER components can be important for skin cancer transformation. Several studies show that 1,25VD3 protects DNA against damage induced by UV, but the exact mechanism of this protection is not completely clear. 1,25VD3 was also shown to affect cell cycle regulation and apoptosis in several signaling pathways, so it can be considered as a potential modulator of the cellular DNA damage response, which is crucial for mutagenesis and cancer transformation. 1,25VD3 was shown to affect DNA repair and potentially NER through decreasing nitrosylation of DNA repair enzymes by NO overproduction by UV, but other mechanisms of the interaction between 1,25VD3 and NER machinery also are suggested. Therefore, the array of NER gene functioning could be analyzed and an appropriate amount of 1.25VD3 could be recommended to decrease UV-induced DNA damage important for skin cancer transformation.

Expanded CAG/CTG Repeat DNA Induces a Checkpoint Response That Impacts Cell Proliferation in Saccharomyces cerevisiae

PubMed Central

Sundararajan, Rangapriya; Freudenreich, Catherine H.

2011-01-01

Repetitive DNA elements are mutational hotspots in the genome, and their instability is linked to various neurological disorders and cancers. Although it is known that expanded trinucleotide repeats can interfere with DNA replication and repair, the cellular response to these events has not been characterized. Here, we demonstrate that an expanded CAG/CTG repeat elicits a DNA damage checkpoint response in budding yeast. Using microcolony and single cell pedigree analysis, we found that cells carrying an expanded CAG repeat frequently experience protracted cell division cycles, persistent arrests, and morphological abnormalities. These phenotypes were further exacerbated by mutations in DSB repair pathways, including homologous recombination and end joining, implicating a DNA damage response. Cell cycle analysis confirmed repeat-dependent S phase delays and G2/M arrests. Furthermore, we demonstrate that the above phenotypes are due to the activation of the DNA damage checkpoint, since expanded CAG repeats induced the phosphorylation of the Rad53 checkpoint kinase in a rad52Δ recombination deficient mutant. Interestingly, cells mutated for the MRX complex (Mre11-Rad50-Xrs2), a central component of DSB repair which is required to repair breaks at CAG repeats, failed to elicit repeat-specific arrests, morphological defects, or Rad53 phosphorylation. We therefore conclude that damage at expanded CAG/CTG repeats is likely sensed by the MRX complex, leading to a checkpoint response. Finally, we show that repeat expansions preferentially occur in cells experiencing growth delays. Activation of DNA damage checkpoints in repeat-containing cells could contribute to the tissue degeneration observed in trinucleotide repeat expansion diseases. PMID:21437275

REC-2006-A Fractionated Extract of Podophyllum hexandrum Protects Cellular DNA from Radiation-Induced Damage by Reducing the Initial Damage and Enhancing Its Repair In Vivo.

PubMed

Chaudhary, Pankaj; Shukla, Sandeep Kumar; Sharma, Rakesh Kumar

2011-01-01

Podophyllum hexandrum, a perennial herb commonly known as the Himalayan May Apple, is well known in Indian and Chinese traditional systems of medicine. P. hexandrum has been widely used for the treatment of venereal warts, skin infections, bacterial and viral infections, and different cancers of the brain, lung and bladder. This study aimed at elucidating the effect of REC-2006, a bioactive fractionated extract from the rhizome of P. hexandrum, on the kinetics of induction and repair of radiation-induced DNA damage in murine thymocytes in vivo. We evaluated its effect on non-specific radiation-induced DNA damage by the alkaline halo assay in terms of relative nuclear spreading factor (RNSF) and gene-specific radiation-induced DNA damage via semi-quantitative polymerase chain reaction. Whole body exposure of animals with gamma rays (10 Gy) caused a significant amount of DNA damage in thymocytes (RNSF values 17.7 ± 0.47, 12.96 ± 1.64 and 3.3 ± 0.014) and a reduction in the amplification of β-globin gene to 0, 28 and 43% at 0, 15 and 60 min, respectively. Administrating REC-2006 at a radioprotective concentration (15 mg kg(-1) body weight) 1 h before irradiation resulted in time-dependent reduction of DNA damage evident as a decrease in RNSF values 6.156 ± 0.576, 1.647 ± 0.534 and 0.496 ± 0.012, and an increase in β-globin gene amplification 36, 95 and 99%, at 0, 15 and 60 min, respectively. REC-2006 scavenged radiation-induced hydroxyl radicals in a dose-dependent manner stabilized DPPH free radicals and also inhibited superoxide anions. Various polyphenols and flavonoides present in REC-2006 might contribute to scavenging of radiation-induced free radicals, thereby preventing DNA damage and stimulating its repair.

REC-2006—A Fractionated Extract of Podophyllum hexandrum Protects Cellular DNA from Radiation-Induced Damage by Reducing the Initial Damage and Enhancing Its Repair In Vivo

PubMed Central

Chaudhary, Pankaj; Shukla, Sandeep Kumar; Sharma, Rakesh Kumar

2011-01-01

Podophyllum hexandrum, a perennial herb commonly known as the Himalayan May Apple, is well known in Indian and Chinese traditional systems of medicine. P. hexandrum has been widely used for the treatment of venereal warts, skin infections, bacterial and viral infections, and different cancers of the brain, lung and bladder. This study aimed at elucidating the effect of REC-2006, a bioactive fractionated extract from the rhizome of P. hexandrum, on the kinetics of induction and repair of radiation-induced DNA damage in murine thymocytes in vivo. We evaluated its effect on non-specific radiation-induced DNA damage by the alkaline halo assay in terms of relative nuclear spreading factor (RNSF) and gene-specific radiation-induced DNA damage via semi-quantitative polymerase chain reaction. Whole body exposure of animals with gamma rays (10 Gy) caused a significant amount of DNA damage in thymocytes (RNSF values 17.7 ± 0.47, 12.96 ± 1.64 and 3.3 ± 0.014) and a reduction in the amplification of β-globin gene to 0, 28 and 43% at 0, 15 and 60 min, respectively. Administrating REC-2006 at a radioprotective concentration (15 mg kg−1 body weight) 1 h before irradiation resulted in time-dependent reduction of DNA damage evident as a decrease in RNSF values 6.156 ± 0.576, 1.647 ± 0.534 and 0.496 ± 0.012, and an increase in β-globin gene amplification 36, 95 and 99%, at 0, 15 and 60 min, respectively. REC-2006 scavenged radiation-induced hydroxyl radicals in a dose-dependent manner stabilized DPPH free radicals and also inhibited superoxide anions. Various polyphenols and flavonoides present in REC-2006 might contribute to scavenging of radiation-induced free radicals, thereby preventing DNA damage and stimulating its repair. PMID:20008078

DNA Damage by Ionizing Radiation: Tandem Double Lesions by Charged Particles

NASA Technical Reports Server (NTRS)

Huo, Winifred M.; Chaban, Galina M.; Wang, Dunyou; Dateo, Christopher E.

2005-01-01

Oxidative damages by ionizing radiation are the source of radiation-induced carcinogenesis, damage to the central nervous system, lowering of the immune response, as well as other radiation-induced damages to human health. Monte Carlo track simulations and kinetic modeling of radiation damages to the DNA employ available molecular and cellular data to simulate the biological effect of high and low LET radiation io the DNA. While the simulations predict single and double strand breaks and base damages, so far all complex lesions are the result of stochastic coincidence from independent processes. Tandem double lesions have not yet been taken into account. Unlike the standard double lesions that are produced by two separate attacks by charged particles or radicals, tandem double lesions are produced by one single attack. The standard double lesions dominate at the high dosage regime. On the other hand, tandem double lesions do not depend on stochastic coincidences and become important at the low dosage regime of particular interest to NASA. Tandem double lesions by hydroxyl radical attack of guanine in isolated DNA have been reported at a dosage of radiation as low as 10 Gy. The formation of two tandem base lesions was found to be linear with the applied doses, a characteristic of tandem lesions. However, tandem double lesions from attack by a charged particle have not been reported.

The use of comet assay in plant toxicology: recent advances

PubMed Central

Santos, Conceição L. V.; Pourrut, Bertrand; Ferreira de Oliveira, José M. P.

2015-01-01

The systematic study of genotoxicity in plants induced by contaminants and other stress agents has been hindered to date by the lack of reliable and robust biomarkers. The comet assay is a versatile and sensitive method for the evaluation of DNA damages and DNA repair capacity at single-cell level. Due to its simplicity and sensitivity, and the small number of cells required to obtain robust results, the use of plant comet assay has drastically increased in the last decade. For years its use was restricted to a few model species, e.g., Allium cepa, Nicotiana tabacum, Vicia faba, or Arabidopsis thaliana but this number largely increased in the last years. Plant comet assay has been used to study the genotoxic impact of radiation, chemicals including pesticides, phytocompounds, heavy metals, nanoparticles or contaminated complex matrices. Here we will review the most recent data on the use of this technique as a standard approach for studying the genotoxic effects of different stress conditions on plants. Also, we will discuss the integration of information provided by the comet assay with other DNA-damage indicators, and with cellular responses including oxidative stress, cell division or cell death. Finally, we will focus on putative relations between transcripts related with DNA damage pathways, DNA replication and repair, oxidative stress and cell cycle progression that have been identified in plant cells with comet assays demonstrating DNA damage. PMID:26175750

A dual mechanism involved in membrane and nucleic acid disruption of AvBD103b, a new avian defensin from the king penguin, against Salmonella enteritidis CVCC3377.

PubMed

Teng, Da; Wang, Xiumin; Xi, Di; Mao, Ruoyu; Zhang, Yong; Guan, Qingfeng; Zhang, Jun; Wang, Jianhua

2014-10-01

The food-borne bacterial gastrointestinal infection is a serious public health threat. Defensins are evolutionarily conserved innate immune components with broad-spectrum antibacterial activity that do not easily induce resistance. AvBD103b, an avian defensin with potent activity against Salmonella enteritidis, was isolated from the stomach contents of the king penguin (Aptenodytes patagonicus). To elucidate further the antibacterial mechanism of AvBD103b, its effect on the S. enteritidis CVCC3377 cell membrane and intracellular DNA was researched. The cell surface hydrophobicity and a N-phenyl-1-naphthylamine uptake assay demonstrated that AvBD103b treatment increased the cell surface hydrophobicity and outer membrane permeability. Atomic absorption spectrometry, ultraviolet spectrophotometry, flow cytometry, and transmission electron microscopy (TEM) indicated that AvBD103b treatment can lead to the release of the cellular contents and cell death through damage of the membrane. DNA gel retardation and circular dichroism analysis demonstrated that AvBD103b interacted with DNA and intercalated into the DNA base pairs. A cell cycle assay demonstrated that AvBD103b affected cellular functions, such as DNA synthesis. Our results confirmed that AvBD103b exerts its antibacterial activity by damaging the cell membrane and interfering with intracellular DNA, ultimately causing cell death, and suggested that AvBD103b may be a promising candidate as an alternative to antibiotics against S. enteritidis.

Genotoxic effects of vinclozolin on the aquatic insect Chironomus riparius (Diptera, Chironomidae).

PubMed

Aquilino, Mónica; Sánchez-Argüello, Paloma; Martínez-Guitarte, José-Luis

2018-01-01

Vinclozolin (Vz) is a pollutant found in aquatic environments whose antiandrogenic effects in reproduction are well known in mammals. Although its reproductive effects have been less studied in invertebrates, other effects, including genotoxicity, have been described. Therefore, in this work, we studied the genotoxic effects of Vz in the freshwater benthic invertebrate Chironomus riparius. DNA damage was evaluated with the comet assay (tail area, olive moment, tail moment and % DNA in tail), and the transcriptional levels of different genes involved in DNA repair (ATM, NLK and XRCC1) and apoptosis (DECAY) were measured by RT-PCR. Fourth instar larvae of C. riparius, were exposed to Vz for 24 h at 20 and 200 μg/L. The Vz exposures affected the DNA integrity in this organism, since a dose-response relationship occurred, with DNA strand breaks significantly increased with increased dose for tail area, olive moment and tail moment parameters. Additionally, the lower concentration of Vz produced a significant induction of the transcripts of three genes under study (ATM, NLK and XRCC1) showing the activation of the cellular repair mechanism. In contrast, the expression of these genes with the highest concentration were downregulated, indicating failure of the cellular repair mechanism, which would explain the higher DNA damage. These data report for the first time the alterations of Vz on gene transcription of an insect and confirm the potential genotoxicity of this compound on freshwater invertebrates. Copyright © 2017 Elsevier Ltd. All rights reserved.

Ribosome Synthesis and MAPK Activity Modulate Ionizing Radiation-Induced Germ Cell Apoptosis in Caenorhabditis elegans

PubMed Central

Eberhard, Ralf; Stergiou, Lilli; Hofmann, E. Randal; Hofmann, Jen; Haenni, Simon; Teo, Youjin; Furger, André; Hengartner, Michael O.

