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

Directory of Open Access Journals (Sweden)

Anthony Skipper

2016-01-01

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

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

International Nuclear Information System (INIS)

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

2005-01-01

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

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

Science.gov (United States)

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

2011-11-01

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

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

Science.gov (United States)

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 edaravone offers protection from radiation-induced cytogenetic alterations.

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

Directory of Open Access Journals (Sweden)

Luca A Petruccelli

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

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

Science.gov (United States)

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

2011-01-01

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

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

Directory of Open Access Journals (Sweden)

Liwei Dong

2015-06-01

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

Oxidative stress induction by T-2 toxin causes DNA damage and triggers apoptosis via caspase pathway in human cervical cancer cells

International Nuclear Information System (INIS)

Chaudhari, Manjari; Jayaraj, R.; Bhaskar, A.S.B.; Lakshmana Rao, P.V.

2009-01-01

T-2 toxin is the most toxic trichothecene and both humans and animals suffer from several pathological conditions after consumption of foodstuffs contaminated with trichothecenes. We investigated the molecular mechanism of T-2 toxin induced cytotoxicity and cell death in HeLa cells. T-2 toxin at LC50 of 10 ng/ml caused time dependent increase in cytotoxicity as assessed by dye uptake, lactatedehydrogenase leakage and MTT assay. The toxin caused generation of reactive oxygen species as early as 30 min followed by significant depletion of glutathione levels and increased lipid peroxidation. The results indicate oxidative stress as underlying mechanism of cytotoxicity. Single stranded DNA damage after T-2 treatment was observed as early as 2 and 4 h by DNA diffusion assay. The cells exhibited apoptotic morphology like condensed chromatin and nuclear fragmentation after 4 h of treatment. Downstream of T-2 induced oxidative stress and DNA damage a time dependent increase in expression level of p53 protein was observed. The increase in Bax/Bcl2 ratio indicated shift in response, in favour of apoptotic process in T-2 toxin treated cells. Western blot analysis showed increase in levels of mitochondrial apoptogenic factors Bax, Bcl-2, cytochrome-c followed by activation of caspases-9, -3 and -7 leading to DNA fragmentation and apoptosis. In addition to caspase-dependent pathway, our results showed involvement of caspase-independent AIF pathway in T-2 induced apoptosis. Broad spectrum caspase inhibitor z-VAD-fmk could partially protect the cells from DNA damage but could not inhibit AIF induced oligonucleosomal DNA fragmentation beyond 4 h. Results of the study clearly show that oxidative stress is the underlying mechanism by which T-2 toxin causes DNA damage and apoptosis.

Autophagy in DNA Damage Response

Directory of Open Access Journals (Sweden)

Piotr Czarny

2015-01-01

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

Epidermal Rac1 regulates the DNA damage response and protects from UV-light-induced keratinocyte apoptosis and skin carcinogenesis

Science.gov (United States)

Deshmukh, Jayesh; Pofahl, Ruth; Haase, Ingo

2017-01-01

Non-melanoma skin cancer (NMSC) is the most common type of cancer. Increased expression and activity of Rac1, a small Rho GTPase, has been shown previously in NMSC and other human cancers; suggesting that Rac1 may function as an oncogene in skin. DMBA/TPA skin carcinogenesis studies in mice have shown that Rac1 is required for chemically induced skin papilloma formation. However, UVB radiation by the sun, which causes DNA damage, is the most relevant cause for NMSC. A potential role of Rac1 in UV-light-induced skin carcinogenesis has not been investigated so far. To investigate this, we irradiated mice with epidermal Rac1 deficiency (Rac1-EKO) and their controls using a well-established protocol for long-term UV-irradiation. Most of the Rac1-EKO mice developed severe skin erosions upon long-term UV-irradiation, unlike their controls. These skin erosions in Rac1-EKO mice healed subsequently. Surprisingly, we observed development of squamous cell carcinomas (SCCs) within the UV-irradiation fields. This shows that the presence of Rac1 in the epidermis protects from UV-light-induced skin carcinogenesis. Short-term UV-irradiation experiments revealed increased UV-light-induced apoptosis of Rac1-deficient epidermal keratinocytes in vitro as well as in vivo. Further investigations using cyclobutane pyrimidine dimer photolyase transgenic mice revealed that the observed increase in UV-light-induced keratinocyte apoptosis in Rac1-EKO mice is DNA damage dependent and correlates with caspase-8 activation. Furthermore, Rac1-deficient keratinocytes showed reduced levels of p53, γ-H2AX and p-Chk1 suggesting an attenuated DNA damage response upon UV-irradiation. Taken together, our data provide direct evidence for a protective role of Rac1 in UV-light-induced skin carcinogenesis and keratinocyte apoptosis probably through regulating mechanisms of the DNA damage response and repair pathways. PMID:28277539

Epidermal Rac1 regulates the DNA damage response and protects from UV-light-induced keratinocyte apoptosis and skin carcinogenesis.

Science.gov (United States)

Deshmukh, Jayesh; Pofahl, Ruth; Haase, Ingo

2017-03-09

Non-melanoma skin cancer (NMSC) is the most common type of cancer. Increased expression and activity of Rac1, a small Rho GTPase, has been shown previously in NMSC and other human cancers; suggesting that Rac1 may function as an oncogene in skin. DMBA/TPA skin carcinogenesis studies in mice have shown that Rac1 is required for chemically induced skin papilloma formation. However, UVB radiation by the sun, which causes DNA damage, is the most relevant cause for NMSC. A potential role of Rac1 in UV-light-induced skin carcinogenesis has not been investigated so far. To investigate this, we irradiated mice with epidermal Rac1 deficiency (Rac1-EKO) and their controls using a well-established protocol for long-term UV-irradiation. Most of the Rac1-EKO mice developed severe skin erosions upon long-term UV-irradiation, unlike their controls. These skin erosions in Rac1-EKO mice healed subsequently. Surprisingly, we observed development of squamous cell carcinomas (SCCs) within the UV-irradiation fields. This shows that the presence of Rac1 in the epidermis protects from UV-light-induced skin carcinogenesis. Short-term UV-irradiation experiments revealed increased UV-light-induced apoptosis of Rac1-deficient epidermal keratinocytes in vitro as well as in vivo. Further investigations using cyclobutane pyrimidine dimer photolyase transgenic mice revealed that the observed increase in UV-light-induced keratinocyte apoptosis in Rac1-EKO mice is DNA damage dependent and correlates with caspase-8 activation. Furthermore, Rac1-deficient keratinocytes showed reduced levels of p53, γ-H2AX and p-Chk1 suggesting an attenuated DNA damage response upon UV-irradiation. Taken together, our data provide direct evidence for a protective role of Rac1 in UV-light-induced skin carcinogenesis and keratinocyte apoptosis probably through regulating mechanisms of the DNA damage response and repair pathways.

Inhibition of autophagy enhances DNA damage-induced apoptosis by disrupting CHK1-dependent S phase arrest

Energy Technology Data Exchange (ETDEWEB)

Liou, Jong-Shian; Wu, Yi-Chen; Yen, Wen-Yen; Tang, Yu-Shuan [Institute of Cellular and Organismic Biology, Academia Sinica, Taipei 115, Taiwan, ROC (China); Kakadiya, Rajesh B.; Su, Tsann-Long [Institute of Biomedical Sciences, Academia Sinica, Taipei 115, Taiwan, ROC (China); Yih, Ling-Huei, E-mail: lhyih@gate.sinica.edu.tw [Institute of Cellular and Organismic Biology, Academia Sinica, Taipei 115, Taiwan, ROC (China)

2014-08-01

DNA damage has been shown to induce autophagy, but the role of autophagy in the DNA damage response and cell fate is not fully understood. BO-1012, a bifunctional alkylating derivative of 3a-aza-cyclopenta[a]indene, is a potent DNA interstrand cross-linking agent with anticancer activity. In this study, BO-1012 was found to reduce DNA synthesis, inhibit S phase progression, and induce phosphorylation of histone H2AX on serine 139 (γH2AX) exclusively in S phase cells. Both CHK1 and CHK2 were phosphorylated in response to BO-1012 treatment, but only depletion of CHK1, but not CHK2, impaired BO-1012-induced S phase arrest and facilitated the entry of γH2AX-positive cells into G2 phase. CHK1 depletion also significantly enhanced BO-1012-induced cell death and apoptosis. These results indicate that BO-1012-induced S phase arrest is a CHK1-dependent pro-survival response. BO-1012 also resulted in marked induction of acidic vesicular organelle (AVO) formation and microtubule-associated protein 1 light chain 3 (LC3) processing and redistribution, features characteristic of autophagy. Depletion of ATG7 or co-treatment of cells with BO-1012 and either 3-methyladenine or bafilomycin A1, two inhibitors of autophagy, not only reduced CHK1 phosphorylation and disrupted S phase arrest, but also increased cleavage of caspase-9 and PARP, and cell death. These results suggest that cells initiate S phase arrest and autophagy as pro-survival responses to BO-1012-induced DNA damage, and that suppression of autophagy enhances BO-1012-induced apoptosis via disruption of CHK1-dependent S phase arrest. - Highlights: • Autophagy inhibitors enhanced the cytotoxicity of a DNA alkylating agent, BO-1012. • BO-1012-induced S phase arrest was a CHK1-dependent pro-survival response. • Autophagy inhibition enhanced BO-1012 cytotoxicity via disrupting the S phase arrest.

Distinct patterns of DNA damage response and apoptosis correlate with Jak/Stat and PI3kinase response profiles in human acute myelogenous leukemia.

Directory of Open Access Journals (Sweden)

David B Rosen

Full Text Available BACKGROUND: Single cell network profiling (SCNP utilizing flow cytometry measures alterations in intracellular signaling responses. Here SCNP was used to characterize Acute Myeloid Leukemia (AML disease subtypes based on survival, DNA damage response and apoptosis pathways. METHODOLOGY AND PRINCIPAL FINDINGS: Thirty four diagnostic non-M3 AML samples from patients with known clinical outcome were treated with a panel of myeloid growth factors and cytokines, as well as with apoptosis-inducing agents. Analysis of induced Jak/Stat and PI3K pathway responses in blasts from individual patient samples identified subgroups with distinct signaling profiles that were not seen in the absence of a modulator. In vitro exposure of patient samples to etoposide, a DNA damaging agent, revealed three distinct "DNA damage response (DDR/apoptosis" profiles: 1 AML blasts with a defective DDR and failure to undergo apoptosis; 2 AML blasts with proficient DDR and failure to undergo apoptosis; 3 AML blasts with proficiency in both DDR and apoptosis pathways. Notably, AML samples from clinical responders fell within the "DDR/apoptosis" proficient profile and, as well, had low PI3K and Jak/Stat signaling responses. In contrast, samples from clinical non responders had variable signaling profiles often with in vitro apoptotic failure and elevated PI3K pathway activity. Individual patient samples often harbored multiple, distinct, leukemia-associated cell populations identifiable by their surface marker expression, functional performance of signaling pathway in the face of cytokine or growth factor stimulation, as well as their response to apoptosis-inducing agents. CONCLUSIONS AND SIGNIFICANCE: Characterizing and tracking changes in intracellular pathway profiles in cell subpopulations both at baseline and under therapeutic pressure will likely have important clinical applications, potentially informing the selection of beneficial targeted agents, used either alone or in

The Role of Mitochondrial DNA in Mediating Alveolar Epithelial Cell Apoptosis and Pulmonary Fibrosis

Science.gov (United States)

Kim, Seok-Jo; Cheresh, Paul; Jablonski, Renea P.; Williams, David B.; Kamp, David W.

2015-01-01

Convincing evidence has emerged demonstrating that impairment of mitochondrial function is critically important in regulating alveolar epithelial cell (AEC) programmed cell death (apoptosis) that may contribute to aging-related lung diseases, such as idiopathic pulmonary fibrosis (IPF) and asbestosis (pulmonary fibrosis following asbestos exposure). The mammalian mitochondrial DNA (mtDNA) encodes for 13 proteins, including several essential for oxidative phosphorylation. We review the evidence implicating that oxidative stress-induced mtDNA damage promotes AEC apoptosis and pulmonary fibrosis. We focus on the emerging role for AEC mtDNA damage repair by 8-oxoguanine DNA glycosylase (OGG1) and mitochondrial aconitase (ACO-2) in maintaining mtDNA integrity which is important in preventing AEC apoptosis and asbestos-induced pulmonary fibrosis in a murine model. We then review recent studies linking the sirtuin (SIRT) family members, especially SIRT3, to mitochondrial integrity and mtDNA damage repair and aging. We present a conceptual model of how SIRTs modulate reactive oxygen species (ROS)-driven mitochondrial metabolism that may be important for their tumor suppressor function. The emerging insights into the pathobiology underlying AEC mtDNA damage and apoptosis is suggesting novel therapeutic targets that may prove useful for the management of age-related diseases, including pulmonary fibrosis and lung cancer. PMID:26370974

The intersection between DNA damage response and cell death pathways.

Science.gov (United States)

Nowsheen, S; Yang, E S

2012-10-01

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

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

International Nuclear Information System (INIS)

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

2012-01-01

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

Mechanisms of dealing with DNA damage in terminally differentiated cells

Energy Technology Data Exchange (ETDEWEB)

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

2010-03-01

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

Mechanisms of dealing with DNA damage in terminally differentiated cells

International Nuclear Information System (INIS)

Fortini, P.; Dogliotti, E.

2010-01-01

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

Cellular Responses to Cisplatin-Induced DNA Damage

Directory of Open Access Journals (Sweden)

Alakananda Basu

2010-01-01

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

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

Science.gov (United States)

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

2017-08-01

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

The DNA damage response during mitosis

International Nuclear Information System (INIS)

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

2013-01-01

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

The DNA damage response during mitosis

Energy Technology Data Exchange (ETDEWEB)

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

2013-10-15

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

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

Directory of Open Access Journals (Sweden)

Hurng-Wern Huang

2018-04-01

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

MicroRNAs, the DNA damage response and cancer

International Nuclear Information System (INIS)

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

2011-01-01

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

The DNA damage response during mitosis.

Science.gov (United States)

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

2013-10-01

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

Lung Oxidative Stress, DNA Damage, Apoptosis, and Fibrosis in Adenine-Induced Chronic Kidney Disease in Mice

Directory of Open Access Journals (Sweden)

Abderrahim Nemmar

2017-11-01

Full Text Available It is well-established that there is a crosstalk between the lung and the kidney, and several studies have reported association between chronic kidney disease (CKD and pulmonary pathophysiological changes. Experimentally, CKD can be caused in mice by dietary intake of adenine. Nevertheless, the consequence of such intervention on the lung received only scant attention. Here, we assessed the pulmonary effects of adenine (0.2% w/w in feed for 4 weeks-induced CKD in mice by assessing various physiological histological and biochemical endpoints. Adenine treatment induced a significant increase in urine output, urea and creatinine concentrations, and it decreased the body weight and creatinine clearance. It also increased proteinuria and the urinary levels of kidney injury molecule-1 and neutrophil gelatinase-associated lipocalin. Compared with control group, the histopathological evaluation of lungs from adenine-treated mice showed polymorphonuclear leukocytes infiltration in alveolar and bronchial walls, injury, and fibrosis. Moreover, adenine caused a significant increase in lung lipid peroxidation and reactive oxygen species and decreased the antioxidant catalase. Adenine also induced DNA damage assessed by COMET assay. Similarly, adenine caused apoptosis in the lung characterized by a significant increase of cleaved caspase-3. Moreover, adenine induced a significant increase in the expression of nuclear factor erythroid 2–related factor 2 (Nrf2 in the lung. We conclude that administration of adenine in mice induced CKD is accompanied by lung oxidative stress, DNA damage, apoptosis, and Nrf2 expression and fibrosis.