2013-01-01

Synthesis of ribosomal RNA by RNA polymerase I (RNA pol I) is an elemental biological process and is key for cellular homeostasis. In a forward genetic screen in C. elegans designed to identify DNA damage-response factors, we isolated a point mutation of RNA pol I, rpoa-2(op259), that leads to altered rRNA synthesis and a concomitant resistance to ionizing radiation (IR)-induced germ cell apoptosis. This weak apoptotic IR response could be phenocopied when interfering with other factors of ribosome synthesis. Surprisingly, despite their resistance to DNA damage, rpoa-2(op259) mutants present a normal CEP-1/p53 response to IR and increased basal CEP-1 activity under normal growth conditions. In parallel, rpoa-2(op259) leads to reduced Ras/MAPK pathway activity, which is required for germ cell progression and physiological germ cell death. Ras/MAPK gain-of-function conditions could rescue the IR response defect in rpoa-2(op259), pointing to a function for Ras/MAPK in modulating DNA damage-induced apoptosis downstream of CEP-1. Our data demonstrate that a single point mutation in an RNA pol I subunit can interfere with multiple key signalling pathways. Ribosome synthesis and growth-factor signalling are perturbed in many cancer cells; such an interplay between basic cellular processes and signalling might be critical for how tumours evolve or respond to treatment. PMID:24278030

Single-molecule localization microscopy reveals molecular transactions during RAD51 filament assembly at cellular DNA damage sites

PubMed Central

Haas, Kalina T; Lee, MiYoung; Esposito, Alessandro; Venkitaraman, Ashok R

2018-01-01

Abstract RAD51 recombinase assembles on single-stranded (ss)DNA substrates exposed by DNA end-resection to initiate homologous recombination (HR), a process fundamental to genome integrity. RAD51 assembly has been characterized using purified proteins, but its ultrastructural topography in the cell nucleus is unexplored. Here, we combine cell genetics with single-molecule localization microscopy and a palette of bespoke analytical tools, to visualize molecular transactions during RAD51 assembly in the cellular milieu at resolutions approaching 30–40 nm. In several human cell types, RAD51 focalizes in clusters that progressively extend into long filaments, which abut—but do not overlap—with globular bundles of replication protein A (RPA). Extended filaments alter topographically over time, suggestive of succeeding steps in HR. In cells depleted of the tumor suppressor protein BRCA2, or overexpressing its RAD51-binding BRC repeats, RAD51 fails to assemble at damage sites, although RPA accumulates unhindered. By contrast, in cells lacking a BRCA2 carboxyl (C)-terminal region targeted by cancer-causing mutations, damage-induced RAD51 assemblies initiate but do not extend into filaments. We suggest a model wherein RAD51 assembly proceeds concurrently with end-resection at adjacent sites, via an initiation step dependent on the BRC repeats, followed by filament extension through the C-terminal region of BRCA2. PMID:29309696

Delineating the requirements for spontaneous DNA damage resistance pathways in genome maintenance and viability in Saccharomyces cerevisiae.

PubMed

Morey, Natalie J; Doetsch, Paul W; Jinks-Robertson, Sue

2003-06-01

Cellular metabolic processes constantly generate reactive species that damage DNA. To counteract this relentless assault, cells have developed multiple pathways to resist damage. The base excision repair (BER) and nucleotide excision repair (NER) pathways remove damage whereas the recombination (REC) and postreplication repair (PRR) pathways bypass the damage, allowing deferred removal. Genetic studies in yeast indicate that these pathways can process a common spontaneous lesion(s), with mutational inactivation of any pathway increasing the burden on the remaining pathways. In this study, we examine the consequences of simultaneously compromising three or more of these pathways. Although the presence of a functional BER pathway alone is able to support haploid growth, retention of the NER, REC, or PRR pathway alone is not, indicating that BER is the key damage resistance pathway in yeast and may be responsible for the removal of the majority of either spontaneous DNA damage or specifically those lesions that are potentially lethal. In the diploid state, functional BER, NER, or REC alone can support growth, while PRR alone is insufficient for growth. In diploids, the presence of PRR alone may confer a lethal mutation load or, alternatively, PRR alone may be insufficient to deal with potentially lethal, replication-blocking lesions.

Two sides of the same coin: TFIIH complexes in transcription and DNA repair.

PubMed

Zhovmer, Alexander; Oksenych, Valentyn; Coin, Frédéric

2010-04-13

TFIIH is organized into a seven-subunit core associated with a three-subunit Cdk-activating kinase (CAK) module. TFIIH has roles in both transcription initiation and DNA repair. During the last 15 years, several studies have been conducted to identify the composition of the TFIIH complex involved in DNA repair. Recently, a new technique combining chromatin immunoprecipitation and western blotting resolved the hidden nature of the TFIIH complex participating in DNA repair. Following the recruitment of TFIIH to the damaged site, the CAK module is released from the core TFIIH, and the core subsequently associates with DNA repair factors. The release of the CAK is specifically driven by the recruitment of the DNA repair factor XPA and is required to promote the incision/excision of the damaged DNA. Once the DNA lesions have been repaired, the CAK module returns to the core TFIIH on the chromatin, together with the release of the repair factors. These data highlight the dynamic composition of a fundamental cellular factor that adapts its subunit composition to the cell needs.

Atorvastatin Downregulates In Vitro Methyl Methanesulfonate and Cyclophosphamide Alkylation-Mediated Cellular and DNA Injuries

PubMed Central

Christoni, Larissa S. A.; Justo, Graça; Soeiro, Maria N. C.

2018-01-01

Statins are 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors, and this class of drugs has been studied as protective agents against DNA damages. Alkylating agents (AAs) are able to induce alkylation in macromolecules, causing DNA damage, as DNA methylation. Our objective was to evaluate atorvastatin (AVA) antimutagenic, cytoprotective, and antigenotoxic potentials against DNA lesions caused by AA. AVA chemopreventive ability was evaluated using antimutagenicity assays (Salmonella/microsome assay), cytotoxicity, cell cycle, and genotoxicity assays in HepG2 cells. The cells were cotreated with AVA and the AA methyl methanesulfonate (MMS) or cyclophosphamide (CPA). Our datum showed that AVA reduces the alkylation-mediated DNA damage in different in vitro experimental models. Cytoprotection of AVA at low doses (0.1–1.0 μM) was observed after 24 h of cotreatment with MMS or CPA at their LC50, causing an increase in HepG2 survival rates. After all, AVA at 10 μM and 25 μM had decreased effect in micronucleus formation in HepG2 cells and restored cell cycle alterations induced by MMS and CPA. This study supports the hypothesis that statins can be chemopreventive agents, acting as antimutagenic, antigenotoxic, and cytoprotective components, specifically against alkylating agents of DNA. PMID:29849914

Effects of corexit oil dispersants and the WAF of dispersed oil on DNA damage and repair in cultured human bronchial airway cells, BEAS-2B

PubMed Central

Major, Danielle; Derbes, Rebecca S.; Wang, He; Roy-Engel, Astrid M.

2016-01-01

Large quantities of dispersants were used as a method to disperse the roughly 210 million gallons of spilled crude oil that consumed the Gulf of Mexico. Little is known if the oil-dispersant and oil-dispersant mixtures on human airway BEAS-2B epithelial cells. Here we present the cytotoxic and genotoxic in vitro effects on the human lung cells BEAS-2B following exposure to and oil-dispersant mixtures on human airway BEAS-2B epithelial cells. Here we present the cytotoxic and genotoxic in vitro effects on the human lung cells BEAS-2B following exposure to Corexit dispersants EC9500 and EC9527, Water Accommodated Fraction (WAF) -crude, WAF-9500 + Oil, and WAF-9527 + Oil. Cellular cytotoxicity to WAF-dispersed oil samples was observed at concentrations greater than 1000 ppm with over 70% of observed cellular death. At low concentration exposures (100 and 300 ppm) DNA damage was evidenced by the detection of single strand breaks (SSBs) and double strand breaks (DSBs) as measured by alkaline and neutral comet assay analyses. Immunoblot analyses of the phosphorylated histone H2A.X (ɣ-H2A.X) and tumor suppressor p53 protein confirmed activation of the DNA damage response due to the exposure-induced DNA breaks. Although, many xenobiotics interfere with DNA repair pathways, in vitro evaluation of the nucleotide excision repair (NER) and DSB repair pathways appear to be unaffected by the oil-dispersant mixtures tested. Overall, this study supports that oil-dispersant mixtures induce genotoxic effects in culture. PMID:27563691

Silver nanoparticles: correlating nanoparticle size and cellular uptake with genotoxicity

PubMed Central

Butler, Kimberly S.; Peeler, David J.; Casey, Brendan J.; Dair, Benita J.; Elespuru, Rosalie K.

2015-01-01

The focus of this research was to develop a better understanding of the pertinent physico-chemical properties of silver nanoparticles (AgNPs) that affect genotoxicity, specifically how cellular uptake influences a genotoxic cell response. The genotoxicity of AgNPs was assessed for three potential mechanisms: mutagenicity, clastogenicity and DNA strand-break-based DNA damage. Mutagenicity (reverse mutation assay) was assessed in five bacterial strains of Salmonella typhimurium and Echerichia coli, including TA102 that is sensitive to oxidative DNA damage. AgNPs of all sizes tested (10, 20, 50 and 100nm), along with silver nitrate (AgNO3), were negative for mutagenicity in bacteria. No AgNPs could be identified within the bacteria cells using transmission electron microscopy (TEM), indicating these bacteria lack the ability to actively uptake AgNPs 10nm or larger. Clastogenicity (flow cytometry-based micronucleus assay) and intermediate DNA damage (DNA strand breaks as measured in the Comet assay) were assessed in two mammalian white blood cell lines: Jurkat Clone E6-1 and THP-1. It was observed that micronucleus and Comet assay end points were inversely correlated with AgNP size, with smaller NPs inducing a more genotoxic response. TEM results indicated that AgNPs were confined within intracellular vesicles of mammalian cells and did not penetrate the nucleus. The genotoxicity test results and the effect of AgNO3 controls suggest that silver ions may be the primary, and perhaps only, cause of genotoxicity. Furthermore, since AgNO3 was not mutagenic in the gram-negative bacterial Ames strains tested, the lack of bacterial uptake of the AgNPs may not be the major reason for the lack of genotoxicity observed. PMID:25964273

Effects of Replication and Transcription on DNA Structure-Related Genetic Instability.

PubMed

Wang, Guliang; Vasquez, Karen M

2017-01-05

Many repetitive sequences in the human genome can adopt conformations that differ from the canonical B-DNA double helix (i.e., non-B DNA), and can impact important biological processes such as DNA replication, transcription, recombination, telomere maintenance, viral integration, transposome activation, DNA damage and repair. Thus, non-B DNA-forming sequences have been implicated in genetic instability and disease development. In this article, we discuss the interactions of non-B DNA with the replication and/or transcription machinery, particularly in disease states (e.g., tumors) that can lead to an abnormal cellular environment, and how such interactions may alter DNA replication and transcription, leading to potential conflicts at non-B DNA regions, and eventually result in genetic stability and human disease.

Effects of Replication and Transcription on DNA Structure-Related Genetic Instability

PubMed Central

Wang, Guliang; Vasquez, Karen M.

2017-01-01

Many repetitive sequences in the human genome can adopt conformations that differ from the canonical B-DNA double helix (i.e., non-B DNA), and can impact important biological processes such as DNA replication, transcription, recombination, telomere maintenance, viral integration, transposome activation, DNA damage and repair. Thus, non-B DNA-forming sequences have been implicated in genetic instability and disease development. In this article, we discuss the interactions of non-B DNA with the replication and/or transcription machinery, particularly in disease states (e.g., tumors) that can lead to an abnormal cellular environment, and how such interactions may alter DNA replication and transcription, leading to potential conflicts at non-B DNA regions, and eventually result in genetic stability and human disease. PMID:28067787

DNA damage response curtails detrimental replication stress and chromosomal instability induced by the dietary carcinogen PhIP

PubMed Central

Mimmler, Maximilian; Peter, Simon; Kraus, Alexander; Stroh, Svenja; Nikolova, Teodora; Seiwert, Nina; Hasselwander, Solveig; Neitzel, Carina; Haub, Jessica; Monien, Bernhard H.; Nicken, Petra; Steinberg, Pablo; Shay, Jerry W.; Kaina, Bernd; Fahrer, Jörg

2016-01-01

PhIP is an abundant heterocyclic aromatic amine (HCA) and important dietary carcinogen. Following metabolic activation, PhIP causes bulky DNA lesions at the C8-position of guanine. Although C8-PhIP-dG adducts are mutagenic, their interference with the DNA replication machinery and the elicited DNA damage response (DDR) have not yet been studied. Here, we analyzed PhIP-triggered replicative stress and elucidated the role of the apical DDR kinases ATR, ATM and DNA-PKcs in the cellular defense response. First, we demonstrate that PhIP induced C8-PhIP-dG adducts and DNA strand breaks. This stimulated ATR-CHK1 signaling, phosphorylation of histone 2AX and the formation of RPA foci. In proliferating cells, PhIP treatment increased the frequency of stalled replication forks and reduced fork speed. Inhibition of ATR in the presence of PhIP-induced DNA damage strongly promoted the formation of DNA double-strand breaks, activation of the ATM-CHK2 pathway and hyperphosphorylation of RPA. The abrogation of ATR signaling potentiated the cell death response and enhanced chromosomal aberrations after PhIP treatment, while ATM and DNA-PK inhibition had only marginal effects. These results strongly support the notion that ATR plays a key role in the defense against cancer formation induced by PhIP and related HCAs. PMID:27599846

DNA Repair Deficiency in Neurodegeneration

PubMed Central

Jeppesen, Dennis Kjølhede; Bohr, Vilhelm A.; Stevnsner, Tinna

2011-01-01

Deficiency in repair of nuclear and mitochondrial DNA damage has been linked to several neurodegenerative disorders. Many recent experimental results indicate that the post-mitotic neurons are particularly prone to accumulation of unrepaired DNA lesions potentially leading to progressive neurodegeneration. Nucleotide excision repair is the cellular pathway responsible for removing helix-distorting DNA damage and deficiency in such repair is found in a number of diseases with neurodegenerative phenotypes, including Xeroderma Pigmentosum and Cockayne syndrome. The main pathway for repairing oxidative base lesions is base excision repair, and such repair is crucial for neurons given their high rates of oxygen metabolism. Mismatch repair corrects base mispairs generated during replication and evidence indicates that oxidative DNA damage can cause this pathway to expand trinucleotide repeats, thereby causing Huntington’s disease. Single-strand breaks are common DNA lesions and are associated with the neurodegenerative diseases, ataxia-oculomotor apraxia-1 and spinocerebellar ataxia with axonal neuropathy-1. DNA double-strand breaks are toxic lesions and two main pathways exist for their repair: homologous recombination and non-homologous end-joining. Ataxia telangiectasia and related disorders with defects in these pathways illustrate that such defects can lead to early childhood neurodegeneration. Aging is a risk factor for neurodegeneration and accumulation of oxidative mitochondrial DNA damage may be linked with the age-associated neurodegenerative disorders Alzheimer’s disease, Parkinson’s disease and amyotrophic lateral sclerosis. Mutation in the WRN protein leads to the premature aging disease Werner syndrome, a disorder that features neurodegeneration. In this article we review the evidence linking deficiencies in the DNA repair pathways with neurodegeneration. PMID:21550379

Genomic and post-genomic effects of anti-glaucoma drugs preservatives in trabecular meshwork.