DNA damage signaling and apoptosis in preinvasive tubal lesions of ovarian carcinoma.

Science.gov (United States)

Chene, Gautier; Ouellet, Veronique; Rahimi, Kurosh; Barres, Veronique; Caceres, Katia; Meunier, Liliane; Cyr, Louis; De Ladurantaye, Manon; Provencher, Diane; Mes Masson, Anne Marie

2015-06-01

High-grade serous ovarian cancer (HGSC) is the most life-threatening gynecological malignancy despite surgery and chemotherapy. A better understanding of the molecular basis of the preinvasive stages might be helpful in early detection and diagnosis. Genetic instability is 1 of the characteristics shared by most human cancers, and its level is variable through precancerous lesions to advanced cancer. Because DNA damage response (DDR) has been described as 1 of the first phases in genomic instability, we investigated the level of DDR activation and the apoptosis pathway in serous tubal intraepithelial carcinoma (STIC), the potential precursor of HGSC. A tissue microarray including 21 benign fallopian tubes, 21 STICs, 17 HGSCs from patients with STICs (associated ovarian cancer [AOC]) from the same individuals, and 30 HGSCs without STICs (non-AOC) was used in this study.Immunohistochemistry was performed to evaluate the level of DDR proteins (pATM, pChk2, γH2AX, 53BP1, and TRF2), apoptosis proteins (Bcl2, BAX, and BIM), and cyclin E. The expression of all DDR proteins increased from benign fallopian tubes to STICs. The level of expression of pATM, pChk2, γH2AX, and TRF2 was also increased in STICs in comparison with AOC. BAX, BIM, and cyclin E expressions were high in STICs, whereas Bcl2 expression was low. Immunohistochemical profiles of AOC and non-AOC were also different. These results suggest an activation of the DDR and apoptosis pathways in STICs, indicating that genomic instability may occur early in the precancerous lesions of HGSC.

Evaluating In Vitro DNA Damage Using Comet Assay.

Science.gov (United States)

Lu, Yanxin; Liu, Yang; Yang, Chunzhang

2017-10-11

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

DNA apoptosis and stability in B-cell chronic lymphoid leukaemia: implication of the DNA double-strand breaks repair system by non homologous recombination

International Nuclear Information System (INIS)

Deriano, L.

2005-01-01

After an introduction presenting the diagnosis and treatment of chronic lymphoid leukaemia, its molecular and genetic characteristics, and its cellular origin and clonal evolution, this research thesis describes the apoptosis (definition and characteristics, cancer and chemotherapy, apoptotic ways induced by gamma irradiation), the genotoxic stresses, the different repair mechanisms for different damages, and the DNA repair processes. It reports how human chronic lymphocytic leukaemia B cells can escape DNA damage-induced apoptosis through the non-homologous end-joining DNA repair pathway, and presents non-homologous end-joining DNA repair as a potent mutagenic process in human chronic lymphocytic leukaemia B cells

Detection on emamectin benzoate-induced apoptosis and DNA damage in Spodoptera frugiperda Sf-9 cell line.

Science.gov (United States)

Wu, Xiwei; Zhang, Lei; Yang, Chao; Zong, Mimi; Huang, Qingchun; Tao, Liming

2016-01-01

Emamectin benzoate (EMB), an important macrocyclic lactone insecticide that belongs to the avermectin family and possesses excellent potency in controlling pests, is non-carcinogenic and non-mutagenic conducted in rats and mice, but EMB-induced cytotoxicity and genotoxicity in arthropod insect have been seldom reported yet. In the present paper, we quantified the cytotoxicity of EMB through the detections on cell viability, DNA damage, and cell apoptosis in Spodoptera frugiperda Sf-9 cells in vitro. The results showed that EMB caused a concentration- and time-dependent reduction on the viability of Sf-9 cells, and the median inhibitory concentrations (IC50) were 3.34μM at 72h of exposure. The dual acridine orange/ethidium bromide staining showed that exposure to EMB induced a significant time- and concentration-dependent increase on cell apoptosis. The alkaline comet assay revealed that EMB induced significant increases on single-strand DNA breaks, and the percentage of γH2AX-positive cells represented a time- and concentration-dependent formation of DNA double-strand breaks in Sf-9 cells. Interestingly, the similar cytotoxic actions of EMB also went for the human cancerous HeLa cells as a control cell group. Data demonstrated the potential cytotoxic effect of EMB on Sf-9 cells that was significantly greater than the effect of hydrogen peroxide at the same concentrations. Copyright © 2015 Elsevier Inc. All rights reserved.

Piper nigrum ethanolic extract rich in piperamides causes ROS overproduction, oxidative damage in DNA leading to cell cycle arrest and apoptosis in cancer cells.

Science.gov (United States)

de Souza Grinevicius, Valdelúcia Maria Alves; Kviecinski, Maicon Roberto; Santos Mota, Nádia Sandrini Ramos; Ourique, Fabiana; Porfirio Will Castro, Luiza Sheyla Evenni; Andreguetti, Rafaela Rafognato; Gomes Correia, João Francisco; Filho, Danilo Wilhem; Pich, Claus Tröger; Pedrosa, Rozangela Curi

2016-08-02

Ayurvedic and Chinese traditional medicine and tribal people use herbal preparations containing Piper nigrum fruits for the treatment of many health disorders like inflammation, fever, asthma and cancer. In Brazil, traditional maroon culture associates the spice Piper nigrum to health recovery and inflammation attenuation. The aim of the current work was to evaluate the relationship between reactive oxygen species (ROS) overproduction, DNA fragmentation, cell cycle arrest and apoptosis induced by Piper nigrum ethanolic extract and its antitumor activity. The plant was macerated in ethanol. Extract constitution was assessed by TLC, UV-vis and ESI-IT-MS/MS spectrometry. The cytotoxicity, proliferation and intracellular ROS generation was evaluated in MCF-7 cells. DNA damage effects were evaluated through intercalation into CT-DNA, plasmid DNA cleavage and oxidative damage in CT-DNA. Tumor growth inhibition, survival time increase, apoptosis, cell cycle arrest and oxidative stress were assessed in Ehrlich ascites carcinoma-bearing mice. Extraction yielded 64mg/g (36% piperine and 4.2% piperyline). Treatments caused DNA damage and reduced cell viability (EC50=27.1±2.0 and 80.5±6.6µg/ml in MCF-7 and HT-29 cells, respectively), inhibiting cell proliferation by 57% and increased ROS generation in MCF-7 cells (65%). Ehrlich carcinoma was inhibited by the extract, which caused reduction of tumor growth (60%), elevated survival time (76%), cell cycle arrest and induced apoptosis. The treatment with extract increased Bax and p53 and inhibited Bcl-xL and cyclin A expression. It also induced an oxidative stress in vivo verified as enhanced lipid peroxidation and carbonyl proteins content and increased activities of glutathione reductase, superoxide dismutase and catalase. GSH concentration was decreased in tumor tissue from mice. The ethanolic extract has cytotoxic and antiproliferative effect on MCF-7 cells and antitumor effect in vivo probably due to ROS overproduction

Common genomic signaling among initial DNA damage and radiation-induced apoptosis in peripheral blood lymphocytes from locally advanced breast cancer patients

DEFF Research Database (Denmark)

Henríquez-Hernández, Luis Alberto; Pinar, Beatriz; Carmona-Vigo, Ruth

2013-01-01

PURPOSE: To investigate the genomic signaling that defines sensitive lymphocytes to radiation and if such molecular profiles are consistent with clinical toxicity; trying to disclose the radiobiology mechanisms behind these cellular processes. PATIENTS AND METHODS: Twelve consecutive patients...... suffering from locally advanced breast cancer and treated with high-dose hyperfractionated radiotherapy were recruited. Initial DNA damage was measured by pulsed-field gel electrophoresis and radiation-induced apoptosis was measured by flow cytometry. Gene expression was assessed by DNA microarray. RESULTS...

DNA damage assessment by visualization and quantification of DNA damage response

International Nuclear Information System (INIS)

Matsuda, Shun; Matsuda, Tomonari; Ikura, Tsuyoshi

2017-01-01

DNA damage response (DDR) carries out signal transduction for DNA repair, activation of cell cycle checkpoint, and apoptosis to maintain genome integrity, in response to DNA damage. Many proteins and their post-translational modifications participate in the process. Especially, S139-phosphorylated histone H2AX (γH2AX), which is formed by DNA double-strand breaks (DSBs), is an important factor to bring and retain other DDR proteins to DSB sites, Thus, γH2AX is used as a good indicator of DSBs in clinical study and pharmacology for efficacy evaluation of chemotherapy and radiotherapy, detection of precancerous regions, and others. In regulatory science, γH2AX is also a useful biomarker of genotoxicity of chemicals, since a wide range of genotoxic chemicals induce γH2AX. However, conventional detection methods of γH2AX absolutely require anti-γH2AX antibody whose staining is burdensome and time-consuming, and some of these methods are not so superior in quantitativity. In this review, we introduce two new methods to overcome these limitations, involving an easy-to-use genotoxicity assay using DDR-visualizing cells and an absolute quantification method of γH2AX using liquid chromatography-tandem mass spectrometry (LC/MS/MS). (author)

Viral single-strand DNA induces p53-dependent apoptosis in human embryonic stem cells.

Science.gov (United States)

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.

Viral single-strand DNA induces p53-dependent apoptosis in human embryonic stem cells.

Directory of Open Access Journals (Sweden)

Matthew L Hirsch

Full Text Available 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.

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

Science.gov (United States)

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

2016-01-01

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

DNA damage-inducible transcript 4 (DDIT4) mediates methamphetamine-induced autophagy and apoptosis through mTOR signaling pathway in cardiomyocytes

International Nuclear Information System (INIS)

Chen, Rui; Wang, Bin; Chen, Ling; Cai, Dunpeng; Li, Bing; Chen, Chuanxiang; Huang, Enping; Liu, Chao; Lin, Zhoumeng; Xie, Wei-Bing; Wang, Huijun

2016-01-01

Methamphetamine (METH) is an amphetamine-like psychostimulant that is commonly abused. Previous studies have shown that METH can induce damages to the nervous system and recent studies suggest that METH can also cause adverse and potentially lethal effects on the cardiovascular system. Recently, we demonstrated that DNA damage-inducible transcript 4 (DDIT4) regulates METH-induced neurotoxicity. However, the role of DDIT4 in METH-induced cardiotoxicity remains unknown. We hypothesized that DDIT4 may mediate METH-induced autophagy and apoptosis in cardiomyocytes. To test the hypothesis, we examined DDIT4 protein expression in cardiomyocytes and in heart tissues of rats exposed to METH with Western blotting. We also determined the effects on METH-induced autophagy and apoptosis after silencing DDIT4 expression with synthetic siRNA with or without pretreatment of a mTOR inhibitor rapamycin in cardiomyocytes using Western blot analysis, fluorescence microscopy and TUNEL staining. Our results showed that METH exposure increased DDIT4 expression and decreased phosphorylation of mTOR that was accompanied with increased autophagy and apoptosis both in vitro and in vivo. These effects were normalized after silencing DDIT4. On the other hand, rapamycin promoted METH-induced autophagy and apoptosis in DDIT4 knockdown cardiomyocytes. These results suggest that DDIT4 mediates METH-induced autophagy and apoptosis through mTOR signaling pathway in cardiomyocytes. - Highlights: • METH exposure increases DDIT4 expression in cardiomyocytes. • DDIT4 mediates METH-induced autophagy and apoptosis in cardiomyocytes. • DDIT4 silencing protects cardiomyocytes against METH-caused autophagy and apoptosis.

DNA damage-inducible transcript 4 (DDIT4) mediates methamphetamine-induced autophagy and apoptosis through mTOR signaling pathway in cardiomyocytes

Energy Technology Data Exchange (ETDEWEB)

Chen, Rui [Department of Forensic Medicine, School of Basic Medical Science, Southern Medical University, Guangzhou 510515 (China); Department of Forensic Medicine, Guangdong Medical University, Dongguan 523808 (China); Wang, Bin; Chen, Ling; Cai, Dunpeng; Li, Bing; Chen, Chuanxiang; Huang, Enping [Department of Forensic Medicine, School of Basic Medical Science, Southern Medical University, Guangzhou 510515 (China); Liu, Chao [Guangzhou Forensic Science Institute, Guangzhou 510030 (China); Lin, Zhoumeng [Institute of Computational Comparative Medicine and Department of Anatomy and Physiology, College of Veterinary Medicine, Kansas State University, Manhattan, KS 66506 (United States); Xie, Wei-Bing, E-mail: xieweib@126.com [Department of Forensic Medicine, School of Basic Medical Science, Southern Medical University, Guangzhou 510515 (China); Wang, Huijun, E-mail: hjwang711@yahoo.cn [Department of Forensic Medicine, School of Basic Medical Science, Southern Medical University, Guangzhou 510515 (China)

2016-03-15

Methamphetamine (METH) is an amphetamine-like psychostimulant that is commonly abused. Previous studies have shown that METH can induce damages to the nervous system and recent studies suggest that METH can also cause adverse and potentially lethal effects on the cardiovascular system. Recently, we demonstrated that DNA damage-inducible transcript 4 (DDIT4) regulates METH-induced neurotoxicity. However, the role of DDIT4 in METH-induced cardiotoxicity remains unknown. We hypothesized that DDIT4 may mediate METH-induced autophagy and apoptosis in cardiomyocytes. To test the hypothesis, we examined DDIT4 protein expression in cardiomyocytes and in heart tissues of rats exposed to METH with Western blotting. We also determined the effects on METH-induced autophagy and apoptosis after silencing DDIT4 expression with synthetic siRNA with or without pretreatment of a mTOR inhibitor rapamycin in cardiomyocytes using Western blot analysis, fluorescence microscopy and TUNEL staining. Our results showed that METH exposure increased DDIT4 expression and decreased phosphorylation of mTOR that was accompanied with increased autophagy and apoptosis both in vitro and in vivo. These effects were normalized after silencing DDIT4. On the other hand, rapamycin promoted METH-induced autophagy and apoptosis in DDIT4 knockdown cardiomyocytes. These results suggest that DDIT4 mediates METH-induced autophagy and apoptosis through mTOR signaling pathway in cardiomyocytes. - Highlights: • METH exposure increases DDIT4 expression in cardiomyocytes. • DDIT4 mediates METH-induced autophagy and apoptosis in cardiomyocytes. • DDIT4 silencing protects cardiomyocytes against METH-caused autophagy and apoptosis.

DNA Damage and Pulmonary Hypertension

Science.gov (United States)

Ranchoux, Benoît; Meloche, Jolyane; Paulin, Roxane; Boucherat, Olivier; Provencher, Steeve; Bonnet, Sébastien

2016-01-01

Pulmonary hypertension (PH) is defined by a mean pulmonary arterial pressure over 25 mmHg at rest and is diagnosed by right heart catheterization. Among the different groups of PH, pulmonary arterial hypertension (PAH) is characterized by a progressive obstruction of distal pulmonary arteries, related to endothelial cell dysfunction and vascular cell proliferation, which leads to an increased pulmonary vascular resistance, right ventricular hypertrophy, and right heart failure. Although the primary trigger of PAH remains unknown, oxidative stress and inflammation have been shown to play a key role in the development and progression of vascular remodeling. These factors are known to increase DNA damage that might favor the emergence of the proliferative and apoptosis-resistant phenotype observed in PAH vascular cells. High levels of DNA damage were reported to occur in PAH lungs and remodeled arteries as well as in animal models of PH. Moreover, recent studies have demonstrated that impaired DNA-response mechanisms may lead to an increased mutagen sensitivity in PAH patients. Finally, PAH was linked with decreased breast cancer 1 protein (BRCA1) and DNA topoisomerase 2-binding protein 1 (TopBP1) expression, both involved in maintaining genome integrity. This review aims to provide an overview of recent evidence of DNA damage and DNA repair deficiency and their implication in PAH pathogenesis. PMID:27338373

Chalcone-imidazolone conjugates induce apoptosis through DNA damage pathway by affecting telomeres

Directory of Open Access Journals (Sweden)

Kamal Ahmed

2011-04-01

Full Text Available Abstract Background Breast cancer is one of the most prevalent cancers in the world and more than one million women are diagnosed leading to 410,000 deaths every year. In our previous studies new chalcone-imidazolone conjugates were prepared and evaluated for their anticancer activity in a panel of 53 human tumor cell lines and the lead compounds identified were 6 and 8. This prompted us to investigate the mechanism of apoptotic event. Results Involvement of pro-apoptotic protein (Bax, active caspase-9 and cleavage of retinoblastoma protein was studied. Interestingly, the compounds caused upregulation of p21, check point proteins (Chk1, Chk2 and as well as their phosphorylated forms which are known to regulate the DNA damage pathway. Increased p53BP1 foci by immunolocalisation studies and TRF1 suggested the possible involvement of telomere and associated proteins in the apoptotic event. The telomeric protein such as TRF2 which is an important target for anticancer therapy against human breast cancer was extensively studied along with proteins involved in proper functioning of telomeres. Conclusions The apoptotic proteins such as Bax, active caspase-9 and cleaved RB are up-regulated in the compound treated cells revealing the apoptotic nature of the compounds. Down regulation of TRF2 and upregulation of the TRF1 as well as telomerase assay indicated the decrease in telomeric length revealing telomeric dysfunction and thereby controlling the rapid rate of cell proliferation. In summary, chalcone-imidazolone conjugates displayed significant DNA damage activity particularly at telomeres and caused both apoptosis and senescence-like growth arrest which suggested that these compounds have potential activity against breast carcinoma.

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

Directory of Open Access Journals (Sweden)

Xiaoguang Zhou

2014-12-01

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

Primary DNA damage in chrome-plating workers.

Science.gov (United States)

Gambelunghe, A; Piccinini, R; Ambrogi, M; Villarini, M; Moretti, M; Marchetti, C; Abbritti, G; Muzi, G

2003-06-30

In order to evaluate the primary DNA damage due to occupational exposure to chromium (VI), DNA strand-breaks and apoptosis in peripheral lymphocytes were measured in a group of 19 chrome-plating workers. DNA strand-breaks was assessed by alkaline (pH>13) single-cell microgel electrophoresis ('comet') assay, while apoptosis was measured by flow-cytometry after propidium iodide staining of the cells. Concentrations of chromium in urine, erythrocytes and lymphocytes were investigated as biological indicators of exposure. A group of 18 hospital workers (control group I) and another 20 university personnel (control group II) without exposure to chromium were also studied as controls. The results of the study show that chrome-plating workers have higher levels of chromium in urine, erythrocytes and lymphocytes than unexposed workers. Comet tail moment values, assumed as index of DNA damage, are increased in chromium-exposed workers and results are significantly correlated to chromium lymphocyte concentrations. No difference emerged in the percentage of apoptotic nuclei in exposed and unexposed workers. The study confirms that measurements of chromium in erythrocytes and lymphocytes may provide useful information about recent and past exposure to hexavalent chromium at the workplace. The increase in DNA strand-breaks measured by comet assay suggests this test is valid for the biological monitoring of workers exposed to genotoxic compounds such as chromium (VI).

Administration of the optimized β-Lapachone-poloxamer-cyclodextrin ternary system induces apoptosis, DNA damage and reduces tumor growth in a human breast adenocarcinoma xenograft mouse model.

Science.gov (United States)

Seoane, Samuel; Díaz-Rodríguez, Patricia; Sendon-Lago, Juan; Gallego, Rosalia; Pérez-Fernández, Román; Landin, Mariana

2013-08-01

β-Lapachone (β-Lap) is a 1,2-orthonaphthoquinone that selectively induces cell death in human cancer cells through NAD(P)H:quinone oxidoreductase-1 (NQO1). NQO1 is overexpressed in a variety of tumors, as compared to normal adjacent tissue. However, the low solubility and non-specific distribution of β-Lap limit its suitability for clinical assays. We formulated β-Lap in an optimal random methylated-β-cyclodextrin/poloxamer 407 mixture (i.e., β-Lap ternary system) and, using human breast adenocarcinoma MCF-7 cells and immunodeficient mice, performed in vitro and in vivo evaluation of its anti-tumor effects on proliferation, cell cycle, apoptosis, DNA damage, and tumor growth. This ternary system is fluid at room temperature, gels over 29 °C, and provides a significant amount of drug, thus facilitating intratumoral delivery, in situ gelation, and the formation of a depot for time-release. Administration of β-Lap ternary system to MCF-7 cells induces an increase in apoptosis and DNA damage, while producing no changes in cell cycle. Moreover, in a mouse xenograft tumor model, intratumoral injection of the system significantly reduces tumor volume, while increasing apoptosis and DNA damage without visible toxicity to liver or kidney. These anti-tumoral effects and lack of visible toxicity make this system a promising new therapeutic agent for breast cancer treatment. Copyright © 2013 Elsevier B.V. All rights reserved.

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

Science.gov (United States)

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

2016-12-01

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

Regulation of the DNA Damage Response by DNA-PKcs Inhibitory Phosphorylation of ATM.

Science.gov (United States)

Zhou, Yi; Lee, Ji-Hoon; Jiang, Wenxia; Crowe, Jennie L; Zha, Shan; Paull, Tanya T

2017-01-05

Ataxia-telangiectasia mutated (ATM) regulates the DNA damage response as well as DNA double-strand break repair through homologous recombination. Here we show that ATM is hyperactive when the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) is chemically inhibited or when the DNA-PKcs gene is deleted in human cells. Pre-incubation of ATM protein with active DNA-PKcs also significantly reduces ATM activity in vitro. We characterize several phosphorylation sites in ATM that are targets of DNA-PKcs and show that phospho-mimetic mutations at these residues significantly inhibit ATM activity and impair ATM signaling upon DNA damage. In contrast, phospho-blocking mutations at one cluster of sites increase the frequency of apoptosis during normal cell growth. DNA-PKcs, which is integral to the non-homologous end joining pathway, thus negatively regulates ATM activity through phosphorylation of ATM. These observations illuminate an important regulatory mechanism for ATM that also controls DNA repair pathway choice. Copyright © 2017 Elsevier Inc. All rights reserved.

The roles of DNA damage-dependent signals and MAPK cascades in tributyltin-induced germline apoptosis in Caenorhabditis elegans.

Science.gov (United States)

Wang, Yun; Wang, Shunchang; Luo, Xun; Yang, Yanan; Jian, Fenglei; Wang, Xuemin; Xie, Lucheng

2014-08-01

The induction of apoptosis is recognized to be a major mechanism of tributyltin (TBT) toxicity. However, the underlying signaling pathways for TBT-induced apoptosis remain unclear. In this study, using the nematode Caenorhabditis elegans, we examined whether DNA damage response (DDR) pathway and mitogen-activated protein kinase (MAPK) signaling cascades are involved in TBT-induced germline apoptosis and cell cycle arrest. Our results demonstrated that exposing worms to TBT at the dose of 10nM for 6h significantly increased germline apoptosis in N2 strain. Germline apoptosis was absent in strains that carried ced-3 or ced-4 loss-of-function alleles, indicating that both caspase protein CED-3 and Apaf-1 protein CED-4 were required for TBT-induced apoptosis. TBT-induced apoptosis was blocked in the Bcl-2 gain-of-function strain ced-9(n1950), whereas TBT induced a minor increase in the BH3-only protein EGL-1 mutated strain egl-1(n1084n3082). Checkpoint proteins HUS-1 and CLK-2 exerted proapoptotic effects, and the null mutation of cep-1, the homologue of tumor suppressor gene p53, significantly inhibited TBT-induced apoptosis. Apoptosis in the loss-of-function strains of ERK, JNK and p38 MAPK signaling pathways were completely or mildly suppressed under TBT stress. These results were supported by the results of mRNA expression levels of corresponding genes. The present study indicated that TBT-induced apoptosis required the core apoptotic machinery, and that DDR genes and MAPK pathways played essential roles in signaling the processes. Copyright © 2014 Elsevier Ltd. All rights reserved.

Abrus agglutinin promotes irreparable DNA damage by triggering ROS generation followed by ATM-p73 mediated apoptosis in oral squamous cell carcinoma.

Science.gov (United States)

Sinha, Niharika; Panda, Prashanta K; Naik, Prajna P; Das, Durgesh N; Mukhopadhyay, Subhadip; Maiti, Tapas K; Shanmugam, Muthu K; Chinnathambi, Arunachalam; Zayed, M E; Alharbi, Sulaiman A; Sethi, Gautam; Agarwal, Rajesh; Bhutia, Sujit K

2017-11-01

Oral cancer, a type of head and neck cancer, is ranked as one of the top most malignancies in India. Herein, we evaluated the anticancer efficacy of Abrus agglutinin (AGG), a plant lectin, in oral squamous cell carcinoma. AGG selectively inhibited cell growth, and caused cell cycle arrest and mitochondrial apoptosis through a reactive oxygen species (ROS)-mediated ATM-p73 dependent pathway in FaDu cells. AGG-induced ROS accumulation was identified as the major mechanism regulating apoptosis, DNA damage and DNA-damage response, which were significantly reversed by ROS scavenger N-acetylcysteine (NAC). Moreover, AGG was found to interact with mitochondrial manganese-dependent superoxide dismutase that might inhibit its activity and increase ROS in FaDu cells. In oral cancer p53 is mutated, thus we focused on p73; AGG resulted in p73 upregulation and knock down of p73 caused a decrease in AGG-induced apoptosis. Interestingly, AGG-dependent p73 expression was found to be regulated by ROS, which was reversed by NAC treatment. A reduction in the level of p73 in AGG-treated shATM cells was found to be associated with a decreased apoptosis. Moreover, administration of AGG (50 μg/kg body weight) significantly inhibited the growth of FaDu xenografts in athymic nude mice. In immunohistochemical analysis, the xenografts from AGG-treated mice displayed a decrease in PCNA expression and an increase in caspase-3 activation as compared to the controls. In conclusion, we established a connection among ROS, ATM and p73 in AGG-induced apoptosis, which might be useful in enhancing the therapeutic targeting of p53 deficient oral squamous cell carcinoma. © 2017 Wiley Periodicals, Inc.

Hydroxychavicol, a betel leaf component, inhibits prostate cancer through ROS-driven DNA damage and apoptosis

Energy Technology Data Exchange (ETDEWEB)

Gundala, Sushma Reddy; Yang, Chunhua [Department of Biology, Georgia State University, Atlanta, GA 30303 (United States); Mukkavilli, Rao [Advinus Therapeutics, Karnataka (India); Paranjpe, Rutugandha; Brahmbhatt, Meera; Pannu, Vaishali; Cheng, Alice [Department of Biology, Georgia State University, Atlanta, GA 30303 (United States); Reid, Michelle D. [Department of Pathology, Emory University School of Medicine, Atlanta, GA (United States); Aneja, Ritu, E-mail: raneja@gsu.edu [Department of Biology, Georgia State University, Atlanta, GA 30303 (United States)

2014-10-01

Dietary phytochemicals are excellent ROS-modulating agents and have been shown to effectively enhance ROS levels beyond toxic threshold in cancer cells to ensure their selective killing while leaving normal cells unscathed. Here we demonstrate that hydroxychavicol (HC), extracted and purified from Piper betel leaves, significantly inhibits growth and proliferation via ROS generation in human prostate cancer, PC-3 cells. HC perturbed cell-cycle kinetics and progression, reduced clonogenicity and mediated cytotoxicity by ROS-induced DNA damage leading to activation of several pro-apoptotic molecules. In addition, HC treatment elicited a novel autophagic response as evidenced by the appearance of acidic vesicular organelles and increased expression of autophagic markers, LC3-IIb and beclin-1. Interestingly, quenching of ROS with tiron, an antioxidant, offered significant protection against HC-induced inhibition of cell growth and down regulation of caspase-3, suggesting the crucial role of ROS in mediating cell death. The collapse of mitochondrial transmembrane potential by HC further revealed the link between ROS generation and induction of caspase-mediated apoptosis in PC-3 cells. Our data showed remarkable inhibition of prostate tumor xenografts by ∼ 72% upon daily oral administration of 150 mg/kg bw HC by quantitative tumor volume measurements and non-invasive real-time bioluminescent imaging. HC was well-tolerated at this dosing level without any observable toxicity. This is the first report to demonstrate the anti-prostate cancer efficacy of HC in vitro and in vivo, which is perhaps attributable to its selective prooxidant activity to eliminate cancer cells thus providing compelling grounds for future preclinical studies to validate its potential usefulness for prostate cancer management. - Highlights: • HC perturbs cell-cycle progression by induction of reactive oxygen species (ROS). • HC mediated cytotoxicity by ROS-induced DNA damage leading to

Hydroxychavicol, a betel leaf component, inhibits prostate cancer through ROS-driven DNA damage and apoptosis

International Nuclear Information System (INIS)

Gundala, Sushma Reddy; Yang, Chunhua; Mukkavilli, Rao; Paranjpe, Rutugandha; Brahmbhatt, Meera; Pannu, Vaishali; Cheng, Alice; Reid, Michelle D.; Aneja, Ritu

2014-01-01

Dietary phytochemicals are excellent ROS-modulating agents and have been shown to effectively enhance ROS levels beyond toxic threshold in cancer cells to ensure their selective killing while leaving normal cells unscathed. Here we demonstrate that hydroxychavicol (HC), extracted and purified from Piper betel leaves, significantly inhibits growth and proliferation via ROS generation in human prostate cancer, PC-3 cells. HC perturbed cell-cycle kinetics and progression, reduced clonogenicity and mediated cytotoxicity by ROS-induced DNA damage leading to activation of several pro-apoptotic molecules. In addition, HC treatment elicited a novel autophagic response as evidenced by the appearance of acidic vesicular organelles and increased expression of autophagic markers, LC3-IIb and beclin-1. Interestingly, quenching of ROS with tiron, an antioxidant, offered significant protection against HC-induced inhibition of cell growth and down regulation of caspase-3, suggesting the crucial role of ROS in mediating cell death. The collapse of mitochondrial transmembrane potential by HC further revealed the link between ROS generation and induction of caspase-mediated apoptosis in PC-3 cells. Our data showed remarkable inhibition of prostate tumor xenografts by ∼ 72% upon daily oral administration of 150 mg/kg bw HC by quantitative tumor volume measurements and non-invasive real-time bioluminescent imaging. HC was well-tolerated at this dosing level without any observable toxicity. This is the first report to demonstrate the anti-prostate cancer efficacy of HC in vitro and in vivo, which is perhaps attributable to its selective prooxidant activity to eliminate cancer cells thus providing compelling grounds for future preclinical studies to validate its potential usefulness for prostate cancer management. - Highlights: • HC perturbs cell-cycle progression by induction of reactive oxygen species (ROS). • HC mediated cytotoxicity by ROS-induced DNA damage leading to

Transient p53 suppression increases reprogramming of human fibroblasts without affecting apoptosis and DNA damage

DEFF Research Database (Denmark)

Rasmussen, Mikkel Aabech; Holst, Bjørn; Tümer, Zeynep

2014-01-01

The discovery of human-induced pluripotent stem cells (iPSCs) has sparked great interest in the potential treatment of patients with their own in vitro differentiated cells. Recently, knockout of the Tumor Protein 53 (p53) gene was reported to facilitate reprogramming but unfortunately also led...... to genomic instability. Here, we report that transient suppression of p53 during nonintegrative reprogramming of human fibroblasts leads to a significant increase in expression of pluripotency markers and overall number of iPSC colonies, due to downstream suppression of p21, without affecting apoptosis...... and DNA damage. Stable iPSC lines generated with or without p53 suppression showed comparable expression of pluripotency markers and methylation patterns, displayed normal karyotypes, contained between 0 and 5 genomic copy number variations and produced functional neurons in vitro. In conclusion...