PubMed

Izzotti, Alberto; La Maestra, Sebastiano; Micale, Rosanna Tindara; Longobardi, Maria Grazia; Saccà, Sergio Claudio

2015-02-01

Oxidative stress plays an important role in glaucoma. Some preservatives of anti-glaucoma drugs, commonly used in glaucoma therapy, can prevent or induce oxidative stress in the trabecular meshwork. The aim of this study is to evaluate cellular and molecular damage induced in trabecular meshwork by preservatives contained in anti-glaucoma drugs. Cell viability (MTT test), DNA fragmentation (Comet test), oxidative DNA damage (8-oxo-dG), and gene expression (cDNA microarray) have been evaluated in trabecular meshwork specimens and in human trabecular meshwork cells treated with benzalkonium chloride, polyQuad, purite, and sofzia-like mixture. Moreover, antimicrobial effectiveness and safety of preservative contents in drugs was tested. In ex vivo experiments, benzalkonium chloride and polyQuad induced high level of DNA damage in trabecular meshwork specimens, while the effect of purite and sofzia were more attenuated. The level of DNA fragmentation induced by benzalkonium chloride was 2.4-fold higher in subjects older than 50 years than in younger subjects. Benzalkonium chloride, and polyQuad significantly increased oxidative DNA damage as compared to sham-treated specimens. Gene expression was altered by benzalkonium chloride, polyQuad, and purite but not by sofzia. In in vitro experiments, benzalkonium chloride and polyQuad dramatically decreased trabecular meshwork cell viability, increased DNA fragmentation, and altered gene expression. A lesser effect was also exerted by purite and sofzia. Genes targeted by these alterations included Fas and effector caspase-3. The efficacy of the preservatives in inhibiting bacterial growth increased the adverse effects in trabecular meshwork in terms of DNA damage and alteration of gene expression. Presented data indicates the delicate balance between efficacy and safety of drug preservatives as not yet optimized. Copyright © 2014 Elsevier B.V. All rights reserved.

Insulin Resistance in Alzheimer Disease: p53 and MicroRNAs as Important Players.

PubMed

Gasiorowski, Kazimierz; Brokos, Barbara; Leszek, Jerzy; Tarasov, Vadim V; Ashraf, Ghulam Md; Aliev, Gjumrakch

2017-01-01

Glucose homeostasis is crucial for neuronal survival, synaptic plasticity, and is indispensable for learning and memory. Reduced sensitivity of cells to insulin and impaired insulin signaling in brain neurons participate in the pathogenesis of Alzheimer disease (AD). The tumor suppressor protein p53 coordinates with multiple cellular pathways in response to DNA damage and cellular stresses. However, prolonged stress conditions unveil deleterious effects of p53-evoked insulin resistance in neurons; enhancement of transcription of pro-oxidant factors, accumulation of toxic metabolites (e.g. ceramide and products of advanced glycation) and ROS-modified cellular components, together with the activation of proapoptotic genes, could finally induce a suicide death program of autophagy/apoptosis in neurons. Recent studies reveal the impact of p53 on expression and processing of several microRNAs (miRs) under DNA damage-inducing conditions. Additionally, the role of miRs in promotion of insulin resistance and type 2 diabetes mellitus has been well documented. Detailed recognition of the role of p53/miRs crosstalk in driving insulin resistance in AD brains could improve the disease diagnostics and aid future therapy. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Proposed Pharmacological Countermeasures Against Apoptotic Cell Death in Experimental Models Mimicking Space Environment Damage

NASA Astrophysics Data System (ADS)

Lulli, Matteo; Papucci, Laura; Witort, Ewa; Donnini, Martino; Lapucci, Andrea; Lazzarano, Stefano; Mazzoni, Tiziano; Simoncini, Madine; Falciani, Piergiuseppe; Capaccioli, Sergio

2008-06-01

Several damaging agents have been suggested to affect human vision during long term space travels. Recently, apoptosis induced by DNA-damaging agents has emerged as frequent pathogenetic mechanism of ophthalmologic pathologies. Here, we propose two countermeasures: coenzyme Q10 and bcl-2 downregulation preventing antisense oligoribonucleotides (ORNs), aimed to inhibit cellular apoptotic death. Our studies have been carried out on retina and neuronal cultured cells treated with the following apoptotic stimuli mimicking space environment: a several-day exposure to either 3H-labeled tymidine or to the genotoxic drug doxorubicin, UV irradiation, hypoxia and glucose/growth factor starvation (Locke medium). The preliminary results clearly indicate that CoQ10, as well as bcl-2 down-regulation preventing ORNs, significantly counteract apoptosis in response to different DNA damaging agents in cultured eye and in neuronal cells. This supports the possibility that both could be optimal countermeasures against ophthalmologic lesions during space explorations.

Hereditary Disorders with Defective Repair of UV-Induced DNA Damage

PubMed Central

Moriwaki, Shinichi

2013-01-01

Nucleotide excision repair (NER) is an essential system for correcting ultraviolet (UV)—induced DNA damage. Lesions remaining in DNA due to reduced capacity of NER may result in cellular death, premature aging, mutagenesis and carcinogenesis of the skin. So, NER is an important protection against these changes. There are three representative genodermatoses resulting from genetic defects in NER: xeroderma pigmentosum (XP), Cockayne syndrome (CS), and trichothiodystrophy (TTD). In Japan, CS is similarly rare but XP is more common and TTD is less common compared to Western countries. In 1998, we established the system for the diagnosis of these disorders and we have been performing DNA repair and genetic analysis for more than 400 samples since then. At present, there is no cure for any human genetic disorder. Early diagnosis and symptomatic treatment of neurological, ocular and dermatological abnormalities should contribute to prolonging life and elevating QOL in patients. PMID:23966815

Does a role for selenium in DNA damage repair explain apparent controversies in its use in chemoprevention?

PubMed Central

Diamond, Alan M.

2013-01-01

The trace element selenium is an essential micronutrient that has received considerable attention for its potential use in the prevention of cancer. In spite of this interest, the mechanism(s) by which selenium might function as a chemopreventive remain to be determined. Considerable experimental evidence indicates that one possible mechanism by which selenium supplementation may exert its benefits is by enhancing the DNA damage repair response, and this includes data obtained using cultured cells, animal models as well as in human clinical studies. In these studies, selenium supplementation has been shown to be beneficial in reducing the frequency of DNA adducts and chromosome breaks, consequentially reducing the likelihood of detrimental mutations that ultimately contribute to carcinogenesis. The benefits of selenium can be envisioned as being due, at least in part, to it being a critical constituent of selenoproteins such as glutathione peroxidases and thioredoxin reductases, proteins that play important roles in antioxidant defence and maintaining the cellular reducing environment. Selenium, therefore, may be protective by preventing DNA damage from occurring as well as by increasing the activity of repair enzymes such as DNA glycosylases and DNA damage repair pathways that involve p53, BRCA1 and Gadd45. An improved understanding of the mechanism of selenium’s impact on DNA repair processes may help to resolve the apparently contradicting data obtained from decades of animal work, human epidemiology and more recently, clinical supplementation studies. PMID:23204505

Viral single-strand DNA induces p53-dependent apoptosis in human embryonic stem cells.

PubMed

Hirsch, Matthew L; Fagan, B Matthew; Dumitru, Raluca; Bower, Jacquelyn J; Yadav, Swati; Porteus, Matthew H; Pevny, Larysa H; Samulski, R Jude

2011-01-01

Human embryonic stem cells (hESCs) are primed for rapid apoptosis following mild forms of genotoxic stress. A natural form of such cellular stress occurs in response to recombinant adeno-associated virus (rAAV) single-strand DNA genomes, which exploit the host DNA damage response for replication and genome persistence. Herein, we discovered a unique DNA damage response induced by rAAV transduction specific to pluripotent hESCs. Within hours following rAAV transduction, host DNA damage signaling was elicited as measured by increased gamma-H2AX, ser15-p53 phosphorylation, and subsequent p53-dependent transcriptional activation. Nucleotide incorporation assays demonstrated that rAAV transduced cells accumulated in early S-phase followed by the induction of apoptosis. This lethal signaling sequalae required p53 in a manner independent of transcriptional induction of Puma, Bax and Bcl-2 and was not evident in cells differentiated towards a neural lineage. Consistent with a lethal DNA damage response induced upon rAAV transduction of hESCs, empty AAV protein capsids demonstrated no toxicity. In contrast, DNA microinjections demonstrated that the minimal AAV origin of replication and, in particular, a 40 nucleotide G-rich tetrad repeat sequence, was sufficient for hESC apoptosis. Our data support a model in which rAAV transduction of hESCs induces a p53-dependent lethal response that is elicited by a telomeric sequence within the AAV origin of replication.

Chromatibody, a novel non-invasive molecular tool to explore and manipulate chromatin in living cells

PubMed Central

Jullien, Denis; Vignard, Julien; Fedor, Yoann; Béry, Nicolas; Olichon, Aurélien; Crozatier, Michèle; Erard, Monique; Cassard, Hervé; Ducommun, Bernard; Salles, Bernard

2016-01-01

ABSTRACT Chromatin function is involved in many cellular processes, its visualization or modification being essential in many developmental or cellular studies. Here, we present the characterization of chromatibody, a chromatin-binding single-domain, and explore its use in living cells. This non-intercalating tool specifically binds the heterodimer of H2A–H2B histones and displays a versatile reactivity, specifically labeling chromatin from yeast to mammals. We show that this genetically encoded probe, when fused to fluorescent proteins, allows non-invasive real-time chromatin imaging. Chromatibody is a dynamic chromatin probe that can be modulated. Finally, chromatibody is an efficient tool to target an enzymatic activity to the nucleosome, such as the DNA damage-dependent H2A ubiquitylation, which can modify this epigenetic mark at the scale of the genome and result in DNA damage signaling and repair defects. Taken together, these results identify chromatibody as a universal non-invasive tool for either in vivo chromatin imaging or to manipulate the chromatin landscape. PMID:27206857

The bornavirus-derived human protein EBLN1 promotes efficient cell cycle transit, microtubule organisation and genome stability.

PubMed

Myers, Katie N; Barone, Giancarlo; Ganesh, Anil; Staples, Christopher J; Howard, Anna E; Beveridge, Ryan D; Maslen, Sarah; Skehel, J Mark; Collis, Spencer J

2016-10-14

It was recently discovered that vertebrate genomes contain multiple endogenised nucleotide sequences derived from the non-retroviral RNA bornavirus. Strikingly, some of these elements have been evolutionary maintained as open reading frames in host genomes for over 40 million years, suggesting that some endogenised bornavirus-derived elements (EBL) might encode functional proteins. EBLN1 is one such element established through endogenisation of the bornavirus N gene (BDV N). Here, we functionally characterise human EBLN1 as a novel regulator of genome stability. Cells depleted of human EBLN1 accumulate DNA damage both under non-stressed conditions and following exogenously induced DNA damage. EBLN1-depleted cells also exhibit cell cycle abnormalities and defects in microtubule organisation as well as premature centrosome splitting, which we attribute in part, to improper localisation of the nuclear envelope protein TPR. Our data therefore reveal that human EBLN1 possesses important cellular functions within human cells, and suggest that other EBLs present within vertebrate genomes may also possess important cellular functions.

Recruitment of DNA Replication and Damage Response Proteins to Viral Replication Centers during Infection with NS2 Mutants of Minute Virus of Mice (MVM)

PubMed Central

Ruiz, Zandra; Mihaylov, Ivailo S.; Cotmore, Susan F.; Tattersall, Peter

2010-01-01

MVM NS2 is essential for viral DNA amplification, but its mechanism of action is unknown. A classification scheme for autonomous parvovirus-associated replication (APAR) center development, based on NS1 distribution, was used to characterize abnormal APAR body maturation in NS2null mutant infections, and their organization examined for defects in host protein recruitment. Since acquisition of known replication factors appeared normal, we looked for differences in invoked DNA damage responses. We observed widespread association of H2AX/MDC1 damage response foci with viral replication centers, and sequestration and complex hyperphosphorylation of RPA32, which occurred in wildtype and mutant infections. Quantifying these responses by western transfer indicated that both wildtype and NS2 mutant MVM elicited ATM activation, while phosphorylation of ATR, already basally activated in asynchronous A9 cells, was downregulated. We conclude that MVM infection invokes multiple damage responses that influence the APAR environment, but that NS2 does not modify the recruitment of cellular proteins. PMID:21193212

Recruitment of DNA replication and damage response proteins to viral replication centers during infection with NS2 mutants of Minute Virus of Mice (MVM).

PubMed

Ruiz, Zandra; Mihaylov, Ivailo S; Cotmore, Susan F; Tattersall, Peter

2011-02-20

MVM NS2 is essential for viral DNA amplification, but its mechanism of action is unknown. A classification scheme for autonomous parvovirus-associated replication (APAR) center development, based on NS1 distribution, was used to characterize abnormal APAR body maturation in NS2null mutant infections, and their organization examined for defects in host protein recruitment. Since acquisition of known replication factors appeared normal, we looked for differences in invoked DNA damage responses. We observed widespread association of H2AX/MDC1 damage response foci with viral replication centers, and sequestration and complex hyperphosphorylation of RPA(32), which occurred in wildtype and mutant infections. Quantifying these responses by western transfer indicated that both wildtype and NS2 mutant MVM elicited ATM activation, while phosphorylation of ATR, already basally activated in asynchronous A9 cells, was downregulated. We conclude that MVM infection invokes multiple damage responses that influence the APAR environment, but that NS2 does not modify the recruitment of cellular proteins. Copyright © 2010 Elsevier Inc. All rights reserved.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Adhikary, Suraj; Eichman, Brandt F.