Oncogene-induced senescence is part of the tumorigenesis barrier imposed by DNA damage checkpoints

DEFF Research Database (Denmark)

Bartkova, Jirina; Rezaei, Nousin; Liontos, Michalis

2006-01-01

Recent studies have indicated the existence of tumorigenesis barriers that slow or inhibit the progression of preneoplastic lesions to neoplasia. One such barrier involves DNA replication stress, which leads to activation of the DNA damage checkpoint and thereby to apoptosis or cell cycle arrest...... and senescence markers cosegregate closely. Thus, senescence in human preneoplastic lesions is a manifestation of oncogene-induced DNA replication stress and, together with apoptosis, provides a barrier to malignant progression....

DNA damage response in nephrotoxic and ischemic kidney injury

Energy Technology Data Exchange (ETDEWEB)

Yan, Mingjuan; Tang, Chengyuan [Department of Nephrology, The Second Xiangya Hospital, Central South University, Changsha, Hunan 410011 (China); Ma, Zhengwei [Department of Cellular Biology & Anatomy, Medical College of Georgia at Augusta University and Charlie Norwood VA Medical Center, Augusta, GA 30912 (United States); Huang, Shuang [Department of Anatomy and Cell Biology, University of Florida College of Medicine, Gainesville, FL (United States); Dong, Zheng, E-mail: zdong@augusta.edu [Department of Nephrology, The Second Xiangya Hospital, Central South University, Changsha, Hunan 410011 (China); Department of Cellular Biology & Anatomy, Medical College of Georgia at Augusta University and Charlie Norwood VA Medical Center, Augusta, GA 30912 (United States)

2016-12-15

DNA damage activates specific cell signaling cascades for DNA repair, cell cycle arrest, senescence, and/or cell death. Recent studies have demonstrated DNA damage response (DDR) in experimental models of acute kidney injury (AKI). In cisplatin-induced AKI or nephrotoxicity, the DDR pathway of ATR/Chk2/p53 is activated and contributes to renal tubular cell apoptosis. In ischemic AKI, DDR seems more complex and involves at least the ataxia telangiectasia mutated (ATM), a member of the phosphatidylinositol 3-kinase-related kinase (PIKK) family, and p53; however, while ATM may promote DNA repair, p53 may trigger cell death. Targeting DDR for kidney protection in AKI therefore relies on a thorough elucidation of the DDR pathways in various forms of AKI.

Tenuifolide B from Cinnamomum tenuifolium Stem Selectively Inhibits Proliferation of Oral Cancer Cells via Apoptosis, ROS Generation, Mitochondrial Depolarization, and DNA Damage.

Science.gov (United States)

Chen, Chung-Yi; Yen, Ching-Yu; Wang, Hui-Ru; Yang, Hui-Ping; Tang, Jen-Yang; Huang, Hurng-Wern; Hsu, Shih-Hsien; Chang, Hsueh-Wei

2016-11-05

The development of drugs that selectively kill oral cancer cells but are less harmful to normal cells still provide several challenges. In this study, the antioral cancer effects of tenuifolide B (TFB), extracted from the stem of the plant Cinnamomum tenuifolium are evaluated in terms of their effects on cancer cell viability, cell cycle analysis, apoptosis, oxidative stress, and DNA damage. Cell viability of oral cancer cells (Ca9-22 and CAL 27) was found to be significantly inhibited by TFB in a dose-responsive manner in terms of ATP assay, yielding IC 50 = 4.67 and 7.05 μM (24 h), but are less lethal to normal oral cells (HGF-1). Dose-responsive increases in subG1 populations as well as the intensities of flow cytometry-based annexin V/propidium iodide (PI) analysis and pancaspase activity suggested that apoptosis was inducible by TFB in these two types of oral cancer cells. Pretreatment with the apoptosis inhibitor (Z-VAD-FMK) reduced the annexin V intensity of these two TFB-treated oral cancer cells, suggesting that TFB induced apoptosis-mediated cell death to oral cancer cells. Cleaved-poly (ADP-ribose) polymerase (PARP) and cleaved-caspases 3, 8, and 9 were upregulated in these two TFB-treated oral cancer cells over time but less harmful for normal oral HGF-1 cells. Dose-responsive and time-dependent increases in reactive oxygen species (ROS) and decreases in mitochondrial membrane potential (MitoMP) in these two TFB-treated oral cancer cells suggest that TFB may generate oxidative stress as measured by flow cytometry. N -acetylcysteine (NAC) pretreatment reduced the TFB-induced ROS generation and further validated that ROS was relevant to TFB-induced cell death. Both flow cytometry and Western blotting demonstrated that the DNA double strand marker γH2AX dose-responsively increased in TFB-treated Ca9-22 cells and time-dependently increased in two TFB-treated oral cancer cells. Taken together, we infer that TFB can selectively inhibit cell proliferation of

Reactive oxygen species-mediated DNA damage and apoptosis in human skin epidermal cells after exposure to nickel nanoparticles.

Science.gov (United States)

Alarifi, Saud; Ali, Daoud; Alakhtani, Saad; Al Suhaibani, Entissar S; Al-Qahtani, Ahmed A

2014-01-01

Nickel nanoparticles (NiNPs) are increasingly used in various applications due to their unique properties. However, there is little information concerning the toxicity of NiNPs in the human skin cell (A431). The present study was designed to investigate the cytotoxicity, apoptosis, and DNA damage due to NiNPs in A431 cells. A cellular proliferative capacity test showed that NiNPs induce significant cytotoxicity in a dose- and time-dependent manner. NiNPs were also found to induce oxidative stress evidenced by the generation of reactive oxygen species (ROS) and depletion of glutathione (GSH). Further, co-treatment with the antioxidant N-acetylcysteine (NAC) mitigated the ROS generation due to NiNPs, suggesting the potential mechanism of oxidative stress. NiNPs also induced significant elevation of lipid peroxidation, catalase, and superoxide dismutase and caspase-3 activity in A431 cells. In addition, NAC suppressed NiNP-induced caspase-3 activity. DNA fragmentation analysis using the comet assay showed that the NiNPs cause genotoxicity in a dose- and time-dependent manner. Therefore, the study points out the capability of the NiNPs to induce oxidative stress resulting in apoptosis and genotoxicity. This study warrants more careful assessment of NiNPs before their industrial applications.

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

Directory of Open Access Journals (Sweden)

Yanjun Zhang

2014-07-01

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

Estrogen signalling and the DNA damage response in hormone dependent breast cancers

Directory of Open Access Journals (Sweden)

C Elizabeth Caldon

2014-05-01

Full Text Available Estrogen is necessary for the normal growth and development of breast tissue, but high levels of estrogen are a major risk factor for breast cancer. One mechanism by which estrogen could contribute to breast cancer is via the induction of DNA damage. This perspective discusses the mechanisms by which estrogen alters the DNA damage response (DDR and DNA repair through the regulation of key effector proteins including ATM, ATR, CHK1, BRCA1 and p53 and the feedback on estrogen receptor signalling from these proteins. We put forward the hypothesis that estrogen receptor signalling converges to suppress effective DNA repair and apoptosis in favour of proliferation. This is important in hormone-dependent breast cancer as it will affect processing of estrogen-induced DNA damage, as well as other genotoxic insults. DDR and DNA repair proteins are frequently mutated or altered in estrogen responsive breast cancer which will further change the processing of DNA damage. Finally the action of estrogen signalling on DNA damage is also relevant to the therapeutic setting as the suppression of a DNA damage response by estrogen has the potential to alter the response of cancers to anti-hormone treatment or chemotherapy that induces DNA damage.

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

Science.gov (United States)

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

2014-01-01

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

JS-K, a nitric oxide prodrug, induces DNA damage and apoptosis in HBV-positive hepatocellular carcinoma HepG2.2.15 cell.

Science.gov (United States)

Liu, Zhengyun; Li, Guangmin; Gou, Ying; Xiao, Dongyan; Luo, Guo; Saavedra, Joseph E; Liu, Jie; Wang, Huan

2017-08-01

Hepatocellular carcinoma (HCC) is the most important cause of cancer-related death, and 85% of HCC is caused by chronic HBV infection, the prognosis of patients and the reduction of HBV DNA levels remain unsatisfactory. JS-K, a nitric oxide-releasing diazeniumdiolates, is effective against various tumors, but little is known on its effects on HBV positive HCC. We found that JS-K reduced the expression of HBsAg and HBeAg in HBV-positive HepG2.2.15 cells. This study aimed to further examine anti-tumor effects of JS-K on HepG2.2.15 cells. The MTT assay and colony forming assay were used to study the cell growth inhibition of JS-K; scratch assay and transwell assay were performed to detect cell migration. The cell cycle was detected by flow cytometry. The immunofluorescence, flow cytometry analysis, and western blot were used to study DNA damage and cell apoptosis. JS-K inhibited HepG2.2.15 cell growth in a dose-dependent manner, suppressed cell colony formation and migration, arrested cells gather in the G2 phase. JS-K (1-20μM) increased the expression of DNA damage-associated protein phosphorylation H 2 AX (γH 2 AX), phosphorylation of checkpoint kinase 1 (p-Chk1), phosphorylation of checkpoint kinase 2 (p-Chk2), ataxia-telangiectasia mutated (ATM), phosphorylation of ataxia-telangiectasia mutated rad3-related (p-ATR) and apoptotic-associated proteins cleaved caspase-3, cleaved caspase-7, cleaved poly ADP-ribose polymerase (cleaved PARP). The study demonstrated JS-K is effective against HBV-positive HepG2.2.15 cells, the mechanisms are not only related to inhibition of HBsAg and HBeAg secretion, but also related with induction of DNA damage and apoptosis. JS-K is a promising anti-cancer candidate against HBV-positive HCC. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

The chemopreventive activity of the histone deacetylase inhibitor tributyrin in colon carcinogenesis involves the induction of apoptosis and reduction of DNA damage

Energy Technology Data Exchange (ETDEWEB)

Heidor, Renato [Laboratory of Diet, Nutrition and Cancer, Department of Food and Experimental Nutrition, Faculty of Pharmaceutical Sciences, University of São Paulo (Brazil); Advanced Research Center in Food Science and Nutrition (NAPAN) and Food Research Center (FoRC), Faculty of Pharmaceutical Sciences, University of São Paulo (Brazil); Furtado, Kelly Silva; Ortega, Juliana Festa [Laboratory of Diet, Nutrition and Cancer, Department of Food and Experimental Nutrition, Faculty of Pharmaceutical Sciences, University of São Paulo (Brazil); Oliveira, Tiago Franco de [Department of Clinical and Toxicological Analyses, Faculty of Pharmaceutical Sciences, University of São Paulo (Brazil); Tavares, Paulo Eduardo Latorre Martins; Vieira, Alessandra; Miranda, Mayara Lilian Paulino [Laboratory of Diet, Nutrition and Cancer, Department of Food and Experimental Nutrition, Faculty of Pharmaceutical Sciences, University of São Paulo (Brazil); Purgatto, Eduardo [Laboratory of Food Chemistry and Biochemistry, Department of Food and Experimental Nutrition, Faculty of Pharmaceutical Sciences, University of São Paulo (Brazil); Advanced Research Center in Food Science and Nutrition (NAPAN) and Food Research Center (FoRC), Faculty of Pharmaceutical Sciences, University of São Paulo (Brazil); Moreno, Fernando Salvador, E-mail: rmoreno@usp.br [Laboratory of Diet, Nutrition and Cancer, Department of Food and Experimental Nutrition, Faculty of Pharmaceutical Sciences, University of São Paulo (Brazil); Advanced Research Center in Food Science and Nutrition (NAPAN) and Food Research Center (FoRC), Faculty of Pharmaceutical Sciences, University of São Paulo (Brazil)

2014-04-15

The chemopreventive activity of the histone deacetylase inhibitor (HDACi) tributyrin (TB), a prodrug of butyric acid (BA), was evaluated in a rat model of colon carcinogenesis. The animals were treated with TB (TB group: 200 mg/100 g of body weight, b.w.) or maltodextrin (MD isocaloric control group: 300 mg/100 g b.w.) daily for 9 consecutive weeks. In the 3rd and 4th weeks of treatment, the rats in the TB and MD groups were given DMH (40 mg/kg b.w.) twice a week. After 9 weeks, the animals were euthanized, and the distal colon was examined. Compared with the control group (MD group), TB treatment reduced the total number of aberrant crypt foci (ACF; p < 0.05) as well as the ACF with ≥ 4 crypts (p < 0.05), which are considered more aggressive, but not inhibited the formation of DMH-induced O6-methyldeoxyguanosine DNA adducts. The TB group also showed a higher apoptotic index (p < 0.05) and reduced DNA damage (p < 0.05) compared with MD group. TB acted as a HDACi, as rats treated with the prodrug of BA had higher levels of histone H3K9 acetylation compared with the MD group (p < 0.05). TB administration resulted in increased colonic tissue concentrations of BA (p < 0.05) compared with the control animals. These results suggest that TB can be considered a promising chemopreventive agent for colon carcinogenesis because it reduced the number of ACF, including those that were more aggressive. Induction of apoptosis and reduction of DNA damage are cellular mechanisms that appear to be involved in the chemopreventive activity of TB. - Highlights: • Tributyrin is a chemopreventive agent for rat colon aberrant crypt foci. • Tributyrin increased apoptosis in an experimental rat colon carcinogenesis model. • Tributyrin treatment in a rat colon carcinogenesis model decreased DNA damage. • Tributyrin treatment induced H3K9 acetylation in a rat colon carcinogenesis model.