DNA glycosylases specialized for the repair of alkylation damage must identify, with fine specificity, a diverse array of subtle modifications within DNA. The current mechanism involves damage sensing through interrogation of the DNA duplex, followed by more specific recognition of the target base inside the active site pocket. To better understand the physical basis for alkylpurine detection, we determined the crystal structure of Schizosaccharomyces pombe Mag1 (spMag1) in complex with DNA and performed a mutational analysis of spMag1 and the close homologue from Saccharomyces cerevisiae (scMag). Despite strong homology, spMag1 and scMag differ in substrate specificity and cellular alkylation sensitivity,more » although the enzymological basis for their functional differences is unknown. We show that Mag preference for 1,N{sup 6}-ethenoadenine ({var_epsilon}A) is influenced by a minor groove-interrogating residue more than the composition of the nucleobase-binding pocket. Exchanging this residue between Mag proteins swapped their {var_epsilon}A activities, providing evidence that residues outside the extrahelical base-binding pocket have a role in identification of a particular modification in addition to sensing damage.« less

Oxidative stress-induced protein damage inhibits DNA repair and determines mutation risk and anticancer drug effectiveness

PubMed Central

McAdam, Elizabeth; Brem, Reto; Karran, Peter

2016-01-01

The relationship between sun exposure and non-melanoma skin cancer risk is well established. Solar ultraviolet radiation (UV; wavelengths 280-400 nm) is firmly implicated in skin cancer development. Nucleotide excision repair (NER) protects against cancer by removing potentially mutagenic DNA lesions induced by UVB (280-320 nm). How the 20-fold more abundant UVA (320-400 mn) component of solar UV radiation increases skin cancer risk is not understood. We demonstrate here that the contribution of UVA to the effects of UV radiation on cultured human cells is largely independent of its ability to damage DNA. Instead, the effects of UVA reflect the induction of oxidative stress that causes extensive protein oxidation. Because NER proteins are among those damaged, UVA irradiation inhibits NER and increases the cells’ susceptibility to mutation by UVB. NER inhibition is a common consequence of oxidative stress. Exposure to chemical oxidants, treatment with drugs that deplete cellular antioxidants, and interventions that interfere with glucose metabolism to disrupt the supply of cellular reducing power all inhibit NER. Tumor cells are often in a condition of oxidative stress and one effect of the NER inhibition that results from stress-induced protein oxidation is an increased sensitivity to the anticancer drug cisplatin. Statement of implication: Since NER is both a defence against cancer a significant determinant of cell survival after treatment with anticancer drugs, its attenuation by protein damage under conditions of oxidative-stress has implications for both cancer risk and for the effectiveness of anticancer therapy. PMID:27106867

Ageing induced vascular smooth muscle cell senescence in atherosclerosis.

PubMed

Uryga, Anna K; Bennett, Martin R

2016-04-15

Atherosclerosis is a disease of ageing in that its incidence and prevalence increase with age. However, atherosclerosis is also associated with biological ageing, manifest by a number of typical hallmarks of ageing in the atherosclerotic plaque. Thus, accelerated biological ageing may be superimposed on the effects of chronological ageing in atherosclerosis. Tissue ageing is seen in all cells that comprise the plaque, but particularly in vascular smooth muscle cells (VSMCs). Hallmarks of ageing include evidence of cell senescence, DNA damage (including telomere attrition), mitochondrial dysfunction, a pro-inflammatory secretory phenotype, defects in proteostasis, epigenetic changes, deregulated nutrient sensing, and exhaustion of progenitor cells. In this model, initial damage to DNA (genomic, telomeric, mitochondrial and epigenetic changes) results in a number of cellular responses (cellular senescence, deregulated nutrient sensing and defects in proteostasis). Ultimately, ongoing damage and attempts at repair by continued proliferation overwhelm reparative capacity, causing loss of specialised cell functions, cell death and inflammation. This review summarises the evidence for accelerated biological ageing in atherosclerosis, the functional consequences of cell ageing on cells comprising the plaque, and the causal role that VSMC senescence plays in atherogenesis. © 2015 The Authors. The Journal of Physiology © 2015 The Physiological Society.

Nucleolus as an emerging hub in maintenance of genome stability and cancer pathogenesis.

PubMed

Lindström, Mikael S; Jurada, Deana; Bursac, Sladana; Orsolic, Ines; Bartek, Jiri; Volarevic, Sinisa

2018-05-01

The nucleolus is the major site for synthesis of ribosomes, complex molecular machines that are responsible for protein synthesis. A wealth of research over the past 20 years has clearly indicated that both quantitative and qualitative alterations in ribosome biogenesis can drive the malignant phenotype via dysregulation of protein synthesis. However, numerous recent proteomic, genomic, and functional studies have implicated the nucleolus in the regulation of processes that are unrelated to ribosome biogenesis, including DNA-damage response, maintenance of genome stability and its spatial organization, epigenetic regulation, cell-cycle control, stress responses, senescence, global gene expression, as well as assembly or maturation of various ribonucleoprotein particles. In this review, the focus will be on features of rDNA genes, which make them highly vulnerable to DNA damage and intra- and interchromosomal recombination as well as built-in mechanisms that prevent and repair rDNA damage, and how dysregulation of this interplay affects genome-wide DNA stability, gene expression and the balance between euchromatin and heterochromatin. We will also present the most recent insights into how malfunction of these cellular processes may be a central driving force of human malignancies, and propose a promising new therapeutic approach for the treatment of cancer.

A novel type of cellular senescence that can be enhanced in mouse models and human tumor xenografts to suppress prostate tumorigenesis

PubMed Central

Alimonti, Andrea; Nardella, Caterina; Chen, Zhenbang; Clohessy, John G.; Carracedo, Arkaitz; Trotman, Lloyd C.; Cheng, Ke; Varmeh, Shohreh; Kozma, Sara C.; Thomas, George; Rosivatz, Erika; Woscholski, Rudiger; Cognetti, Francesco; Scher, Howard I.; Pandolfi, Pier Paolo

2010-01-01

Irreversible cell growth arrest, a process termed cellular senescence, is emerging as an intrinsic tumor suppressive mechanism. Oncogene-induced senescence is thought to be invariably preceded by hyperproliferation, aberrant replication, and activation of a DNA damage checkpoint response (DDR), rendering therapeutic enhancement of this process unsuitable for cancer treatment. We previously demonstrated in a mouse model of prostate cancer that inactivation of the tumor suppressor phosphatase and tensin homolog deleted on chromosome 10 (Pten) elicits a senescence response that opposes tumorigenesis. Here, we show that Pten-loss–induced cellular senescence (PICS) represents a senescence response that is distinct from oncogene-induced senescence and can be targeted for cancer therapy. Using mouse embryonic fibroblasts, we determined that PICS occurs rapidly after Pten inactivation, in the absence of cellular proliferation and DDR. Further, we found that PICS is associated with enhanced p53 translation. Consistent with these data, we showed that in mice p53-stabilizing drugs potentiated PICS and its tumor suppressive potential. Importantly, we demonstrated that pharmacological inhibition of PTEN drives senescence and inhibits tumorigenesis in vivo in a human xenograft model of prostate cancer. Taken together, our data identify a type of cellular senescence that can be triggered in nonproliferating cells in the absence of DNA damage, which we believe will be useful for developing a “pro-senescence” approach for cancer prevention and therapy. PMID:20197621

Radiation cataracts: mechanisms involved in their long delayed occurrence but then rapid progression.

PubMed

Wolf, Norman; Pendergrass, William; Singh, Narendra; Swisshelm, Karen; Schwartz, Jeffrey

2008-02-05

This study was directed to assess the DNA damage and DNA repair response to X-ray inflicted lens oxidative damage and to investigate the subsequent changes in lens epithelial cell (LEC) behavior in vivo that led to long delayed but then rapidly developing cataracts. Two-month-old C57Bl/6 female mice received 11 Grays (Gy) of soft x-irradiation to the head only. The animals' eyes were examined for cataract status in 30 day intervals by slit lamp over an 11 month period post-irradiation. LEC migration, DNA fragment, free DNA retention, and reactive oxygen species (ROS) presence were established in the living lenses with fluorescent dyes using laser scanning confocal microscopy (LSCM). The extent and removal of initial LEC DNA damage were determined by comet assay. Immunohistochemistry was used to determine the presence of oxidized DNA and the response of a DNA repair protein in the lenses. This treatment resulted in advanced cortical cataracts that developed 5-11 months post-irradiation but then appeared suddenly within a 30 day period. The initially incurred DNA strand breaks were repaired within 30 min, but DNA damage remained as shown 72 h post-irradiation by the presence of the DNA adduct, 8-hydroxyguanosine (8-OHG), and a DNA repair protein, XRCC1. This was followed months later by abnormal behavior by LEC descendant cells with abnormal differentiation and migration patterns as seen with LSCM and fluorescent dyes. The sudden development of cortical cataracts several months post-irradiation coupled with the above findings suggests an accumulation of damaged descendants from the initially x-irradiated LECs. As these cells migrate abnormally and leave acellular lens surface sites, eventually a crisis point may arrive for lens entry of environmental O(2) with resultant ROS formation that overwhelms protection by resident antioxidant enzymes and results in the coagulation of lens proteins. The events seen in this study indicate the retention and transmission of progenitor cell DNA damage in descendant LEC. The cellular and molecular events parallel those previously reported for LSCM observations in age-related cataracts.

Primer-Independent DNA Synthesis by a Family B DNA Polymerase from Self-Replicating Mobile Genetic Elements.

PubMed

Redrejo-Rodríguez, Modesto; Ordóñez, Carlos D; Berjón-Otero, Mónica; Moreno-González, Juan; Aparicio-Maldonado, Cristian; Forterre, Patrick; Salas, Margarita; Krupovic, Mart

2017-11-07

Family B DNA polymerases (PolBs) play a central role during replication of viral and cellular chromosomes. Here, we report the discovery of a third major group of PolBs, which we denote primer-independent PolB (piPolB), that might be a link between the previously known protein-primed and RNA/DNA-primed PolBs. PiPolBs are encoded by highly diverse mobile genetic elements, pipolins, integrated in the genomes of diverse bacteria and also present as circular plasmids in mitochondria. Biochemical characterization showed that piPolB displays efficient DNA polymerization activity that can use undamaged and damaged templates and is endowed with proofreading and strand displacement capacities. Remarkably, the protein is also capable of template-dependent de novo DNA synthesis, i.e., DNA-priming activity, thereby breaking the long-standing dogma that replicative DNA polymerases require a pre-existing primer for DNA synthesis. We suggest that piPolBs are involved in self-replication of pipolins and may also contribute to bacterial DNA damage tolerance. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Aprataxin resolves adenylated RNA–DNA junctions to maintain genome integrity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tumbale, Percy; Williams, Jessica S.; Schellenberg, Matthew J.

2013-12-22

Faithful maintenance and propagation of eukaryotic genomes is ensured by three-step DNA ligation reactions used by ATP-dependent DNA ligases. Paradoxically, when DNA ligases encounter nicked DNA structures with abnormal DNA termini, DNA ligase catalytic activity can generate and/or exacerbate DNA damage through abortive ligation that produces chemically adducted, toxic 5'-adenylated (5'-AMP) DNA lesions. Aprataxin (APTX) reverses DNA adenylation but the context for deadenylation repair is unclear. Here we examine the importance of APTX to RNase-H2-dependent excision repair (RER) of a lesion that is very frequently introduced into DNA, a ribonucleotide. We show that ligases generate adenylated 5' ends containing amore » ribose characteristic of RNase H2 incision. APTX efficiently repairs adenylated RNA–DNA, and acting in an RNA–DNA damage response (RDDR), promotes cellular survival and prevents S-phase checkpoint activation in budding yeast undergoing RER. Structure–function studies of human APTX–RNA–DNA–AMP–Zn complexes define a mechanism for detecting and reversing adenylation at RNA–DNA junctions. This involves A-form RNA binding, proper protein folding and conformational changes, all of which are affected by heritable APTX mutations in ataxia with oculomotor apraxia 1. Together, these results indicate that accumulation of adenylated RNA–DNA may contribute to neurological disease.« less

Protective effects of melatonin-loaded lipid-core nanocapsules on paraquat-induced cytotoxicity and genotoxicity in a pulmonary cell line.

PubMed

Charão, Mariele F; Baierle, Marília; Gauer, Bruna; Goethel, Gabriela; Fracasso, Rafael; Paese, Karina; Brucker, Natália; Moro, Angela M; Bubols, Guilherme B; Dias, Bruna B; Matte, Ursula S; Guterres, Silvia S; Pohlmann, Adriana R; Garcia, Solange C

2015-06-01

Many acute poisonings lack effective and specific antidotes. Due to both intentional and accidental exposures, paraquat (PQ) causes thousands of deaths annually, especially by pulmonary fibrosis. Melatonin (Mel), when incorporated into lipid-core nanocapsules (Mel-LNC), has enhanced antioxidant properties. The effects of such a formulation have not yet been studied with respect to mitigation of PQ- induced cytotoxicity and DNA damage. Here, we have tested whether Mel-LNC can ameliorate PQ-induced toxicity in the A549 alveolar epithelial cell line. Physicochemical characterization of the formulations was performed. Cellular uptake was measured using nanocapsules marked with rhodamine B. Cell viability was determined by the MTT assay and DNA damage was assessed by the comet assay. The enzyme-modified comet assay with endonuclease III (Endo III) and formamidopyrimidine glycosylase (FPG) were used to investigate oxidative DNA damage. Incubation with culture medium for 24h did not alter the granulometric profile of Mel-LNC formulations. Following treatment (3 and 24h), red fluorescence was detected around the cell nucleus, indicating internalization of the formulation. Melatonin solution (Mel), Mel-LNC, and LNC did not have significant effects on cell viability or DNA damage. Pre-treatment with Mel-LNC enhanced cell viability and showed a remarkable reduction in % DNA in tail compared to the PQ group; this was not observed in cells pre-treated with Mel. PQ induces oxidative DNA damage detected with the enzyme-modified comet assay. Mel-LNC reduced this damage more effectively than did Mel. In summary, Mel-LNC is better than Mel at protecting A549 cells from the cytotoxic and genotoxic effects of PQ. Copyright © 2015 Elsevier B.V. All rights reserved.