The chemopreventive activity of the histone deacetylase inhibitor tributyrin in colon carcinogenesis involves the induction of apoptosis and reduction of DNA damage

International Nuclear Information System (INIS)

Heidor, Renato; Furtado, Kelly Silva; Ortega, Juliana Festa; Oliveira, Tiago Franco de; Tavares, Paulo Eduardo Latorre Martins; Vieira, Alessandra; Miranda, Mayara Lilian Paulino; Purgatto, Eduardo; Moreno, Fernando Salvador

2014-01-01

The chemopreventive activity of the histone deacetylase inhibitor (HDACi) tributyrin (TB), a prodrug of butyric acid (BA), was evaluated in a rat model of colon carcinogenesis. The animals were treated with TB (TB group: 200 mg/100 g of body weight, b.w.) or maltodextrin (MD isocaloric control group: 300 mg/100 g b.w.) daily for 9 consecutive weeks. In the 3rd and 4th weeks of treatment, the rats in the TB and MD groups were given DMH (40 mg/kg b.w.) twice a week. After 9 weeks, the animals were euthanized, and the distal colon was examined. Compared with the control group (MD group), TB treatment reduced the total number of aberrant crypt foci (ACF; p < 0.05) as well as the ACF with ≥ 4 crypts (p < 0.05), which are considered more aggressive, but not inhibited the formation of DMH-induced O6-methyldeoxyguanosine DNA adducts. The TB group also showed a higher apoptotic index (p < 0.05) and reduced DNA damage (p < 0.05) compared with MD group. TB acted as a HDACi, as rats treated with the prodrug of BA had higher levels of histone H3K9 acetylation compared with the MD group (p < 0.05). TB administration resulted in increased colonic tissue concentrations of BA (p < 0.05) compared with the control animals. These results suggest that TB can be considered a promising chemopreventive agent for colon carcinogenesis because it reduced the number of ACF, including those that were more aggressive. Induction of apoptosis and reduction of DNA damage are cellular mechanisms that appear to be involved in the chemopreventive activity of TB. - Highlights: • Tributyrin is a chemopreventive agent for rat colon aberrant crypt foci. • Tributyrin increased apoptosis in an experimental rat colon carcinogenesis model. • Tributyrin treatment in a rat colon carcinogenesis model decreased DNA damage. • Tributyrin treatment induced H3K9 acetylation in a rat colon carcinogenesis model

Resistance to bleomycin in cancer cell lines is characterized by prolonged doubling time, reduced DNA damage and evasion of G2/M arrest and apoptosis.

Directory of Open Access Journals (Sweden)

Qi Wang

Full Text Available To establish, characterize and elucidate potential mechanisms of acquired bleomycin (BLM resistance using human cancer cell lines. Seven BLM-resistant cell lines were established by exposure to escalating BLM concentrations over a period of 16-24 months. IC50 values and cell doubling times were quantified using a real time cytotoxicity assay. COMET and γ-H2AX assays, cell cycle analysis, and apoptosis assessment further investigated the mechanisms of BLM resistance in these cell lines.Compared with parental cell lines, real time cytotoxicity assays revealed 7 to 49 fold increases in IC50 and a mean doubling time increase of 147 % (range 64 %-352% in BLM-resistant sub-clones (p<0.05 for both. Higher maintenance BLM concentrations were associated with higher IC50 and increased doubling times (p<0.05. Significantly reduced DNA damage (COMET and γ-H2AX assays, G2/M arrest, and apoptosis (p<0.05 for each set of comparison following high-dose acute BLM exposure was observed in resistant sub-clones, compared with their BLM-sensitive parental counterparts. Three weeks of BLM-free culturing resulted in a partial return to BLM sensitivity in 3/7 BLM-resistant sub-clones (p<0.05.Bleomycin resistance may be associated with reduced DNA damage after bleomycin exposure, resulting in reduced G2/M arrest, and reduced apoptosis.

Systems biology approach identifies the kinase Csnk1a1 as a regulator of the DNA damage response in embryonic stem cells.

Science.gov (United States)

Carreras Puigvert, Jordi; von Stechow, Louise; Siddappa, Ramakrishnaiah; Pines, Alex; Bahjat, Mahnoush; Haazen, Lizette C J M; Olsen, Jesper V; Vrieling, Harry; Meerman, John H N; Mullenders, Leon H F; van de Water, Bob; Danen, Erik H J

2013-01-22

In pluripotent stem cells, DNA damage triggers loss of pluripotency and apoptosis as a safeguard to exclude damaged DNA from the lineage. An intricate DNA damage response (DDR) signaling network ensures that the response is proportional to the severity of the damage. We combined an RNA interference screen targeting all kinases, phosphatases, and transcription factors with global transcriptomics and phosphoproteomics to map the DDR in mouse embryonic stem cells treated with the DNA cross-linker cisplatin. Networks derived from canonical pathways shared in all three data sets were implicated in DNA damage repair, cell cycle and survival, and differentiation. Experimental probing of these networks identified a mode of DNA damage-induced Wnt signaling that limited apoptosis. Silencing or deleting the p53 gene demonstrated that genotoxic stress elicited Wnt signaling in a p53-independent manner. Instead, this response occurred through reduced abundance of Csnk1a1 (CK1α), a kinase that inhibits β-catenin. Together, our findings reveal a balance between p53-mediated elimination of stem cells (through loss of pluripotency and apoptosis) and Wnt signaling that attenuates this response to tune the outcome of the DDR.

DNA-repair, cell killing and normal tissue damage

International Nuclear Information System (INIS)

Dahm-Daphi, J.; Dikomey, E.; Brammer, I.

1998-01-01

Background: Side effects of radiotherapy in normal tissue is determined by a variety of factors of which cellular and genetic contributions are described here. Material and methods: Review. Results: Normal tissue damage after irradiation is largely due to loss of cellular proliferative capacity. This can be due to mitotic cell death, apoptosis, or terminal differentiation. Dead or differentiated cells release cytokines which additionally modulate the tissue response. DNA damage, in particular non-reparable or misrepaired double-strand breaks are considered the basic lesion leading to G1-arrest and ultimately to cell inactivation. Conclusion: Evidence for genetic bases of normal tissue response, cell killing and DNA-repair capacity is presented. However, a direct link of all 3 endpoints has not yet been proved directly. (orig.) [de

DNA damage response pathway in radioadaptive response.

Science.gov (United States)

Sasaki, Masao S; Ejima, Yosuke; Tachibana, Akira; Yamada, Toshiko; Ishizaki, Kanji; Shimizu, Takashi; Nomura, Taisei

2002-07-25

Radioadaptive response is a biological defense mechanism in which low-dose ionizing irradiation elicits cellular resistance to the genotoxic effects of subsequent irradiation. However, its molecular mechanism remains largely unknown. We previously demonstrated that the dose recognition and adaptive response could be mediated by a feedback signaling pathway involving protein kinase C (PKC), p38 mitogen activated protein kinase (p38MAPK) and phospholipase C (PLC). Further, to elucidate the downstream effector pathway, we studied the X-ray-induced adaptive response in cultured mouse and human cells with different genetic background relevant to the DNA damage response pathway, such as deficiencies in TP53, DNA-PKcs, ATM and FANCA genes. The results showed that p53 protein played a key role in the adaptive response while DNA-PKcs, ATM and FANCA were not responsible. Wortmannin, a specific inhibitor of phosphatidylinositol 3-kinase (PI3K), mimicked the priming irradiation in that the inhibitor alone rendered the cells resistant against the induction of chromosome aberrations and apoptosis by the subsequent X-ray irradiation. The adaptive response, whether it was afforded by low-dose X-rays or wortmannin, occurred in parallel with the reduction of apoptotic cell death by challenging doses. The inhibitor of p38MAPK which blocks the adaptive response did not suppress apoptosis. These observations indicate that the adaptive response and apoptotic cell death constitute a complementary defense system via life-or-death decisions. The p53 has a pivotal role in channeling the radiation-induced DNA double-strand breaks (DSBs) into an adaptive legitimate repair pathway, where the signals are integrated into p53 by a circuitous PKC-p38MAPK-PLC damage sensing pathway, and hence turning off the signals to an alternative pathway to illegitimate repair and apoptosis. A possible molecular mechanism of adaptive response to low-dose ionizing irradiation has been discussed in relation to

Radiation-induced apoptosis

International Nuclear Information System (INIS)

Ohyama, Harumi

1995-01-01

Apoptosis is an active process of gene-directed cellular self-destruction that can be induced in many cell types via numerous physiological and pathological stimuli. We found that interphasedeath of thymocytes is a typical apoptosis showing the characteristic features of apoptosis including cell shrinkage, chromatin condensation and DNA degradation. Moderate dose of radiation induces extensive apoptosis in rapidly proliferating cell population such as the epithelium of intestinal crypt. Recent reports indicate that the ultimate form of radiation-induced mitotic death in several cells is also apoptosis. One of the hallmarks of apoptosis is the enzymatic internucleosomal degradation of chromatin DNA. We identified an endonuclease responsible for the radiation-induced DNA degradation in rat thymocytes. The death-sparing effects of interrupting RNA and protein synthesis suggested a cell genetic program for apoptosis. Apoptosis of thymocytes initiated by DNA damage, such as radiation and radio mimetic substance, absolutely requires the protein of p53 cancer suppresser gene. The cell death induced by glucocorticoid, or aging, has no such requirement. Expression of oncogene bcl-2 rescues cells from the apoptosis. Massive apoptosis in radiosensitive cells induced by higher dose radiation may be fatal. It is suggested that selective apoptotic elimination of cells would play an important role for protection against carcinogenesis and malformation through removal of cells with unrepaired radiation-induced DNA damages. Data to evaluate the significance of apoptosis in the radiation risk are still poor. Further research should be done in order to clarify the roles of the cell death on the acute and late effects of irradiation. (author)

The presence of acylated ghrelin during in vitro maturation of bovine oocytes induces cumulus cell DNA damage and apoptosis, and impairs early embryo development.

Science.gov (United States)

Sirini, Matias A; Anchordoquy, Juan Mateo; Anchordoquy, Juan Patricio; Pascua, Ana M; Nikoloff, Noelia; Carranza, Ana; Relling, Alejandro E; Furnus, Cecilia C

2017-10-01

The aim of this study was to investigate the effects of acylated ghrelin supplementation during in vitro maturation (IVM) of bovine oocytes. IVM medium was supplemented with 20, 40 or 60 pM acylated ghrelin concentrations. Cumulus expansion area and oocyte nuclear maturation were studied as maturation parameters. Cumulus-oocyte complexes (COC) were assessed with the comet, apoptosis and viability assays. The in vitro effects of acylated ghrelin on embryo developmental capacity and embryo quality were also evaluated. Results demonstrated that acylated ghrelin did not affect oocyte nuclear maturation and cumulus expansion area. However, it induced cumulus cell (CC) death, apoptosis and DNA damage. The damage increased as a function of the concentration employed. Additionally, the percentages of blastocyst yield, hatching and embryo quality decreased with all acylated ghrelin concentrations tested. Our study highlights the importance of acylated ghrelin in bovine reproduction, suggesting that this metabolic hormone could function as a signal that prevents the progress to reproductive processes.

Carnosine attenuates cyclophosphamide-induced bone marrow suppression by reducing oxidative DNA damage

Directory of Open Access Journals (Sweden)

Jie Deng

2018-04-01

Full Text Available Oxidative DNA damage in bone marrow cells is the main side effect of chemotherapy drugs including cyclophosphamide (CTX. However, not all antioxidants are effective in inhibiting oxidative DNA damage. In this study, we report the beneficial effect of carnosine (β-alanyl-l-histidine, a special antioxidant with acrolein-sequestering ability, on CTX-induced bone marrow cell suppression. Our results show that carnosine treatment (100 and 200 mg/kg, i.p. significantly inhibited the generation of reactive oxygen species (ROS and 8-hydroxy-2′-deoxyguanosine (8-oxo-dG, and decreased chromosomal abnormalities in the bone marrow cells of mice treated with CTX (20 mg/kg, i.v., 24 h. Furthermore, carnosine evidently mitigated CTX-induced G2/M arrest in murine bone marrow cells, accompanied by reduced ratios of p-Chk1/Chk1 and p-p53/p53 as well as decreased p21 expression. In addition, cell apoptosis caused by CTX was also suppressed by carnosine treatment, as assessed by decreased TUNEL-positive cell counts, down-regulated expressions of Bax and Cyt c, and reduced ratios of cleaved Caspase-3/Caspase-3. These results together suggest that carnosine can protect murine bone marrow cells from CTX-induced DNA damage via its antioxidant activity. Keywords: Carnosine, Cyclophosphamide, Oxidative DNA damage, Sister chromatid exchange, Apoptosis, Cell cycle arrest

DNA damage and autophagy

International Nuclear Information System (INIS)

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

2011-01-01

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

Role of DNA mismatch repair and p53 in signaling induction of apoptosis by alkylating agents.

Science.gov (United States)

Hickman, M J; Samson, L D

1999-09-14

All cells are unavoidably exposed to chemicals that can alkylate DNA to form genotoxic damage. Among the various DNA lesions formed, O(6)-alkylguanine lesions can be highly cytotoxic, and we recently demonstrated that O(6)-methylguanine (O(6)MeG) and O(6)-chloroethylguanine (O(6)CEG) specifically initiate apoptosis in hamster cells. Here we show, in both hamster and human cells, that the MutSalpha branch of the DNA mismatch repair pathway (but not the MutSbeta branch) is absolutely required for signaling the initiation of apoptosis in response to O(6)MeGs and is partially required for signaling apoptosis in response to O(6)CEGs. Further, O(6)MeG lesions signal the stabilization of the p53 tumor suppressor, and such signaling is also MutSalpha-dependent. Despite this, MutSalpha-dependent apoptosis can be executed in a p53-independent manner. DNA mismatch repair status did not influence the response of cells to other inducers of p53 and apoptosis. Thus, it appears that mismatch repair status, rather than p53 status, is a strong indicator of the susceptibility of cells to alkylation-induced apoptosis. This experimental system will allow dissection of the signal transduction events that couple a specific type of DNA base lesion with the final outcome of apoptotic cell death.

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

Directory of Open Access Journals (Sweden)

Guo Zong-Lun

2010-02-01

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

Are glutathione S transferases involved in DNA damage signalling? Interactions with DNA damage and repair revealed from molecular epidemiology studies

International Nuclear Information System (INIS)

Dusinska, Maria; Staruchova, Marta; Horska, Alexandra; Smolkova, Bozena; Collins, Andrew; Bonassi, Stefano; Volkovova, Katarina

2012-01-01

Glutathione S-transferases (GSTs) are members of a multigene family of isoenzymes that are important in the control of oxidative stress and in phase II metabolism. Acting non-enzymically, GSTs can modulate signalling pathways of cell proliferation, cell differentiation and apoptosis. Using a molecular epidemiology approach, we have investigated a potential involvement of GSTs in DNA damage processing, specifically the modulation of DNA repair in a group of 388 healthy adult volunteers; 239 with at least 5 years of occupational exposure to asbestos, stone wool or glass fibre, and 149 reference subjects. We measured DNA damage in lymphocytes using the comet assay (alkaline single cell gel electrophoresis): strand breaks (SBs) and alkali-labile sites, oxidised pyrimidines with endonuclease III, and oxidised purines with formamidopyrimidine DNA glycosylase. We also measured GST activity in erythrocytes, and the capacity for base excision repair (BER) in a lymphocyte extract. Polymorphisms in genes encoding three GST isoenzymes were determined, namely deletion of GSTM1 and GSTT1 and single nucleotide polymorphism Ile105Val in GSTP1. Consumption of vegetables and wine correlated negatively with DNA damage and modulated BER. GST activity correlated with oxidised bases and with BER capacity, and differed depending on polymorphisms in GSTP1, GSTT1 and GSTM1. A significantly lower BER rate was associated with the homozygous GSTT1 deletion in all asbestos site subjects and in the corresponding reference group. Multifactorial analysis revealed effects of sex and exposure in GSTP1 Ile/Val heterozygotes but not in Ile/Ile homozygotes. These variants affected also SBs levels, mainly by interactions of GSTP1 genotype with exposure, with sex, and with smoking habit; and by an interaction between sex and smoking. Our results show that GST polymorphisms and GST activity can apparently influence DNA stability and repair of oxidised bases, suggesting a potential new role for these

Are glutathione S transferases involved in DNA damage signalling? Interactions with DNA damage and repair revealed from molecular epidemiology studies

Energy Technology Data Exchange (ETDEWEB)

Dusinska, Maria, E-mail: Maria.DUSINSKA@nilu.no [CEE-Health Effects Group, NILU - Norwegian Institute for Air Research, Kjeller (Norway); Staruchova, Marta; Horska, Alexandra [Department of Experimental and Applied Genetics, Slovak Medical University, Bratislava (Slovakia); Smolkova, Bozena [Laboratory of Cancer Genetics, Cancer Research Institute of the Slovak Academy of Sciences, Bratislava (Slovakia); Collins, Andrew [Department of Nutrition, Faculty of Medicine, University of Oslo (Norway); Bonassi, Stefano [Unit of Clinical and Molecular Epidemiology, IRCCS San Raffaele Pisana, Rome (Italy); Volkovova, Katarina [Department of Experimental and Applied Genetics, Slovak Medical University, Bratislava (Slovakia)

2012-08-01

Glutathione S-transferases (GSTs) are members of a multigene family of isoenzymes that are important in the control of oxidative stress and in phase II metabolism. Acting non-enzymically, GSTs can modulate signalling pathways of cell proliferation, cell differentiation and apoptosis. Using a molecular epidemiology approach, we have investigated a potential involvement of GSTs in DNA damage processing, specifically the modulation of DNA repair in a group of 388 healthy adult volunteers; 239 with at least 5 years of occupational exposure to asbestos, stone wool or glass fibre, and 149 reference subjects. We measured DNA damage in lymphocytes using the comet assay (alkaline single cell gel electrophoresis): strand breaks (SBs) and alkali-labile sites, oxidised pyrimidines with endonuclease III, and oxidised purines with formamidopyrimidine DNA glycosylase. We also measured GST activity in erythrocytes, and the capacity for base excision repair (BER) in a lymphocyte extract. Polymorphisms in genes encoding three GST isoenzymes were determined, namely deletion of GSTM1 and GSTT1 and single nucleotide polymorphism Ile105Val in GSTP1. Consumption of vegetables and wine correlated negatively with DNA damage and modulated BER. GST activity correlated with oxidised bases and with BER capacity, and differed depending on polymorphisms in GSTP1, GSTT1 and GSTM1. A significantly lower BER rate was associated with the homozygous GSTT1 deletion in all asbestos site subjects and in the corresponding reference group. Multifactorial analysis revealed effects of sex and exposure in GSTP1 Ile/Val heterozygotes but not in Ile/Ile homozygotes. These variants affected also SBs levels, mainly by interactions of GSTP1 genotype with exposure, with sex, and with smoking habit; and by an interaction between sex and smoking. Our results show that GST polymorphisms and GST activity can apparently influence DNA stability and repair of oxidised bases, suggesting a potential new role for these

Effects of chemical-induced DNA damage on male germ cells

Energy Technology Data Exchange (ETDEWEB)

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

1998-12-31

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

Candidate genes for cross-resistance against DNA-damaging drugs

DEFF Research Database (Denmark)

Wittig, Rainer; Nessling, Michelle; Will, Rainer D

2002-01-01

Drug resistance of tumor cells leads to major drawbacks in the treatment of cancer. To identify candidate genes for drug resistance, we compared the expression patterns of the drug-sensitive human malignant melanoma cell line MeWo and three derived sublines with acquired resistance to the DNA...... as several apoptosis-related genes, in particular STK17A and CRYAB. As MPP1 and CRYAB are also among the 14 genes differentially expressed in all three of the drug-resistant sublines, they represent the strongest candidates for resistance against DNA-damaging drugs....

Tyrosine 370 phosphorylation of ATM positively regulates DNA damage response

Science.gov (United States)

Lee, Hong-Jen; Lan, Li; Peng, Guang; Chang, Wei-Chao; Hsu, Ming-Chuan; Wang, Ying-Nai; Cheng, Chien-Chia; Wei, Leizhen; Nakajima, Satoshi; Chang, Shih-Shin; Liao, Hsin-Wei; Chen, Chung-Hsuan; Lavin, Martin; Ang, K Kian; Lin, Shiaw-Yih; Hung, Mien-Chie

2015-01-01

Ataxia telangiectasia mutated (ATM) mediates DNA damage response by controling irradiation-induced foci formation, cell cycle checkpoint, and apoptosis. However, how upstream signaling regulates ATM is not completely understood. Here, we show that upon irradiation stimulation, ATM associates with and is phosphorylated by epidermal growth factor receptor (EGFR) at Tyr370 (Y370) at the site of DNA double-strand breaks. Depletion of endogenous EGFR impairs ATM-mediated foci formation, homologous recombination, and DNA repair. Moreover, pretreatment with an EGFR kinase inhibitor, gefitinib, blocks EGFR and ATM association, hinders CHK2 activation and subsequent foci formation, and increases radiosensitivity. Thus, we reveal a critical mechanism by which EGFR directly regulates ATM activation in DNA damage response, and our results suggest that the status of ATM Y370 phosphorylation has the potential to serve as a biomarker to stratify patients for either radiotherapy alone or in combination with EGFR inhibition. PMID:25601159

Concordant and opposite roles of DNA-PK and the "facilitator of chromatin transcription" (FACT in DNA repair, apoptosis and necrosis after cisplatin

Directory of Open Access Journals (Sweden)

Calkins Anne S

2011-06-01

Full Text Available Abstract Background Platinum-containing chemotherapy produces specific DNA damage and is used to treat several human solid tumors. Tumors initially sensitive to platinum-based drugs frequently become resistant. Inhibition of DNA repair is a potential strategy to enhance cisplatin effectiveness. After cisplatin treatment, a balance between repair and apoptosis determines whether cancer cells proliferate or die. DNA-dependent protein kinase (DNA-PK binds to DNA double strand breaks (DSBs through its Ku subunits and initiates non-homologous end joining. Inhibition of DNA-PK sensitizes cancer cells to cisplatin killing. The goal of this study is to elucidate the mechanism underlying the effects of DNA-PK on cisplatin sensitivity. Results Silencing the expression of the catalytic subunit of DNA-PK (DNA-PKcs increased sensitivity to cisplatin and decreased the appearance of γH2AX after cisplatin treatment. We purified DNA-PK by its Ku86 subunit and identified interactors by tandem mass spectrometry before and after cisplatin treatment. The structure specific recognition protein 1 (SSRP1, Spt16 and γH2AX appeared in the Ku86 complex 5 hours after cisplatin treatment. SSRP1 and Spt16 form the facilitator of chromatin transcription (FACT. The cisplatin-induced association of FACT with Ku86 and γH2AX was abrogated by DNase treatment. In living cells, SSRP1 and Ku86 were recruited at sites of DSBs induced by laser beams. Silencing SSRP1 expression increased sensitivity to cisplatin and decreased γH2AX appearance. However, while silencing SSRP1 in cisplatin-treated cells increased both apoptosis and necrosis, DNA-PKcs silencing, in contrast, favored necrosis over apoptosis. Conclusions DNA-PK and FACT both play roles in DNA repair. Therefore both are putative targets for therapeutic inhibition. Since DNA-PK regulates apoptosis, silencing DNA-PKcs redirects cells treated with cisplatin toward necrosis. Silencing FACT however, allows both apoptosis and

USP22 Induces Cisplatin Resistance in Lung Adenocarcinoma by Regulating γH2AX-Mediated DNA Damage Repair and Ku70/Bax-Mediated Apoptosis

Directory of Open Access Journals (Sweden)

Aman Wang

2017-05-01

Full Text Available Resistance to platinum-based chemotherapy is one of the most important reasons for treatment failure in advanced non-small cell lung cancer, but the underlying mechanism is extremely complex and unclear. The present study aimed to investigate the correlation of ubiquitin-specific peptidase 22 (USP22 with acquired resistance to cisplatin in lung adenocarcinoma. In this study, we found that overexpression of USP22 could lead to cisplatin resistance in A549 cells. USP22 and its downstream proteins γH2AX and Sirt1 levels are upregulated in the cisplatin- resistant A549/CDDP cell line. USP22 enhances DNA damage repair and induce cisplatin resistance by promoting the phosphorylation of histone H2AX via deubiquitinating histone H2A. In addition, USP22 decreases the acetylation of Ku70 by stabilizing Sirt1, thus inhibiting Bax-mediated apoptosis and inducing cisplatin resistance. The cisplatin sensitivity in cisplatin-resistant A549/CDDP cells was restored by USP22 inhibition in vivo and vitro. In summary, our findings reveal the dual mechanism of USP22 involvement in cisplatin resistance that USP22 can regulate γH2AX-mediated DNA damage repair and Ku70/Bax-mediated apoptosis. USP22 is a potential target in cisplatin-resistant lung adenocarcinoma and should be considered in future therapeutic practice.

The possible DNA damage induced by environmental organic compounds: The case of Nonylphenol.

Science.gov (United States)

Noorimotlagh, Zahra; Mirzaee, Seyyed Abbas; Ahmadi, Mehdi; Jaafarzadeh, Neemat; Rahim, Fakher

2018-08-30

Human impact on the environment leads to the release of many pollutants that produce artificial compounds, which can have harmful effects on the body's endocrine system; these are known as endocrine disruptors (EDs). Nonylphenol (NP) is a chemical compound with a nonyl group that is attached to a phenol ring. NP-induced H 2 AX is a sensitive genotoxic biomarker for detecting possible DNA damage; it also causes male infertility and carcinogenesis. We attempt to comprehensively review all the available evidence about the different ways with descriptive mechanisms for explaining the possible DNA damage that is induced by NP. We systematically searched several databases, including PubMed, Scopus, Web of Science, and gray literature, such as Google Scholar by using medical subheading (MeSH) terms and various combinations of selected keywords from January 1970 to August 2017. The initial search identified 62,737 potentially eligible studies; of these studies, 33 were included according to the established inclusion criteria. Thirty-three selected studies, include the topics of animal model (n = 21), cell line (n = 6), human model (n = 4), microorganisms (n = 1), solid DNA (n = 1), infertility (n = 4), apoptosis (n = 6), and carcinogenesis (n = 3). This review highlighted the possible deleterious effects of NP on DNA damage through the ability to produce ROS/RNS. Finally, it is significant to observe caution at this stage with the continued use of environmental pollutants such as NP, which may induce DNA damage and apoptosis. Copyright © 2018 Elsevier Inc. All rights reserved.

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

Science.gov (United States)

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

2018-03-01

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

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

Science.gov (United States)

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

2010-02-18

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

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

International Nuclear Information System (INIS)

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

2010-01-01

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

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

Science.gov (United States)

2010-01-01

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

Sirtuin 7 promotes cellular survival following genomic stress by attenuation of DNA damage, SAPK activation and p53 response

Energy Technology Data Exchange (ETDEWEB)

Kiran, Shashi; Oddi, Vineesha [Laboratory of Cancer Biology, Centre for DNA Fingerprinting and Diagnostics, Hyderabad, Telangana, 500001 (India); Ramakrishna, Gayatri, E-mail: gayatrirama1@gmail.com [Laboratory of Cancer Biology, Centre for DNA Fingerprinting and Diagnostics, Hyderabad, Telangana, 500001 (India); Laboratory of Cancer Cell Biology, Department of Research, Institute of Liver and Biliary Sciences, Delhi 110070 (India)

2015-02-01

Maintaining the genomic integrity is a constant challenge in proliferating cells. Amongst various proteins involved in this process, Sirtuins play a key role in DNA damage repair mechanisms in yeast as well as mammals. In the present work we report the role of one of the least explored Sirtuin viz., SIRT7, under conditions of genomic stress when treated with doxorubicin. Knockdown of SIRT7 sensitized osteosarcoma (U2OS) cells to DNA damage induced cell death by doxorubicin. SIRT7 overexpression in NIH3T3 delayed cell cycle progression by causing delay in G1 to S transition. SIRT7 overexpressing cells when treated with low dose of doxorubicin (0.25 µM) showed delayed onset of senescence, lesser accumulation of DNA damage marker γH2AX and lowered levels of growth arrest markers viz., p53 and p21 when compared to doxorubicin treated control GFP expressing cells. Resistance to DNA damage following SIRT7 overexpression was also evident by EdU incorporation studies where cellular growth arrest was significantly delayed. When treated with higher dose of doxorubicin (>1 µM), SIRT7 conferred resistance to apoptosis by attenuating stress activated kinases (SAPK viz., p38 and JNK) and p53 response thereby shifting the cellular fate towards senescence. Interestingly, relocalization of SIRT7 from nucleolus to nucleoplasm together with its co-localization with SAPK was an important feature associated with DNA damage. SIRT7 mediated resistance to doxorubicin induced apoptosis and senescence was lost when p53 level was restored by nutlin treatment. Overall, we propose SIRT7 attenuates DNA damage, SAPK activation and p53 response thereby promoting cellular survival under conditions of genomic stress. - Highlights: • Knockdown of SIRT7 sensitized cells to DNA damage induced apoptosis. • SIRT7 delayed onset of premature senescence by attenuating DNA damage response. • Overexpression of SIRT7 delayed cell cycle progression by delaying G1/S transition. • Upon DNA damage SIRT

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

Science.gov (United States)

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

2014-03-01

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

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

Science.gov (United States)

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

2012-12-13

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

Umbelliferone arrest cell cycle at G0/G1 phase and induces apoptosis in human oral carcinoma (KB) cells possibly via oxidative DNA damage.