Enhancement of UVB radiation-mediated apoptosis by knockdown of cytosolic NADP+-dependent isocitrate dehydrogenase in HaCaT cells

PubMed Central

Lee, Su Jeong; Park, Jeen-Woo

2014-01-01

Ultraviolet B (UVB) radiation induces the production of reactive oxygen species (ROS) that promote apoptotic cell death. We showed that cytosolic NADP+-dependent isocitrate dehydrogenase (IDPc) plays an essential role in the control of cellular redox balance and defense against oxidative damage, by supplying NADPH for antioxidant systems. In this study, we demonstrated that knockdown of IDPc expression by RNA interference enhances UVB-induced apoptosis of immortalized human HaCaT keratinocytes. This effect manifested as DNA fragmentation, changes in cellular redox status, mitochondrial dysfunction, and modulation of apoptotic marker expression. Based on our findings, we suggest that attenuation of IDPc expression may protect skin from UVB-mediated damage, by inducing the apoptosis of UV-damaged cells. [BMB Reports 2014; 47(4): 209-214] PMID:24286310

Recombinase and translesion DNA polymerase decrease the speed of replication fork progression during the DNA damage response in Escherichia coli cells

PubMed Central

Tan, Kang Wei; Pham, Tuan Minh; Furukohri, Asako; Maki, Hisaji; Akiyama, Masahiro Tatsumi

2015-01-01

The SOS response is a DNA damage response pathway that serves as a general safeguard of genome integrity in bacteria. Extensive studies of the SOS response in Escherichia coli have contributed to establishing the key concepts of cellular responses to DNA damage. However, how the SOS response impacts on the dynamics of DNA replication fork movement remains unknown. We found that inducing the SOS response decreases the mean speed of individual replication forks by 30–50% in E. coli cells, leading to a 20–30% reduction in overall DNA synthesis. dinB and recA belong to a group of genes that are upregulated during the SOS response, and encode the highly conserved proteins DinB (also known as DNA polymerase IV) and RecA, which, respectively, specializes in translesion DNA synthesis and functions as the central recombination protein. Both genes were independently responsible for the SOS-dependent slowdown of replication fork progression. Furthermore, fork speed was reduced when each gene was ectopically expressed in SOS-uninduced cells to the levels at which they are expressed in SOS-induced cells. These results clearly indicate that the increased expression of dinB and recA performs a novel role in restraining the progression of an unperturbed replication fork during the SOS response. PMID:25628359

Copper-redox cycling by coumarin-di(2-picolyl)amine hybrid molecule leads to ROS-mediated DNA damage and apoptosis: A mechanism for cancer chemoprevention.

PubMed

Khan, Saman; Zafar, Atif; Naseem, Imrana

2018-06-25

Coumarin is an important bioactive pharmacophore. It is found in plants as a secondary metabolite and exhibits diverse pharmacological properties including anticancer effects against different malignancies. Therapeutic efficacy of coumarin derivatives depends on the pattern of substitution and conjugation with different moieties. Cancer cells contain elevated copper as compared to normal cells that plays a role in angiogenesis. Thus, targeting copper in malignant cells via copper chelators can serve as an attractive targeted anticancer strategy. Our previous efforts led to the synthesis of di(2-picolyl)amine-3(bromoacetyl)coumarin hybrid molecule (ligand-L) endowed with DNA/Cu(II) binding properties, and ROS generation ability in the presence of copper ions. In the present study, we aimed to validate copper-dependent cytotoxic action of ligand-L against malignant cells. For this, we used a cellular model system of copper (Cu) overloaded lymphocytes (CuOLs) to simulate malignancy-like condition. In CuOLs, lipid peroxidation/protein carbonylation, ROS generation, DNA fragmentation and apoptosis were investigated in the presence of ligand-L. Results showed that ligand-L-Cu(II) interaction leads to ROS generation, lipid peroxidation/protein carbonylation (oxidative stress parameters), DNA damage, up-regulation of p53 and mitochondrial-mediated apoptosis in treated lymphocytes. Further, pre-incubation with neocuproine (membrane permeable copper chelator) and ROS scavengers attenuated the DNA damage and apoptosis. These results suggest that cellular copper acts as molecular target for ligand-L to propagate redox cycling and generation of ROS via Fenton-like reaction leading to DNA damage and apoptosis. Further, we showed that ligand-L targets elevated copper in breast cancer MCF-7 and colon cancer HCT116 cells leading to a pro-oxidant inhibition of proliferation of cancer cells. In conclusion, we propose copper-dependent ROS-mediated mechanism for the cytotoxic action of ligand-L in malignant cells. Thus, targeting elevated copper represents an effective therapeutic strategy for selective cytotoxicity against malignant cells. Copyright © 2018 Elsevier B.V. All rights reserved.

DNA-targeted 2-nitroimidazoles: studies of the influence of the phenanthridine-linked nitroimidazoles, 2-NLP-3 and 2-NLP-4, on DNA damage induced by ionizing radiation.

PubMed

Buchko, Garry W; Weinfeld, Michael

2002-09-01

The nitroimidazole-linked phenanthridines 2-NLP-3 (5-[3-(2-nitro-1-imidazoyl)-propyl]-phenanthridinium bromide) and 2-NLP-4 (5-[3-(2-nitro-1-imidazoyl)-butyl]-phenanthridinium bromide) are composed of the radiosensitizer, 2-nitroimidazole, attached to the DNA intercalator phenanthridine by a 3- and 4-carbon linker, respectively. Previous in vitro assays showed both compounds to be 10-100 times more efficient as hypoxic cell radiosensitizers (based on external drug concentrations) than the untargeted 2-nitroimidazole radiosensitizer, misonidazole (Cowan et al., Radiat. Res. 127, 81-89, 1991). Here we have used a (32)P postlabeling assay and 5'-end-labeled oligonucleotide assay to compare the radiation-induced DNA damage generated in the presence of 2-NLP-3, 2-NLP-4, phenanthridine and misonidazole. After irradiation of the DNA under anoxic conditions, we observed a significantly greater level of 3'-phosphoglycolate DNA damage in the presence of 2-NLP-3 or 2-NLP-4 compared to irradiation of the DNA in the presence of misonidazole. This may account at least in part for the greater cellular radiosensitization shown by the nitroimidazole-linked phenanthridines over misonidazole. Of the two nitroimidazole-linked phenanthridines, the better in vitro radiosensitizer, 2-NLP-4, generated more 3'-phosphoglycolate in DNA than did 2-NLP-3. At all concentrations, phenanthridine had little effect on the levels of DNA damage, suggesting that the enhanced radiosensitization displayed by 2-NLP-3 and 2-NLP-4 is due to the localization of the 2-nitroimidazole to the DNA by the phenanthridine substituent and not to radiosensitization by the phenanthridine moiety itself.

Correlation analyses of phytochemical composition, chemical, and cellular measures of antioxidant activity of broccoli (Brassica oleracea L. Var. italica).

PubMed

Eberhardt, Marian V; Kobira, Kanta; Keck, Anna-Sigrid; Juvik, John A; Jeffery, Elizabeth H

2005-09-21

Chemical measures of antioxidant activity within the plant, such as the oxygen radical absorbance capacity (ORAC) assay, have been reported for many plant-based foods. However, the extent to which chemical measures relate to cellular measures of oxidative stress is unclear. The natural variation in the phytochemical content of 22 broccoli genotypes was used to determine correlations among chemical composition (carotenoids, tocopherols and polyphenolics), chemical antioxidant activity (ORAC), and measures of cellular antioxidation [prevention of DNA oxidative damage and of oxidation of the biomarker dichlorofluorescein (DCFH) in HepG2 cells] using hydrophilic and lipophilic extracts of broccoli. For lipophilic extracts, ORAC (ORAC-L) correlated with inhibition of cellular oxidation of DCFH (DCFH-L, r = 0.596, p = 0.006). Also, DNA damage in the presence of the lipophilic extract was negatively correlated with both chemical and cellular measures of antioxidant activity as measured by ORAC-L (r = -0.705, p = 0.015) and DCFH-L (r = -0.671, p = 0.048), respectively. However, no correlations were observed for hydrophilic (-H) extracts, except between polyphenol content and ORAC (ORAC-H; r = 0.778, p < 0.001). Inhibition of cellular oxidation by hydrophilic extracts (DCFH-H) and ORAC-H were approximately 8- and 4-fold greater than DCFH-L and ORAC-L, respectively. Whether ORAC-H has more biological relevance than ORAC-L because of its magnitude or whether ORAC-L bears more biological relevance because it relates to cellular estimates of antioxidant activity remains to be determined. Chemical estimates of antioxidant capacity within the plant may not accurately reflect the complex nature of the full antioxidant activity of broccoli extracts within cells.

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

PubMed Central

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

2012-01-01

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. PMID:23235459

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

PubMed

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.

The Aurora-B-dependent NoCut checkpoint prevents damage of anaphase bridges after DNA replication stress.

PubMed

Amaral, Nuno; Vendrell, Alexandre; Funaya, Charlotta; Idrissi, Fatima-Zahra; Maier, Michael; Kumar, Arun; Neurohr, Gabriel; Colomina, Neus; Torres-Rosell, Jordi; Geli, María-Isabel; Mendoza, Manuel

2016-05-01

Anaphase chromatin bridges can lead to chromosome breakage if not properly resolved before completion of cytokinesis. The NoCut checkpoint, which depends on Aurora B at the spindle midzone, delays abscission in response to chromosome segregation defects in yeast and animal cells. How chromatin bridges are detected, and whether abscission inhibition prevents their damage, remain key unresolved questions. We find that bridges induced by DNA replication stress and by condensation or decatenation defects, but not dicentric chromosomes, delay abscission in a NoCut-dependent manner. Decatenation and condensation defects lead to spindle stabilization during cytokinesis, allowing bridge detection by Aurora B. NoCut does not prevent DNA damage following condensin or topoisomerase II inactivation; however, it protects anaphase bridges and promotes cellular viability after replication stress. Therefore, the molecular origin of chromatin bridges is critical for activation of NoCut, which plays a key role in the maintenance of genome stability after replicative stress.

Structurally distinct ubiquitin- and sumo-modified PCNA: implications for their distinct roles in the DNA damage response.

PubMed

Tsutakawa, Susan E; Yan, Chunli; Xu, Xiaojun; Weinacht, Christopher P; Freudenthal, Bret D; Yang, Kun; Zhuang, Zhihao; Washington, M Todd; Tainer, John A; Ivanov, Ivaylo

2015-04-07

Proliferating cell nuclear antigen (PCNA) is a pivotal replication protein, which also controls cellular responses to DNA damage. Posttranslational modification of PCNA by SUMO and ubiquitin modulate these responses. How the modifiers alter PCNA-dependent DNA repair and damage tolerance pathways is largely unknown. We used hybrid methods to identify atomic models of PCNAK107-Ub and PCNAK164-SUMO consistent with small-angle X-ray scattering data of these complexes in solution. We show that SUMO and ubiquitin have distinct modes of association to PCNA. Ubiquitin adopts discrete docked binding positions. By contrast, SUMO associates by simple tethering and adopts extended flexible conformations. These structural differences are the result of the opposite electrostatic potentials of SUMO and Ub. The unexpected contrast in conformational behavior of Ub-PCNA and SUMO-PCNA has implications for interactions with partner proteins, interacting surfaces accessibility, and access points for pathway regulation. Copyright © 2015 Elsevier Ltd. All rights reserved.

Structurally Distinct Ubiquitin- and Sumo-Modified PCNA: Implications for Their Distinct Roles in the DNA Damage Response

DOE PAGES

Tsutakawa, Susan E.; Yan, Chunli; Xu, Xiaojun; ...

2015-03-12

Proliferating cell nuclear antigen (PCNA) is a pivotal replication protein, which also controls cellular responses to DNA damage. Posttranslational modification of PCNA by SUMO and ubiquitin modulate these responses. How the modifiers alter PCNA-dependent DNA repair and damage tolerance pathways is largely unknown. Here, we used hybrid methods to identify atomic models of PCNA K107-Ub and PCNA K164-SUMO consistent with small-angle X-ray scattering data of these complexes in solution. We show that SUMO and ubiquitin have distinct modes of association to PCNA. Ubiquitin adopts discrete docked binding positions. By contrast, SUMO associates by simple tethering and adopts extended flexible conformations.more » These structural differences are the result of the opposite electrostatic potentials of SUMO and Ub. In conclusion, the unexpected contrast in conformational behavior of Ub-PCNA and SUMO-PCNA has implications for interactions with partner proteins, interacting surfaces accessibility, and access points for pathway regulation.« less

Hepatitis B virus and hepatocellular carcinoma

PubMed Central

Arbuthnot, Patrick; Kew, Michael

2001-01-01

Chronic hepatitis B virus (HBV) infection is a major global cause of hepatocellular carcinoma (HCC). Individuals who are chronic carriers have a greater than 100-fold increased relative risk of developing the tumour. Several mechanisms of HBV-induced HCC have been proposed. Integration of HBV DNA into the genome of hepatocytes occurs commonly, although integration at cellular sites that are important for regulation of hepatocyte proliferation appears to be a rare event. Functions of the HBx protein are also potentially oncogenic. These include transcriptional activation of cellular growth regulatory genes, modulation of apoptosis and inhibition of nucleotide excision repair of damaged cellular DNA. The effects of HBx are mediated by interaction with cellular proteins and activation of cell signalling pathways. Variations in HBV genome sequences may be important in hepatocarcinogenesis, although their significance has not yet been completely elucidated. Necroinflammatory hepatic disease, which often accompanies chronic HBV infection, may contribute indirectly to hepatocyte transformation in a number of ways, including by facilitating HBV DNA integration, predisposing to the acquisition of cellular mutations and generating mutagenic oxygen reactive species. Although HCC is a malignancy with a poor prognosis, the availability of an effective vaccine against HBV infection, and its inclusion in the Expanded Programme of Immunization of many countries, augurs well for the eventual elimination of HBV-associated HCC. PMID:11454100

Mitochondrial oxidative stress caused by Sod2 deficiency promotes cellular senescence and aging phenotypes in the skin

PubMed Central

Velarde, Michael C.; Flynn, James M.; Day, Nicholas U.; Melov, Simon; Campisi, Judith

2012-01-01

Cellular senescence arrests the proliferation of mammalian cells at risk for neoplastic transformation, and is also associated with aging. However, the factors that cause cellular senescence during aging are unclear. Excessive reactive oxygen species (ROS) have been shown to cause cellular senescence in culture, and accumulated molecular damage due to mitochondrial ROS has long been thought to drive aging phenotypes in vivo. Here, we test the hypothesis that mitochondrial oxidative stress can promote cellular senescence in vivo and contribute to aging phenotypes in vivo, specifically in the skin. We show that the number of senescent cells, as well as impaired mitochondrial (complex II) activity increase in naturally aged mouse skin. Using a mouse model of genetic Sod2 deficiency, we show that failure to express this important mitochondrial anti-oxidant enzyme also impairs mitochondrial complex II activity, causes nuclear DNA damage, and induces cellular senescence but not apoptosis in the epidermis. Sod2 deficiency also reduced the number of cells and thickness of the epidermis, while increasing terminal differentiation. Our results support the idea that mitochondrial oxidative stress and cellular senescence contribute to aging skin phenotypes in vivo. PMID:22278880

Rhodium metalloinsertor binding generates a lesion with selective cytotoxicity for mismatch repair-deficient cells.

PubMed

Bailis, Julie M; Weidmann, Alyson G; Mariano, Natalie F; Barton, Jacqueline K

2017-07-03

The DNA mismatch repair (MMR) pathway recognizes and repairs errors in base pairing and acts to maintain genome stability. Cancers that have lost MMR function are common and comprise an important clinical subtype that is resistant to many standard of care chemotherapeutics such as cisplatin. We have identified a family of rhodium metalloinsertors that bind DNA mismatches with high specificity and are preferentially cytotoxic to MMR-deficient cells. Here, we characterize the cellular mechanism of action of the most potent and selective complex in this family, [Rh(chrysi)(phen)(PPO)] 2+ (Rh-PPO). We find that Rh-PPO binding induces a lesion that triggers the DNA damage response (DDR). DDR activation results in cell-cycle blockade and inhibition of DNA replication and transcription. Significantly, the lesion induced by Rh-PPO is not repaired in MMR-deficient cells, resulting in selective cytotoxicity. The Rh-PPO mechanism is reminiscent of DNA repair enzymes that displace mismatched bases, and is differentiated from other DNA-targeted chemotherapeutics such as cisplatin by its potency, cellular mechanism, and selectivity for MMR-deficient cells.

The prion-like domain of FUS is multiphosphorylated following DNA damage without altering nuclear localization.

PubMed

Rhoads, Shannon N; Monahan, Zachary T; Yee, Debra S; Leung, Andrew Y; Newcombe, Cameron G; O'Meally, Robert N; Cole, Robert N; Shewmaker, Frank P

2018-06-13

FUS is an abundant, predominantly nuclear protein involved in RNA processing. Under various conditions, FUS functionally associates with RNA and other macromolecules to form distinct, reversible phase-separated liquid structures. Persistence of the phase-separated state and increased cytoplasmic localization are both hypothesized to predispose FUS to irreversible aggregation, which is a pathological hallmark of subtypes of amyotrophic lateral sclerosis and frontotemporal dementia. We previously showed that phosphorylation of FUS's prion-like domain suppressed phase separation and toxic aggregation, proportionally to the number of added phosphates. However, phosphorylation of FUS's prion-like domain was previously reported to promote its cytoplasmic localization, potentially favoring pathological behavior. Here, we used mass spectrometry and human cell models to further identify phosphorylation sites within FUS's prion-like domain, specifically following DNA-damaging stress. In total, 28 putative sites have been identified, about half of which are DNA-dependent protein kinase (DNA-PK) consensus sites. Custom antibodies were developed to confirm the phosphorylation of two of these sites (Ser26 and Ser30). Both sites were usually phosphorylated in a sub-population of cellular FUS following a variety of DNA-damaging stresses, but not necessarily equally or simultaneously. Importantly, we found DNA-PK-dependent multi-phosphorylation of FUS's prion-like domain does not cause cytoplasmic localization.

Cancer prevention, the need to preserve the integrity of the genome at all cost.

PubMed

Okafor, M T; Nwagha, T U; Anusiem, C; Okoli, U A; Nubila, N I; Al-Alloosh, F; Udenyia, I J

2018-05-01

The entire genetic information carried by an organism makes up its genome. Genes have a diverse number of functions. They code different proteins for normal proliferation of cells. However, changes in the base sequence of genes affect their protein by-products which act as messengers for normal cellular functions such as proliferation and repairs. Salient processes for maintaining the integrity of the genome are hinged on intricate mechanisms put in place for the evolution to tackle genomic stresses. To discuss how cells sense and repair damage to their deoxyribonucleic acid (DNA) as well as to highlight how defects in the genes involved in DNA repair contribute to cancer development. Methodology: Online searches on the following databases such as Google Scholar, PubMed, Biomed Central, and SciELO were done. Attempt was made to review articles with keywords such as cancer, cell cycle, tumor suppressor genes, and DNA repair. The cell cycle, tumor suppression genes, DNA repair mechanism, as well as their contribution to cancer development, were discussed and reviewed. Knowledge on how cells detect and repair DNA damage through an array of mechanisms should allay our anxiety as regards cancer development. More studies on DNA damage detection and repair processes are important toward a holistic approach to cancer treatment.

ERCC2/XPD Lys751Gln alter DNA repair efficiency of platinum-induced DNA damage through P53 pathway.

PubMed

Zhang, Guopei; Guan, Yangyang; Zhao, Yuejiao; van der Straaten, Tahar; Xiao, Sha; Xue, Ping; Zhu, Guolian; Liu, Qiufang; Cai, Yuan; Jin, Cuihong; Yang, Jinghua; Wu, Shengwen; Lu, Xiaobo

2017-02-01

Platinum-based treatment causes Pt-DNA adducts which lead to cell death. The platinum-induced DNA damage is recognized and repaired by the nucleotide excision repair (NER) system of which ERCC2/XPD is a critical enzyme. Single nucleotide polymorphisms in ERCC2/XPD have been found to be associated with platinum resistance. The aim of the present study was to investigate whether ERCC2/XPD Lys751Gln (rs13181) polymorphism is causally related to DNA repair capacity of platinum-induced DNA damage. First, cDNA clones expressing different genotypes of the polymorphism was transfected to an ERCC2/XPD defective CHO cell line (UV5). Second, all cells were treated with cisplatin. Cellular survival rate were investigated by MTT growth inhibition assay, DNA damage levels were investigated by comet assay and RAD51 staining. The distribution of cell cycle and the change of apoptosis rates were detected by a flow cytometric method (FCM). Finally, P53mRNA and phospho-P53 protein levels were further investigated in order to explore a possible explanation. As expected, there was a significantly increased in viability of UV5 ERCC2 (AA) as compared to UV5 ERCC2 (CC) after cisplatin treatment. The DNA damage level of UV5 ERCC2 (AA) was significant decreased compared to UV5 ERCC2 (CC) at 24 h of treatment. Mutation of ERCC2rs13181 AA to CC causes a prolonged S phase in cell cycle. UV5 ERCC2 (AA) alleviated the apoptosis compared to UV5 ERCC2 (CC) , meanwhile P53mRNA levels in UV ERCC2 (AA) was also lower when compared UV5 ERCC2 (CC) . It co-incides with a prolonged high expression of phospho-P53, which is relevant for cell cycle regulation, apoptosis, and the DNA damage response (DDR). We concluded that ERCC2/XPD rs13181 polymorphism is possibly related to the DNA repair capacity of platinum-induced DNA damage. This functional study provides some clues to clarify the relationship between cisplatin resistance and ERCC2/XPDrs13181 polymorphism. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

The comet moment as a measure of DNA damage in the comet assay.

PubMed

Kent, C R; Eady, J J; Ross, G M; Steel, G G

1995-06-01

The development of rapid assays of radiation-induced DNA damage requires the definition of reliable parameters for the evaluation of dose-response relationships to compare with cellular endpoints. We have used the single-cell gel electrophoresis (SCGE) or 'comet' assay to measure DNA damage in individual cells after irradiation. Both the alkaline and neutral protocols were used. In both cases, DNA was stained with ethidium bromide and viewed using a fluorescence microscope at 516-560 nm. Images of comets were stored as 512 x 512 pixel images using OPTIMAS, an image analysis software package. Using this software we tested various parameters for measuring DNA damage. We have developed a method of analysis that rigorously conforms to the mathematical definition of the moment of inertia of a plane figure. This parameter does not require the identification of separate head and tail regions, but rather calculates a moment of the whole comet image. We have termed this parameter 'comet moment'. This method is simple to calculate and can be performed using most image analysis software packages that support macro facilities. In experiments on CHO-K1 cells, tail length was found to increase linearly with dose, but plateaued at higher doses. Comet moment also increased linearly with dose, but over a larger dose range than tail length and had no tendency to plateau.

On the mechanism of Cr (VI)-induced carcinogenesis: dose dependence of uptake and cellular responses.

PubMed

Liu, K; Husler, J; Ye, J; Leonard, S S; Cutler, D; Chen, F; Wang, S; Zhang, Z; Ding, M; Wang, L; Shi, X

2001-06-01

Cr (VI) compounds are widely used industrial chemicals and are recognized human carcinogens. The mechanisms of carcinogenesis associated with these compounds remain to be investigated. The present study focused on dose-dependence of Cr (VI)-induced uptake and cellular responses. The results show that Cr (VI) is able to enter the cells (human lung epithelial cell line A549) at low concentration (< 10 microM) and that the Cr (VI) uptake appears to be a combination of saturable transport and passive diffusion. Electron spin resonance (ESR) trapping measurements showed that upon stimulation with Cr (VI), A549 cells were able to generate reactive oxygen species (ROS). The amount of ROS generated depended on the Cr (VI) concentration. ROS generation involved NADPH-dependent flavoenzymes. Cr (VI) affected the following cellular parameters in a dose-dependent manner, (a) activation of nuclear transcription factors NF-kappaB, and p53, (b) DNA damage, (c) induction of cell apoptosis, and (d) inhibition of cell proliferation. The activation of transcription factors was assessed by electrophoretic mobility shift assay and western blot analysis, DNA damage by single cell gel electrophoresis assay, cell apoptosis by DNA fragmentation assay, and cell proliferation by a non-radioactive ELISA kit. At the concentration range used in the present study, no thresholds were found in all of these cell responses to Cr (VI). The results may guide further research to better understand and evaluate the risk of Cr (VI)-induced carcinogenesis at low levels of exposure.

Neurotoxic lupus autoantibodies alter brain function through two distinct mechanisms

PubMed Central

Faust, Thomas W.; Chang, Eric H.; Kowal, Czeslawa; Berlin, RoseAnn; Gazaryan, Irina G.; Bertini, Eva; Zhang, Jie; Sanchez-Guerrero, Jorge; Fragoso-Loyo, Hilda E.; Volpe, Bruce T.; Diamond, Betty; Huerta, Patricio T.

2010-01-01

Damaging interactions between antibodies and brain antigenic targets may be responsible for an expanding range of neurological disorders. In the case of systemic lupus erythematosus (SLE), patients generate autoantibodies (AAbs) that frequently bind dsDNA. Although some symptoms of SLE may arise from direct reactivity to dsDNA, much of the AAb-mediated damage originates from cross-reactivity with other antigens. We have studied lupus AAbs that bind dsDNA and cross-react with the NR2A and NR2B subunits of the NMDA receptor (NMDAR). In adult mouse models, when the blood–brain barrier is compromised, these NMDAR-reactive AAbs access the brain and elicit neuronal death with ensuing cognitive dysfunction and emotional disturbance. The cellular mechanisms that underlie these deleterious effects remain incompletely understood. Here, we show that, at low concentration, the NMDAR-reactive AAbs are positive modulators of receptor function that increase the size of NMDAR-mediated excitatory postsynaptic potentials, whereas at high concentration, the AAbs promote excitotoxicity through enhanced mitochondrial permeability transition. Other synaptic receptors are completely unaffected by the AAbs. NMDAR activation is required for producing both the synaptic and the mitochondrial effects. Our study thus reveals the mechanisms by which NMDAR-reactive AAbs trigger graded cellular alterations, which are likely to be responsible for the transient and permanent neuropsychiatric symptoms observed in patients with SLE. Our study also provides a model in which local AAb concentration determines the exact nature of the cellular response. PMID:20921396

Phosphorylation of Nucleotide Excision Repair Factor Xeroderma Pigmentosum Group A by Ataxia Telangiectasia Mutated and Rad3-Related-Dependent Checkpoint Pathway Promotes Cell Survival in Response to UV Irradiation

PubMed Central

Wu, Xiaoming; Shell, Steven M.; Yang, Zhengguan; Zou, Yue

2006-01-01

DNA damage triggers complex cellular responses in eukaryotic cells, including initiation of DNA repair and activation of cell cycle checkpoints. In addition to inducing cell cycle arrest, checkpoint also has been suggested to modulate a variety of other cellular processes in response to DNA damage. In this study, we present evidence showing that the cellular function of xeroderma pigmentosum group A (XPA), a major nucleotide excision repair (NER) factor, could be modulated by checkpoint kinase ataxia-telangiectasia mutated and Rad3-related (ATR) in response to UV irradiation. We observed the apparent interaction and colocalization of XPA with ATR in response to UV irradiation. We showed that XPA was a substrate for in vitro phosphorylation by phosphatidylinositol-3-kinase-related kinase family kinases whereas in cells XPA was phosphorylated in an ATR-dependent manner and stimulated by UV irradiation. The Ser196 of XPA was identified as a biologically significant residue to be phosphorylated in vivo. The XPA-deficient cells complemented with XPA-S196A mutant, in which Ser196 was substituted with an alanine, displayed significantly higher UV sensitivity compared with the XPA cells complemented with wild-type XPA. Moreover, substitution of Ser196 with aspartic acid for mimicking the phosphorylation of XPA increased the cell survival to UV irradiation. Taken together, our results revealed a potential physical and functional link between NER and the ATR-dependent checkpoint pathway in human cells and suggested that the ATR checkpoint pathway could modulate the cellular activity of NER through phosphorylation of XPA at Ser196 on UV irradiation. PMID:16540648

Enhanced Repair of UV-Induced DNA Damage by 1,25-Dihydroxyvitamin D3 in Skin Is Linked to Pathways that Control Cellular Energy.