Science.gov (United States)

Vijayalakshmi, Annamalai; Sindhu, Ganapathy

2017-08-01

Umbelliferone (UMB) has widespread pharmacological activity, comprising anti-inflammatory, anti-oxidant, anti-genotoxic and anti-immunomodulatory but the anticancer activity remains unknown in human oral carcinoma (HOC) KB cells. MTT assay determinations was revealed that treatment of KB cells with UMB, prevent and reduce the cell proliferation with the IC 50 - 200μM as well as induces loss of cell viability, morphology change and internucleosomal DNA fragmentation in a concentration dependent manner. Acridine orange and ethidium bromide dual staining assay established that UMB induced apoptosis in KB cells in a dose dependent manner. Alkaline comet assay determination revealed UMB has the potential to increase oxidative DNA damage in KB cells through DNA tail formation significantly (pKB cells. Similarly, we observed increased DNA damage stimulated apoptotic morphological changes in UMB treated cells. Taken together, the present study suggests that UMB exhibits anticancer effect on KB cell line with the increased generation of intracellular ROS, triggered oxidative stress mediated depolarization of mitochondria, which contributes cell death via DNA damage as well as cell cycle arrest at G0/G1 phase. The results have also provided us insight in the pharmacological backgrounds for the potential use of UMB, to target divergent pathways of cell survival and cell death. To conclude UMB could develop as a novel candidate for cancer chemoprevention and therapy, which is our future focus and to develop a connectivity map between in vivo and in vitro activity. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

ATM-dependent E2F1 accumulation in the nucleolus is an indicator of ribosomal stress in early response to DNA damage.

Science.gov (United States)

Jin, Ya-Qiong; An, Guo-Shun; Ni, Ju-Hua; Li, Shu-Yan; Jia, Hong-Ti

2014-01-01

The nucleolus plays a major role in ribosome biogenesis. Most genotoxic agents disrupt nucleolar structure and function, which results in the stabilization/activation of p53, inducing cell cycle arrest or apoptosis. Likewise, transcription factor E2F1 as a DNA damage responsive protein also plays roles in cell cycle arrest, DNA repair, or apoptosis in response to DNA damage through transcriptional response and protein-protein interaction. Furthermore, E2F1 is known to be involved in regulating rRNA transcription. However, how E2F1 displays in coordinating DNA damage and nucleolar stress is unclear. In this study, we demonstrate that ATM-dependent E2F1 accumulation in the nucleolus is a characteristic feature of nucleolar stress in early response to DNA damage. We found that at the early stage of DNA damage, E2F1 accumulation in the nucleolus was an ATM-dependent and a common event in p53-suficient and -deficient cells. Increased nucleolar E2F1 was sequestered by the nucleolar protein p14ARF, which repressed E2F1-dependent rRNA transcription initiation, and was coupled with S phase. Our data indicate that early accumulation of E2F1 in the nucleolus is an indicator for nucleolar stress and a component of ATM pathway, which presumably buffers elevation of E2F1 in the nucleoplasm and coordinates the diversifying mechanisms of E2F1 acts in cell cycle progression and apoptosis in early response to DNA damage.

Apoptosis-like death, an extreme SOS response in Escherichia coli.

Science.gov (United States)

Erental, Ariel; Kalderon, Ziva; Saada, Ann; Smith, Yoav; Engelberg-Kulka, Hanna

2014-07-15

In bacteria, SOS is a global response to DNA damage, mediated by the recA-lexA genes, resulting in cell cycle arrest, DNA repair, and mutagenesis. Previously, we reported that Escherichia coli responds to DNA damage via another recA-lexA-mediated pathway resulting in programmed cell death (PCD). We called it apoptosis-like death (ALD) because it is characterized by membrane depolarization and DNA fragmentation, which are hallmarks of eukaryotic mitochondrial apoptosis. Here, we show that ALD is an extreme SOS response that occurs only under conditions of severe DNA damage. Furthermore, we found that ALD is characterized by additional hallmarks of eukaryotic mitochondrial apoptosis, including (i) rRNA degradation by the endoribonuclease YbeY, (ii) upregulation of a unique set of genes that we called extensive-damage-induced (Edin) genes, (iii) a decrease in the activities of complexes I and II of the electron transport chain, and (iv) the formation of high levels of OH˙ through the Fenton reaction, eventually resulting in cell death. Our genetic and molecular studies on ALD provide additional insight for the evolution of mitochondria and the apoptotic pathway in eukaryotes. Importance: The SOS response is the first described and the most studied bacterial response to DNA damage. It is mediated by a set of two genes, recA-lexA, and it results in DNA repair and thereby in the survival of the bacterial culture. We have shown that Escherichia coli responds to DNA damage by an additional recA-lexA-mediated pathway resulting in an apoptosis-like death (ALD). Apoptosis is a mode of cell death that has previously been reported only in eukaryotes. We found that E. coli ALD is characterized by several hallmarks of eukaryotic mitochondrial apoptosis. Altogether, our results revealed that recA-lexA is a DNA damage response coordinator that permits two opposite responses: life, mediated by the SOS, and death, mediated by the ALD. The choice seems to be a function of the degree

DNA damage response in monozygotic twins discordant for smoking habits.

Science.gov (United States)

Marcon, Francesca; Carotti, Daniela; Andreoli, Cristina; Siniscalchi, Ester; Leopardi, Paola; Caiola, Stefania; Biffoni, Mauro; Zijno, Andrea; Medda, Emanuela; Nisticò, Lorenza; Rossi, Sabrina; Crebelli, Riccardo

2013-03-01

Previous studies in twins indicate that non-shared environment, beyond genetic factors, contributes substantially to individual variation in mutagen sensitivity; however, the role of specific causative factors (e.g. tobacco smoke, diet) was not elucidated. In this investigation, a population of 22 couples of monozygotic twins with discordant smoking habits was selected with the aim of evaluating the influence of tobacco smoke on individual response to DNA damage. The study design virtually eliminated the contribution of genetic heterogeneity to the intra-pair variation in DNA damage response, and thus any difference in the end-points investigated could directly be attributed to the non-shared environment experienced by co-twins, which included as main factor cigarette smoke exposure. Peripheral lymphocytes of study subjects were challenged ex vivo with γ-rays, and the induction, processing, fixation of DNA damage evaluated through multiple approaches. Folate status of study subjects was considered significant covariate since it is affected by smoking habits and can influence radiosensitivity. Similar responses were elicited by γ-rays in co-twins for all the end-points analysed, despite their discordant smoking habits. Folate status did not modify DNA damage response, even though a combined effect of smoking habits, low-plasma folic acid level, and ionising radiation was observed on apoptosis. A possible modulation of DNA damage response by duration and intensity of tobacco smoke exposure was suggested by Comet assay and micronucleus data, but the effect was quantitatively limited. Overall, the results obtained indicate that differences in smoking habits do not contribute to a large extent to inter-individual variability in the response to radiation-induced DNA damage observed in healthy human populations.

HeLa DNA damage response induced by 12C6+ ions

International Nuclear Information System (INIS)

Chen Jidong; Li Ning; Zhang Hong; Wu Zhenhua

2009-01-01

The aim of this study is to explore the DNA damage response of HeLa irradiated by 12 C 6+ beam and the mechanism of the p53 activation change in this response.In our present study, double strands break(DSB)of HeLa cells irradiated with 12 C 6+ beam were detected through neutral single cell gel electrophoresis, and AO/EB staining was used to detect the apoptosis of irradiated HeLa in 24h irradiation. Moreover, HeLa was pre-treated with caffeine (ATM and ATR inhibiting) or wormannin with certain concentrations (20 μmol/L, ATM and DNA-PK inhibiting) and irradiated with 1Gy of 12 C 6+ beam,and the expression of p53 was detected with Western blot analysis. The results show that DSB of HeLa caused by 12 C 6+ beam increases with absorbed doses and decreases with the time after irradiation. The apoptosis percentage of irradiated HeLa increases with absorbed doses. It has been found that the p53 expression increases after irradiation, but has not significant increment with caffeine or wortmannin pre-treatment in cells.It can be deduced that the p53 activation is ATM-dependent, but not ATR and DNA-PK-dependent in HeLa DNA damage response induced by 12 C 6+ beam. (authors)

Effect of fenofibrate on oxidative DNA damage and on gene expression related to cell proliferation and apoptosis in rats.

Science.gov (United States)

Nishimura, Jihei; Dewa, Yasuaki; Muguruma, Masako; Kuroiwa, Yuichi; Yasuno, Hiroaki; Shima, Tomomi; Jin, Mailan; Takahashi, Miwa; Umemura, Takashi; Mitsumori, Kunitoshi

2007-05-01

To investigate the relationship between fenofibrate (FF) and oxidative stress, enzymatic, histopathological, and molecular biological analyses were performed in the liver of male F344 rats fed 2 doses of FF (Experiment 1; 0 and 6000 ppm) for 3 weeks and 3 doses (Experiment 2; 0, 3000, and 6000 ppm) for 9 weeks. FF treatment increased the activity of enzymes such as carnitine acetyltransferase, carnitine palmitoyltransferase, fatty acyl-CoA oxidizing system, and catalase in the liver. However, it decreased those of superoxide dismutase in the liver in both experiments. Increased 8-hydroxy-2'-deoxyguanosine levels in liver DNA and lipofuscin accumulation were observed in the treated rats of Experiment 2. In vitro measurement of reactive oxygen species (ROS) in rat liver microsomes revealed a dose-dependent increase due to FF treatment. Microarray (only Experiment 1) or real-time reverse transcription-polymerase chain reaction analyses revealed that the expression levels of metabolism and DNA repair-related genes such as Aco, Cyp4a1, Cat, Yc2, Gpx2, Apex1, Xrcc5, Mgmt, Mlh1, Gadd45a, and Nbn were increased in FF-treated rats. These results provide evidence of a direct or indirect relationship between oxidative stress and FF treatment. In addition, increases in the expression levels of cell cycle-related genes such as Chek1, Cdc25a, and Ccdn1; increases in the expression levels of cell proliferation-related genes such as Hdgfrp3 and Vegfb; and fluctuations in the expression levels of apoptosis-related genes such as Casp11 and Trp53inp1 were observed in these rats. This suggests that cell proliferation induction, apoptosis suppression, and DNA damage due to oxidative stresses are probably involved in the mechanism of hepatocarcinogenesis due to FF in rats.

Plasma induced DNA damage: Comparison with the effects of ionizing radiation

Energy Technology Data Exchange (ETDEWEB)

Lazović, S.; Maletić, D.; Puač, N.; Malović, G.; Petrović, Z. Lj. [Institute of Physics, University of Belgrade, Pregrevica 118, 11080 Belgrade (Serbia); Leskovac, A.; Filipović, J.; Joksić, G. [Department of Physical Chemistry, Vinča Institute of Nuclear Sciences, University of Belgrade, 11001 Belgrade (Serbia)

2014-09-22

We use human primary fibroblasts for comparing plasma and gamma rays induced DNA damage. In both cases, DNA strand breaks occur, but of fundamentally different nature. Unlike gamma exposure, contact with plasma predominantly leads to single strand breaks and base-damages, while double strand breaks are mainly consequence of the cell repair mechanisms. Different cell signaling mechanisms are detected confirming this (ataxia telangiectasia mutated - ATM and ataxia telangiectasia and Rad3 related - ATR, respectively). The effective plasma doses can be tuned to match the typical therapeutic doses of 2 Gy. Tailoring the effective dose through plasma power and duration of the treatment enables safety precautions mainly by inducing apoptosis and consequently reduced frequency of micronuclei.

Solar ultraviolet radiation-induced DNA damage in aquatic organisms: potential environmental impact

International Nuclear Information System (INIS)

Haeder, Donat-P.; Sinha, Rajeshwar P.

2005-01-01

Continuing depletion of stratospheric ozone and subsequent increases in deleterious ultraviolet (UV) radiation at the Earth's surface have fueled the interest in its ecological consequences for aquatic ecosystems. The DNA is certainly one of the key targets for UV-induced damage in a variety of aquatic organisms. UV radiation induces two of the most abundant mutagenic and cytotoxic DNA lesions, cyclobutane pyrimidine dimers (CPDs) and pyrimidine pyrimidone photoproducts (6-4PPs) and their Dewar valence isomers. However, aquatic organisms have developed a number of repair and tolerance mechanisms to counteract the damaging effects of UV on DNA. Photoreactivation with the help of the enzyme photolyase is one of the most important and frequently occurring repair mechanisms in a variety of organisms. Excision repair, which can be distinguished into base excision repair (BER) and nucleotide excision repair (NER), also play an important role in DNA repair in several organisms with the help of a number of glycosylases and polymerases, respectively. In addition, mechanisms such as mutagenic repair or dimer bypass, recombinational repair, cell-cycle checkpoints, apoptosis and certain alternative repair pathways are also operative in various organisms. This review deals with the UV-induced DNA damage and repair in a number of aquatic organisms as well as methods of detecting DNA damage

Estimates of DNA strand breakage in bottlenose dolphin (Tursiops truncatus leukocytes measured with the Comet and DNA diffusion assays

Directory of Open Access Journals (Sweden)

Adriana Díaz

2009-01-01

Full Text Available The analysis of DNA damage by mean of Comet or single cell gel electrophoresis (SCGE assay has been commonly used to assess genotoxic impact in aquatic animals being able to detect exposure to low concentrations of contaminants in a wide range of species. The aims of this work were 1 to evaluate the usefulness of the Comet to detect DNA strand breakage in dolphin leukocytes, 2 to use the DNA diffusion assay to determine the amount of DNA strand breakage associated with apoptosis or necrosis, and 3 to determine the proportion of DNA strand breakage that was unrelated to apoptosis and necrosis. Significant intra-individual variation was observed in all of the estimates of DNA damage. DNA strand breakage was overestimated because a considerable amount (~29% of the DNA damage was derived from apoptosis and necrosis. The remaining DNA damage in dolphin leukocytes was caused by factors unrelated to apoptosis and necrosis. These results indicate that the DNA diffusion assay is a complementary tool that can be used together with the Comet assay to assess DNA damage in bottlenose dolphins.

DNA damage and polyploidization.

Science.gov (United States)

Chow, Jeremy; Poon, Randy Y C

2010-01-01

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

Response to DNA damage: why do we need to focus on protein phosphatases?

Directory of Open Access Journals (Sweden)

Midori eShimada

2013-01-01

Full Text Available Eukaryotic cells are continuously threatened by unavoidable errors during normal DNA replication or various sources of genotoxic stresses that cause DNA damage or stalled replication. To maintain genomic integrity, cells have developed a coordinated signaling network, known as the DNA damage response (DDR. Following DNA damage, sensor molecules detect the presence of DNA damage and transmit signals to downstream transducer molecules. This in turn conveys the signals to numerous effectors, which initiate a large number of specific biological responses, including transient cell cycle arrest mediated by checkpoints, DNA repair, and apoptosis. It is recently becoming clear that dephosphorylation events are involved in keeping DDR factors inactive during normal cell growth. Moreover, dephosphorylation is required to shut off checkpoint arrest following DNA damage and has been implicated in the activation of the DDR. Spatial and temporal regulation of phosphorylation events is essential for the DDR, and fine-tuning of phosphorylation is partly mediated by protein phosphatases. While the role of kinases in the DDR has been well documented, the complex roles of protein dephosphorylation have only recently begun to be investigated. Therefore, it is important to focus on the role of phosphatases and to determine how their activity is regulated upon DNA damage. In this work, we summarize current knowledge on the involvement of serine/threonine phosphatases, especially the protein phosphatase 1, protein phosphatase 2A, and protein phosphatase Mg2+/Mn2+-dependent families, in the DDR.