PubMed

Rybchyn, Mark Stephen; De Silva, Warusavithana Gunawardena Manori; Sequeira, Vanessa Bernadette; McCarthy, Bianca Yuko; Dilley, Anthony Vincent; Dixon, Katie Marie; Halliday, Gary Mark; Mason, Rebecca Sara

2018-05-01

Inadequately repaired post-UV DNA damage results in skin cancers. DNA repair requires energy but skin cells have limited capacity to produce energy after UV insult. We examined whether energy supply is important for DNA repair after UV exposure, in the presence of 1α,25-dihydroxyvitamin D 3 (1,25(OH) 2 D 3 ), which reduces UV-induced DNA damage and photocarcinogenesis in a variety of models. After UV exposure of primary human keratinocytes, the addition of 1,25(OH) 2 D 3 increased unscheduled DNA synthesis, a measure of DNA repair. Oxidative phosphorylation was depleted in UV-irradiated keratinocytes to undetectable levels within an hour of UV irradiation. Treatment with 1,25(OH) 2 D 3 but not vehicle increased glycolysis after UV. 2-Deoxyglucose-dependent inhibition of glycolysis abolished the reduction in cyclobutane pyrimidine dimers by 1,25(OH) 2 D 3 , whereas inhibition of oxidative phosphorylation had no effect. 1,25(OH) 2 D 3 increased autophagy and modulated PINK1/Parkin consistent with enhanced mitophagy. These data confirm that energy availability is limited in keratinocytes after exposure to UV. In the presence of 1,25(OH) 2 D 3 , glycolysis is enhanced along with energy-conserving processes such as autophagy and mitophagy, resulting in increased repair of cyclobutane pyrimidine dimers and decreased oxidative DNA damage. Increased energy availability in the presence of 1,25(OH) 2 D 3 is an important contributor to DNA repair in skin after UV exposure. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Concentration effects of grape seed extracts in anti-oral cancer cells involving differential apoptosis, oxidative stress, and DNA damage.

PubMed

Yen, Ching-Yu; Hou, Ming-Feng; Yang, Zhi-Wen; Tang, Jen-Yang; Li, Kun-Tzu; Huang, Hurng-Wern; Huang, Yu-Hsuan; Lee, Sheng-Yang; Fu, Tzu-Fun; Hsieh, Che-Yu; Chen, Bing-Hung; Chang, Hsueh-Wei

2015-03-29

Grape seeds extract (GSE) is a famous health food supplement for its antioxidant property. Different concentrations of GSE may have different impacts on cellular oxidative/reduction homeostasis. Antiproliferative effect of GSE has been reported in many cancers but rarely in oral cancer. The aim of this study is to examine the antioral cancer effects of different concentrations of GSE in terms of cell viability, apoptosis, reactive oxygen species (ROS), mitochondrial function, and DNA damage. High concentrations (50-400 μg/ml) of GSE dose-responsively inhibited proliferation of oral cancer Ca9-22 cells but low concentrations (1-10 μg/ml) of GSE showed a mild effect in a MTS assay. For apoptosis analyses, subG1 population and annexin V intensity in high concentrations of GSE-treated Ca9-22 cells was increased but less so at low concentrations. ROS generation and mitochondrial depolarization increased dose-responsively at high concentrations but showed minor changes at low concentrations of GSE in Ca9-22 cells. Additionally, high concentrations of GSE dose-responsively induced more γH2AX-based DNA damage than low concentrations. Differential concentrations of GSE may have a differentially antiproliferative function against oral cancer cells via differential apoptosis, oxidative stress and DNA damage.

Recovery from DNA damage-induced G2 arrest requires actin-binding protein filamin-A/actin-binding protein 280.

PubMed

Meng, Xiangbing; Yuan, Yuan; Maestas, Adrian; Shen, Zhiyuan

2004-02-13

Filamin-A (filamin-1) is an actin-binding protein involved in the organization of actin networks. Our previous study shows that filamin-A interacts with BRCA2, and lack of filamin-A expression results in increased cellular sensitivity to several DNA damaging agents in melanoma cells (Yuan, Y., and Shen, Z. (2001) J. Biol. Chem. 276, 48318-48324), suggesting a role of filamin-A in DNA damage response. In this report, we demonstrated that deficiency of filamin-A results in an 8-h delay in the recovery from G2 arrest in response to ionizing radiation. However, filamin-A deficiency does not affect the initial activation of the G2/M checkpoint. We also found that filamin-A deficiency results in sustained activation of Chk1 and Chk2 after irradiation. This in turn causes a delay in the dephosphorylation of phospho-Cdc2, which is inhibitory to the G2/M transition. In addition, filamin-A-deficient M2 cells undergo mitotic catastrophe-related nuclear fragmentation after they are released from the G2 arrest. Together, these data suggest a functional role of filamin-A in the recovery from G2 arrest and subsequent mitotic cell death after DNA damage.

Chemotherapeutic-Induced Cardiovascular Dysfunction: Physiological Effects, Early Detection—The Role of Telomerase to Counteract Mitochondrial Defects and Oxidative Stress

PubMed Central

Quryshi, Nabeel; Norwood Toro, Laura E.; Ait-Aissa, Karima; Kong, Amanda; Beyer, Andreas M.

2018-01-01

Although chemotherapeutics can be highly effective at targeting malignancies, their ability to trigger cardiovascular morbidity is clinically significant. Chemotherapy can adversely affect cardiovascular physiology, resulting in the development of cardiomyopathy, heart failure and microvascular defects. Specifically, anthracyclines are known to cause an excessive buildup of free radical species and mitochondrial DNA damage (mtDNA) that can lead to oxidative stress-induced cardiovascular apoptosis. Therefore, oncologists and cardiologists maintain a network of communication when dealing with patients during treatment in order to treat and prevent chemotherapy-induced cardiovascular damage; however, there is a need to discover more accurate biomarkers and therapeutics to combat and predict the onset of cardiovascular side effects. Telomerase, originally discovered to promote cellular proliferation, has recently emerged as a potential mechanism to counteract mitochondrial defects and restore healthy mitochondrial vascular phenotypes. This review details mechanisms currently used to assess cardiovascular damage, such as C-reactive protein (CRP) and troponin levels, while also unearthing recently researched biomarkers, including circulating mtDNA, telomere length and telomerase activity. Further, we explore a potential role of telomerase in the mitigation of mitochondrial reactive oxygen species and maintenance of mtDNA integrity. Telomerase activity presents a promising indicator for the early detection and treatment of chemotherapy-derived cardiac damage. PMID:29534446

A decrease in cyclin B1 levels leads to polyploidization in DNA damage-induced senescence.

PubMed

Kikuchi, Ikue; Nakayama, Yuji; Morinaga, Takao; Fukumoto, Yasunori; Yamaguchi, Naoto

2010-05-04

Adriamycin, an anthracycline antibiotic, has been used for the treatment of various types of tumours. Adriamycin induces at least two distinct types of growth repression, such as senescence and apoptosis, in a concentration-dependent manner. Cellular senescence is a condition in which cells are unable to proliferate further, and senescent cells frequently show polyploidy. Although abrogation of cell division is thought to correlate with polyploidization, the mechanisms underlying induction of polyploidization in senescent cells are largely unclear. We wished, therefore, to explore the role of cyclin B1 level in polyploidization of Adriamycin-induced senescent cells. A subcytotoxic concentration of Adriamycin induced polyploid cells having the features of senescence, such as flattened and enlarged cell shape and activated beta-galactosidase activity. In DNA damage-induced senescent cells, the levels of cyclin B1 were transiently increased and subsequently decreased. The decrease in cyclin B1 levels occurred in G2 cells during polyploidization upon treatment with a subcytotoxic concentration of Adriamycin. In contrast, neither polyploidy nor a decrease in cyclin B1 levels was induced by treatment with a cytotoxic concentration of Adriamycin. These results suggest that a decrease in cyclin B1 levels is induced by DNA damage, resulting in polyploidization in DNA damage-induced senescence.

Novel TDP2-ubiquitin interactions and their importance for the repair of topoisomerase II-mediated DNA damage

PubMed Central

Rao, Timsi; Gao, Rui; Takada, Saeko; Al Abo, Muthana; Chen, Xiang; Walters, Kylie J.; Pommier, Yves; Aihara, Hideki

2016-01-01

Tyrosyl DNA phosphodiesterase 2 (TDP2) is a multifunctional protein implicated in DNA repair, signal transduction and transcriptional regulation. In its DNA repair role, TDP2 safeguards genome integrity by hydrolyzing 5′-tyrosyl DNA adducts formed by abortive topoisomerase II (Top2) cleavage complexes to allow error-free repair of DNA double-strand breaks, thereby conferring cellular resistance against Top2 poisons. TDP2 consists of a C-terminal catalytic domain responsible for its phosphodiesterase activity, and a functionally uncharacterized N-terminal region. Here, we demonstrate that this N-terminal region contains a ubiquitin (Ub)-associated (UBA) domain capable of binding multiple forms of Ub with distinct modes of interactions and preference for either K48- or K63-linked polyUbs over monoUb. The structure of TDP2 UBA bound to monoUb shows a canonical mode of UBA-Ub interaction. However, the absence of the highly conserved MGF motif and the presence of a fourth α-helix make TDP2 UBA distinct from other known UBAs. Mutations in the TDP2 UBA-Ub binding interface do not affect nuclear import of TDP2, but severely compromise its ability to repair Top2-mediated DNA damage, thus establishing the importance of the TDP2 UBA–Ub interaction in DNA repair. The differential binding to multiple Ub forms could be important for responding to DNA damage signals under different contexts or to support the multi-functionality of TDP2. PMID:27543075

Redundancy of mammalian Y family DNA polymerases in cellular responses to genomic DNA lesions induced by ultraviolet light

PubMed Central

Jansen, Jacob G.; Temviriyanukul, Piya; Wit, Niek; Delbos, Frédéric; Reynaud, Claude-Agnès; Jacobs, Heinz; de Wind, Niels

2014-01-01

Short-wave ultraviolet light induces both mildly helix-distorting cyclobutane pyrimidine dimers (CPDs) and severely distorting (6–4) pyrimidine pyrimidone photoproducts ((6–4)PPs). The only DNA polymerase (Pol) that is known to replicate efficiently across CPDs is Polη, a member of the Y family of translesion synthesis (TLS) DNA polymerases. Phenotypes of Polη deficiency are transient, suggesting redundancy with other DNA damage tolerance pathways. Here we performed a comprehensive analysis of the temporal requirements of Y-family Pols ι and κ as backups for Polη in (i) bypassing genomic CPD and (6–4)PP lesions in vivo, (ii) suppressing DNA damage signaling, (iii) maintaining cell cycle progression and (iv) promoting cell survival, by using mouse embryonic fibroblast lines with single and combined disruptions in these Pols. The contribution of Polι is restricted to TLS at a subset of the photolesions. Polκ plays a dominant role in rescuing stalled replication forks in Polη-deficient mouse embryonic fibroblasts, both at CPDs and (6–4)PPs. This dampens DNA damage signaling and cell cycle arrest, and results in increased survival. The role of relatively error-prone Pols ι and κ as backups for Polη contributes to the understanding of the mutator phenotype of xeroderma pigmentosum variant, a syndrome caused by Polη defects. PMID:25170086

CRN13 candidate effectors from plant and animal eukaryotic pathogens are DNA-binding proteins which trigger host DNA damage response.

PubMed

Ramirez-Garcés, Diana; Camborde, Laurent; Pel, Michiel J C; Jauneau, Alain; Martinez, Yves; Néant, Isabelle; Leclerc, Catherine; Moreau, Marc; Dumas, Bernard; Gaulin, Elodie

2016-04-01

To successfully colonize their host, pathogens produce effectors that can interfere with host cellular processes. Here we investigated the function of CRN13 candidate effectors produced by plant pathogenic oomycetes and detected in the genome of the amphibian pathogenic chytrid fungus Batrachochytrium dendrobatidis (BdCRN13). When expressed in Nicotiana, AeCRN13, from the legume root pathogen Aphanomyces euteiches, increases the susceptibility of the leaves to the oomycete Phytophthora capsici. When transiently expressed in amphibians or plant cells, AeCRN13 and BdCRN13 localize to the cell nuclei, triggering aberrant cell development and eventually causing cell death. Using Förster resonance energy transfer experiments in plant cells, we showed that both CRN13s interact with nuclear DNA and trigger plant DNA damage response (DDR). Mutating key amino acid residues in a predicted HNH-like endonuclease motif abolished the interaction of AeCRN13 with DNA, the induction of DDR and the enhancement of Nicotiana susceptibility to P. capsici. Finally, H2AX phosphorylation, a marker of DNA damage, and enhanced expression of genes involved in the DDR were observed in A. euteiches-infected Medicago truncatula roots. These results show that CRN13 from plant and animal eukaryotic pathogens promotes host susceptibility by targeting nuclear DNA and inducing DDR. © 2015 The Authors. New Phytologist © 2015 New Phytologist Trust.

Cellular Dynamics of Rad51 and Rad54 in Response to Postreplicative Stress and DNA Damage in HeLa Cells.

PubMed

Choi, Eui-Hwan; Yoon, Seobin; Hahn, Yoonsoo; Kim, Keun P

2017-02-01

Homologous recombination (HR) is necessary for maintenance of genomic integrity and prevention of various mutations in tumor suppressor genes and proto-oncogenes. Rad51 and Rad54 are key HR factors that cope with replication stress and DNA breaks in eukaryotes. Rad51 binds to single-stranded DNA (ssDNA) to form the presynaptic filament that promotes a homology search and DNA strand exchange, and Rad54 stimulates the strand-pairing function of Rad51. Here, we studied the molecular dynamics of Rad51 and Rad54 during the cell cycle of HeLa cells. These cells constitutively express Rad51 and Rad54 throughout the entire cell cycle, and the formation of foci immediately increased in response to various types of DNA damage and replication stress, except for caffeine, which suppressed the Rad51-dependent HR pathway. Depletion of Rad51 caused severe defects in response to postreplicative stress. Accordingly, HeLa cells were arrested at the G2-M transition although a small amount of Rad51 was steadily maintained in HeLa cells. Our results suggest that cell cycle progression and proliferation of HeLa cells can be tightly controlled by the abundance of HR proteins, which are essential for the rapid response to postreplicative stress and DNA damage stress.

Recruitment of Fanconi Anemia and Breast Cancer Proteins to DNA Damage Sites is differentially Governed by Replication

PubMed Central

Shen, Xi; Do, Huong; Li, Yongjian; Chung, Woo-Hyun; Tomasz, Maria; de Winter, Johan P.; Xia, Bing; Elledge, Stephen J.; Wang, Weidong; Li, Lei

2009-01-01

Summary Fanconi anemia (FA) is characterized by cellular hypersensivity to DNA crosslinking agents, but how the Fanconi pathway protects cells from DNA crosslinks and whether FA proteins act directly on crosslinks remains unclear. We developed a chromatin-IP-based strategy termed eChIP and detected association of multiple FA proteins with DNA crosslinks in vivo. Inter-dependence analyses revealed that crosslink-specific enrichment of various FA proteins is controlled by distinct mechanisms. BRCA-related FA proteins (BRCA2, FANCJ/BACH1, and FANCN/PALB2), but not FA core and I/D2 complexes, require replication for their crosslink association. FANCD2, but not FANCJ and FANCN, requires the FA core complex for its recruitment. FA core complex requires nucleotide excision repair proteins XPA and XPC for its association. Consistent with the distinct recruitment mechanism, recombination-independent crosslink repair was inversely affected in cells deficient of FANC-core versus BRCA-related FA proteins. Thus, FA proteins participate in distinct DNA damage response mechanisms governed by DNA replication status. PMID:19748364

Nuclear depletion of apurinic/apyrimidinic endonuclease 1 (Ape1/Ref-1) is an indicator of energy disruption in neurons.

PubMed

Singh, Shilpee; Englander, Ella W

2012-11-01

Apurinic/apyrimidinic endonuclease 1 (Ape1/Ref-1) is a multifunctional protein critical for cellular survival. Its involvement in adaptive survival responses includes key roles in redox sensing, transcriptional regulation, and repair of DNA damage via the base excision repair (BER) pathway. Ape1 is abundant in most cell types and central in integrating the first BER step catalyzed by different DNA glycosylases. BER is the main process for removal of oxidative DNA lesions in postmitotic brain cells, and after ischemic brain injury preservation of Ape1 coincides with neuronal survival, while its loss has been associated with neuronal death. Here, we report that in cultured primary neurons, diminution of cellular ATP by either oligomycin or H(2)O(2) is accompanied by depletion of nuclear Ape1, while other BER proteins are unaffected and retain their nuclear localization under these conditions. Importantly, while H(2)O(2) induces γH2AX phosphorylation, indicative of chromatin rearrangements in response to DNA damage, oligomycin does not. Furthermore, despite comparable diminution of ATP content, H(2)O(2) and oligomycin differentially affect critical parameters of mitochondrial respiration that ultimately determine cellular ATP content. Taken together, our findings demonstrate that in neurons, nuclear compartmentalization of Ape1 depends on ATP and loss of nuclear Ape1 reflects disruption of neuronal energy homeostasis. Energy crisis is a hallmark of stroke and other ischemic/hypoxic brain injuries. In vivo studies have shown that Ape1 deficit precedes neuronal loss in injured brain regions. Thus, our findings bring to light the possibility that energy failure-induced Ape1 depletion triggers neuronal death in ischemic brain injuries. Copyright © 2012 Elsevier Inc. All rights reserved.

Emerging links between the biological clock and the DNA damage response.

PubMed

Collis, Spencer J; Boulton, Simon J

2007-08-01

For life forms to survive, they must adapt to their environmental conditions. One such factor that impacts on both prokaryotic and eukaryotic organisms is the light-dark cycle, a consequence of planetary rotation in relation to our sun. In mammals, the daily light cycle has affected the regulation of many cellular processes such as sleep-wake and calorific intake activities, hormone secretion, blood pressure and immune system responses. Such rhythmic behaviour is the consequence of circadian rhythm/biological clock (BC) systems which are controlled in a light stimulus-dependent manner by a master clock called the suprachiasmatic nucleus (SCN) situated within the anterior hypothalamus. Peripheral clocks located in other organs such as the liver and kidneys relay signals from the SCN, which ultimately leads to tightly controlled expression of several protein families that in turn act on a broad range of cellular functions. Work in lower organisms has demonstrated a link between aging processes and BC factors, and studies in both animal models and clinical trials have postulated a role for certain BC-associated proteins in tumourigenesis and cancer progression. Recent exciting data reported within the last year or so have now established a molecular link between specific BC proteins and factors that control the mammalian cell cycle and DNA damage checkpoints. This mini review will focus on these discoveries and emphasise how such BC proteins may be involved, through their interplay with cell cycle/DNA damage response pathways, in the development of human disease such as cancer.

Cellular evaluation of diselenonicotinamide (DSNA) as a radioprotector against cell death and DNA damage.

PubMed

Raghuraman, M; Verma, Prachi; Kunwar, Amit; Phadnis, Prasad P; Jain, V K; Priyadarsini, K Indira

2017-06-01

Diselenonicotinamide (DSNA), a synthetic organoselenium compound, was evaluated for its radioprotective effect in cellular models. A clonogenic assay in Chinese Hamster Ovary (CHO) cells and an apoptosis assay in murine splenic lymphocytes indicated that pre-treatment with DSNA at a concentration of 25 μM significantly protected them from radiation-induced cell death. Upon irradiation (1-12 Gy), dose-response studies were carried out under similar treatment conditions, and its dose modification factor (DMF) was estimated to be 1.26. Furthermore, DSNA showed its radioprotective effect, even when administered after exposure to radiation. Mechanistic investigation revealed that DSNA increased the intracellular levels of GPx and GSH in irradiated cells. In line with this observation, the addition of a pharmacological inhibitor of GPx cycle, abrogated the activity of DSNA. The radioprotective effect of DSNA was also complemented by its ability to prevent radiation-induced DNA damage as monitored by micronucleus and γ-H2AX assays. Furthermore, treatment with DSNA did not show much change in the expressions of Nrf2 dependent genes (γ-GCL and HO-1), but the presence of a pharmacological inhibitor of Nrf2 abrogated the radioprotective activity of DSNA against cell death and DNA damage. Additionally, ATRA treatment also inhibited the DSNA-mediated up-regulation of a repair gene RAD51, suggesting possible involvement of basal Nrf2 in the anti-genotoxic effect of DSNA. In conclusion, the present study demonstrates radioprotection by a synthetic organoselenium compound containing nutritionally important moieties like selenium and nicotinamide.

Radiation-induced oxidative damage to the DNA-binding domain of the lactose repressor

PubMed Central

Gillard, Nathalie; Goffinont, Stephane; Buré, Corinne; Davidkova, Marie; Maurizot, Jean-Claude; Cadene, Martine; Spotheim-Maurizot, Melanie

2007-01-01

Understanding the cellular effects of radiation-induced oxidation requires the unravelling of key molecular events, particularly damage to proteins with important cellular functions. The Escherichia coli lactose operon is a classical model of gene regulation systems. Its functional mechanism involves the specific binding of a protein, the repressor, to a specific DNA sequence, the operator. We have shown previously that upon irradiation with γ-rays in solution, the repressor loses its ability to bind the operator. Water radiolysis generates hydroxyl radicals (OH· radicals) which attack the protein. Damage of the repressor DNA-binding domain, called the headpiece, is most likely to be responsible of this loss of function. Using CD, fluorescence spectroscopy and a combination of proteolytic cleavage with MS, we have examined the state of the irradiated headpiece. CD measurements revealed a dose-dependent conformational change involving metastable intermediate states. Fluorescence measurements showed a gradual degradation of tyrosine residues. MS was used to count the number of oxidations in different regions of the headpiece and to narrow down the parts of the sequence bearing oxidized residues. By calculating the relative probabilities of reaction of each amino acid with OH· radicals, we can predict the most probable oxidation targets. By comparing the experimental results with the predictions we conclude that Tyr7, Tyr12, Tyr17, Met42 and Tyr47 are the most likely hotspots of oxidation. The loss of repressor function is thus correlated with chemical modifications and conformational changes of the headpiece. PMID:17263689

Mutagenic repair of double-stranded DNA breaks in vaccinia virus genomes requires cellular DNA ligase IV activity in the cytosol.

PubMed

Luteijn, Rutger David; Drexler, Ingo; Smith, Geoffrey L; Lebbink, Robert Jan; Wiertz, Emmanuel J H J

2018-06-01

Poxviruses comprise a group of large dsDNA viruses that include members relevant to human and animal health, such as variola virus, monkeypox virus, cowpox virus and vaccinia virus (VACV). Poxviruses are remarkable for their unique replication cycle, which is restricted to the cytoplasm of infected cells. The independence from the host nucleus requires poxviruses to encode most of the enzymes involved in DNA replication, transcription and processing. Here, we use the CRISPR/Cas9 genome engineering system to induce DNA damage to VACV (strain Western Reserve) genomes. We show that targeting CRISPR/Cas9 to essential viral genes limits virus replication efficiently. Although VACV is a strictly cytoplasmic pathogen, we observed extensive viral genome editing at the target site; this is reminiscent of a non-homologous end-joining DNA repair mechanism. This pathway was not dependent on the viral DNA ligase, but critically involved the cellular DNA ligase IV. Our data show that DNA ligase IV can act outside of the nucleus to allow repair of dsDNA breaks in poxvirus genomes. This pathway might contribute to the introduction of mutations within the genome of poxviruses and may thereby promote the evolution of these viruses.

One-electron oxidation reactions of purine and pyrimidine bases in cellular DNA

PubMed Central

Cadet, Jean; Wagner, J. Richard; Shafirovich, Vladimir; Geacintov, Nicholas E.

2014-01-01

Purpose The aim of this survey is to critically review the available information on one-electron oxidation reactions of nucleobases in cellular DNA with emphasis on damage induced through the transient generation of purine and pyrimidine radical cations. Since the indirect effect of ionizing radiation mediated by hydroxyl radical is predominant in cells, efforts have been made to selectively ionize bases using suitable one-electron oxidants that consist among others of high intensity UVC laser pulses. Thus, the main oxidation product in cellular DNA was found to be 8-oxo-7,8-dihydroguanine as a result of direct bi-photonic ionization of guanine bases and indirect formation of guanine radical cations through hole transfer reactions from other base radical cations. The formation of 8-oxo-7,8-dihydroguanine and other purine and pyrimidine degradation products was rationalized in terms of the initial generation of related radical cations followed by either hydration or deprotonation reactions in agreement with mechanistic pathways inferred from detailed mechanistic studies. The guanine radical cation has been shown to be implicated in three other nucleophilic additions that give rise to DNA-protein and DNA-DNA cross-links in model systems. Evidence was recently provided for the occurrence of these three reactions in cellular DNA. Conclusion There is growing evidence that one-electron oxidation reactions of nucleobases whose mechanisms have been characterized in model studies involving aqueous solutions take place in a similar way in cells. It may also be pointed out that the above cross-linked lesions are only produced from the guanine radical cation and may be considered as diagnostic products of the direct effect of ionizing radiation. PMID:24369822

One-electron oxidation reactions of purine and pyrimidine bases in cellular DNA.

PubMed

Cadet, Jean; Wagner, J Richard; Shafirovich, Vladimir; Geacintov, Nicholas E

2014-06-01

The aim of this survey is to critically review the available information on one-electron oxidation reactions of nucleobases in cellular DNA with emphasis on damage induced through the transient generation of purine and pyrimidine radical cations. Since the indirect effect of ionizing radiation mediated by hydroxyl radical is predominant in cells, efforts have been made to selectively ionize bases using suitable one-electron oxidants that consist among others of high intensity UVC laser pulses. Thus, the main oxidation product in cellular DNA was found to be 8-oxo-7,8-dihydroguanine as a result of direct bi-photonic ionization of guanine bases and indirect formation of guanine radical cations through hole transfer reactions from other base radical cations. The formation of 8-oxo-7,8-dihydroguanine and other purine and pyrimidine degradation products was rationalized in terms of the initial generation of related radical cations followed by either hydration or deprotonation reactions in agreement with mechanistic pathways inferred from detailed mechanistic studies. The guanine radical cation has been shown to be implicated in three other nucleophilic additions that give rise to DNA-protein and DNA-DNA cross-links in model systems. Evidence was recently provided for the occurrence of these three reactions in cellular DNA. There is growing evidence that one-electron oxidation reactions of nucleobases whose mechanisms have been characterized in model studies involving aqueous solutions take place in a similar way in cells. It may also be pointed out that the above cross-linked lesions are only produced from the guanine radical cation and may be considered as diagnostic products of the direct effect of ionizing radiation.