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

Science.gov (United States)

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

2013-01-01

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

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

Science.gov (United States)

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.

Dynamic changes to survivin subcellular localization are initiated by DNA damage

Directory of Open Access Journals (Sweden)

Maritess Gay Asumen

2010-07-01

Full Text Available Maritess Gay Asumen1, Tochukwu V Ifeacho2, Luke Cockerham3, Christina Pfandl4, Nathan R Wall31Touro University’s College of Osteopathic Medicine, Vallejo, CA, USA; 2University of Southern California, Los Angeles, CA, USA; 3Center for Health Disparities Research and Molecular Medicine, Loma Linda University, CA, USA; 4Green Mountain Antibodies, Burlington, VT, USAAbstract: Subcellular distribution of the apoptosis inhibitor survivin and its ability to relocalize as a result of cell cycle phase or therapeutic insult has led to the hypothesis that these subcellular pools may coincide with different survivin functions. The PIK kinases (ATM, ATR and DNA-PK phosphorylate a variety of effector substrates that propagate DNA damage signals, resulting in various biological outputs. Here we demonstrate that subcellular repartitioning of survivin in MCF-7 cells as a result of UV light-mediated DNA damage is dependent upon DNA damage-sensing proteins as treatment with the pan PIK kinase inhibitor wortmannin repartitioned survivin in the mitochondria and diminished it from the cytosol and nucleus. Mitochondrial redistribution of survivin, such as was recorded after wortmannin treatment, occurred in cells lacking any one of the three DNA damage sensing protein kinases: DNA-PK, ATM or ATR. However, failed survivin redistribution from the mitochondria in response to low-dose UV occurred only in the cells lacking ATM, implying that ATM may be the primary kinase involved in this process. Taken together, this data implicates survivian’s subcellular distribution is a dynamic physiological process that appears responsive to UV light- initiated DNA damage and that its distribution may be responsible for its multifunctionality.Keywords: survivin, PIK kinases, ATM, ATR, DNA-PK

Critical involvement of the ATM-dependent DNA damage response in the apoptotic demise of HIV-1-elicited syncytia.

Directory of Open Access Journals (Sweden)

Jean-Luc Perfettini

Full Text Available DNA damage can activate the oncosuppressor protein ataxia telangiectasia mutated (ATM, which phosphorylates the histone H2AX within characteristic DNA damage foci. Here, we show that ATM undergoes an activating phosphorylation in syncytia elicited by the envelope glycoprotein complex (Env of human immunodeficiency virus-1 (HIV-1 in vitro. This was accompanied by aggregation of ATM in discrete nuclear foci that also contained phospho-histone H2AX. DNA damage foci containing phosphorylated ATM and H2AX were detectable in syncytia present in the brain or lymph nodes from patients with HIV-1 infection, as well as in a fraction of blood leukocytes, correlating with viral status. Knockdown of ATM or of its obligate activating factor NBS1 (Nijmegen breakage syndrome 1 protein, as well as pharmacological inhibition of ATM with KU-55933, inhibited H2AX phosphorylation and prevented Env-elicited syncytia from undergoing apoptosis. ATM was found indispensable for the activation of MAP kinase p38, which catalyzes the activating phosphorylation of p53 on serine 46, thereby causing p53 dependent apoptosis. Both wild type HIV-1 and an HIV-1 mutant lacking integrase activity induced syncytial apoptosis, which could be suppressed by inhibiting ATM. HIV-1-infected T lymphoblasts from patients with inactivating ATM or NBS1 mutations also exhibited reduced syncytial apoptosis. Altogether these results indicate that apoptosis induced by a fusogenic HIV-1 Env follows a pro-apoptotic pathway involving the sequential activation of ATM, p38MAPK and p53.

Comparison of DNA Damage and Apoptosis Induced By Silver Nanoparticle-containing Dressing Materials During Wound Healing.

Science.gov (United States)

Choi, Young Suk; Gwak, Heui-Chul; Park, Jae Keun; Lim, Ji Yun; Yeo, Eui Dong; Park, Eunseok; Kim, Junyong; Lee, Young Koo

2018-04-13

than products A-C. Dressing materials containing AgNP have an antimicrobial effect. However, the authors observed that some AgNP dressings induced DNA damage and apoptosis. Although AgNP dressings did not cause significant acute apoptotic effects, they should be examined for cytotoxic effects in chronic wounds and should be used with caution when treating chronic wounds and those with low bacteria counts.

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

Science.gov (United States)

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.

Ultrasound-induced DNA damage and signal transductions indicated by gammaH2AX

Science.gov (United States)

Furusawa, Yukihiro; Fujiwara, Yoshisada; Zhao, Qing-Li; Hassan, Mariame Ali; Ogawa, Ryohei; Tabuchi, Yoshiaki; Takasaki, Ichiro; Takahashi, Akihisa; Ohnishi, Takeo; Kondo, Takashi

2011-09-01

Ultrasound (US) has been shown to induce cancer cell death via different forms including apoptosis. Here, we report the potential of low-intensity pulsed US (LIPUS) to induce genomic DNA damage and subsequent DNA damage response. Using the ionizing radiation-induced DNA double-strand breaks (DSBs) as the positive control, we were able to observe the induction of DSBs (as neutral comet tails) and the subsequent formation of gammaH2AX-positive foci (by immunofluorescence detection) in human leukemia cells following exposure to LIPUS. The LIPUS-induced DNA damage arose most likely from the mechanical, but not sonochemical, effect of cavitation, based on our observation that the suppression of inertial cavitation abrogated the gammH2AX foci formation, whereas scavenging of free radical formation (e.g., hydroxyl radical) had no protective effect on it. Treatment with the specific kinase inhibitor of ATM or DNA-PKcs, which can phosphorylate H2AX Ser139, revealed that US-induced gammaH2AX was inhibited more effectively by the DNA-PK inhibitor than ATM kinase inhibitor. Notably, these inhibitor effects were opposite to those with radiation-induced gammH2AX. In conclusion, we report, for the first time that US can induce DNA damage and the DNA damage response as indicated by gammaH2AX was triggered by the cavitational mechanical effects. Thus, it is expected that the data shown here may provide a better understanding of the cellular responses to US.

In Vivo Bystander Effect: Cranial X-Irradiation Leads to Elevated DNA Damage, Altered Cellular Proliferation and Apoptosis, and Increased p53 Levels in Shielded Spleen

International Nuclear Information System (INIS)

Koturbash, Igor; Loree, Jonathan; Kutanzi, Kristy; Koganow, Clayton; Pogribny, Igor; Kovalchuk, Olga

2008-01-01

Purpose: It is well accepted that irradiated cells may 'forward' genome instability to nonirradiated neighboring cells, giving rise to the 'bystander effect' phenomenon. Although bystander effects were well studied by using cell cultures, data for somatic bystander effects in vivo are relatively scarce. Methods and Materials: We set out to analyze the existence and molecular nature of bystander effects in a radiation target-organ spleen by using a mouse model. The animal's head was exposed to X-rays while the remainder of the body was completely protected by a medical-grade shield. Using immunohistochemistry, we addressed levels of DNA damage, cellular proliferation, apoptosis, and p53 protein in the spleen of control animals and completely exposed and head-exposed/body bystander animals. Results: We found that localized head radiation exposure led to the induction of bystander effects in the lead-shielded distant spleen tissue. Namely, cranial irradiation led to increased levels of DNA damage and p53 expression and also altered levels of cellular proliferation and apoptosis in bystander spleen tissue. The observed bystander changes were not caused by radiation scattering and were observed in two different mouse strains; C57BL/6 and BALB/c. Conclusion: Our study proves that bystander effects occur in the distant somatic organs on localized exposures. Additional studies are required to characterize the nature of an enigmatic bystander signal and analyze the long-term persistence of these effects and possible contribution of radiation-induced bystander effects to secondary radiation carcinogenesis

Estimates of DNA damage by the comet assay in the direct-developing frog Eleutherodactylus johnstonei (Anura, Eleutherodactylidae

Directory of Open Access Journals (Sweden)

Laura Carolina Valencia

2011-01-01

Full Text Available The aim of this study was to use the Comet assay to assess genetic damage in the direct-developing frog Eleutherodactylus johnstonei. A DNA diffusion assay was used to evaluate the effectiveness of alkaline, enzymatic and alkaline/enzymatic treatments for lysing E. johnstonei blood cells and to determine the amount of DNA strand breakage associated with apoptosis and necrosis. Cell sensitivity to the mutagens bleomycin (BLM and 4-nitroquinoline-1-oxide (4NQO was also assessed using the Comet assay, as was the assay reproducibility. Alkaline treatment did not lyse the cytoplasmic and nuclear membranes of E. johnstonei blood cells, whereas enzymatic digestion with proteinase K (40 !g/mL yielded naked nuclei. The contribution of apoptosis and necrosis (assessed by the DNA diffusion assay to DNA damage was estimated to range from 0% to 8%. BLM and 4NQO induced DNA damage in E. johnstonei blood cells at different concentrations and exposure times. Dose-effect curves with both mutagens were highly reproducible and showed consistently low coefficients of variation (CV < 10%. The results are discussed with regard to the potential use of the modified Comet assay for assessing the exposure of E. johnstonei to herbicides in ecotoxicological studies.

Estimates of DNA damage by the comet assay in the direct-developing frog Eleutherodactylus johnstonei (Anura, Eleutherodactylidae).

Science.gov (United States)

Valencia, Laura Carolina; García, Adriana; Ramírez-Pinilla, Martha Patricia; Fuentes, Jorge Luis

2011-10-01

The aim of this study was to use the Comet assay to assess genetic damage in the direct-developing frog Eleutherodactylus johnstonei. A DNA diffusion assay was used to evaluate the effectiveness of alkaline, enzymatic and alkaline/enzymatic treatments for lysing E. johnstonei blood cells and to determine the amount of DNA strand breakage associated with apoptosis and necrosis. Cell sensitivity to the mutagens bleomycin (BLM) and 4-nitro-quinoline-1-oxide (4NQO) was also assessed using the Comet assay, as was the assay reproducibility. Alkaline treatment did not lyse the cytoplasmic and nuclear membranes of E. johnstonei blood cells, whereas enzymatic digestion with proteinase K (40 μg/mL) yielded naked nuclei. The contribution of apoptosis and necrosis (assessed by the DNA diffusion assay) to DNA damage was estimated to range from 0% to 8%. BLM and 4NQO induced DNA damage in E. johnstonei blood cells at different concentrations and exposure times. Dose-effect curves with both mutagens were highly reproducible and showed consistently low coefficients of variation (CV ≤ 10%). The results are discussed with regard to the potential use of the modified Comet assay for assessing the exposure of E. johnstonei to herbicides in ecotoxicological studies.

Role of DNA mismatch repair and p53 in signaling induction of apoptosis by alkylating agents

OpenAIRE

Hickman, Mark J.; Samson, Leona D.

1999-01-01

All cells are unavoidably exposed to chemicals that can alkylate DNA to form genotoxic damage. Among the various DNA lesions formed, O6-alkylguanine lesions can be highly cytotoxic, and we recently demonstrated that O6-methylguanine (O6MeG) and O6-chloroethylguanine (O6CEG) specifically initiate apoptosis in hamster cells. Here we show, in both hamster and human cells, that the MutSα branch of the DNA mismatch repair pathway (but not the MutSβ branch) is absolutely required for signaling the ...

Response of thyroid follicular cells to gamma irradiation compared to proton irradiation. I. Initial characterization of DNA damage, micronucleus formation, apoptosis, cell survival, and cell cycle phase redistribution

Science.gov (United States)

Green, L. M.; Murray, D. K.; Bant, A. M.; Kazarians, G.; Moyers, M. F.; Nelson, G. A.; Tran, D. T.

2001-01-01

The RBE of protons has been assumed to be equivalent to that of photons. The objective of this study was to determine whether radiation-induced DNA and chromosome damage, apoptosis, cell killing and cell cycling in organized epithelial cells was influenced by radiation quality. Thyroid-stimulating hormone-dependent Fischer rat thyroid cells, established as follicles, were exposed to gamma rays or proton beams delivered acutely over a range of physical doses. Gamma-irradiated cells were able to repair DNA damage relatively rapidly so that by 1 h postirradiation they had approximately 20% fewer exposed 3' ends than their counterparts that had been irradiated with proton beams. The persistence of free ends of DNA in the samples irradiated with the proton beam implies that either more initial breaks or a quantitatively different type of damage had occurred. These results were further supported by an increased frequency of chromosomal damage as measured by the presence of micronuclei. Proton-beam irradiation induced micronuclei at a rate of 2.4% per gray, which at 12 Gy translated to 40% more micronuclei than in comparable gamma-irradiated cultures. The higher rate of micronucleus formation and the presence of larger micronuclei in proton-irradiated cells was further evidence that a qualitatively more severe class of damage had been induced than was induced by gamma rays. Differences in the type of damage produced were detected in the apoptosis assay, wherein a significant lag in the induction of apoptosis occurred after gamma irradiation that did not occur with protons. The more immediate expression of apoptotic cells in the cultures irradiated with the proton beam suggests that the damage inflicted was more severe. Alternatively, the cell cycle checkpoint mechanisms required for recovery from such damage might not have been invoked. Differences based on radiation quality were also evident in the alpha components of cell survival curves (0.05 Gy(-1) for gamma rays, 0

DNA damage in neurodegenerative diseases

Energy Technology Data Exchange (ETDEWEB)

Coppedè, Fabio, E-mail: fabio.coppede@med.unipi.it; Migliore, Lucia, E-mail: lucia.migliore@med.unipi.it

2015-06-15

Highlights: • Oxidative DNA damage is one of the earliest detectable events in the neurodegenerative process. • The mitochondrial DNA is more vulnerable to oxidative attack than the nuclear DNA. • Cytogenetic damage has been largely documented in Alzheimer's disease patients. • The question of whether DNA damage is cause or consequence of neurodegeneration is still open. • Increasing evidence links DNA damage and repair with epigenetic phenomena. - Abstract: Following the observation of increased oxidative DNA damage in nuclear and mitochondrial DNA extracted from post-mortem brain regions of patients affected by neurodegenerative diseases, the last years of the previous century and the first decade of the present one have been largely dedicated to the search of markers of DNA damage in neuronal samples and peripheral tissues of patients in early, intermediate or late stages of neurodegeneration. Those studies allowed to demonstrate that oxidative DNA damage is one of the earliest detectable events in neurodegeneration, but also revealed cytogenetic damage in neurodegenerative conditions, such as for example a tendency towards chromosome 21 malsegregation in Alzheimer's disease. As it happens for many neurodegenerative risk factors the question of whether DNA damage is cause or consequence of the neurodegenerative process is still open, and probably both is true. The research interest in markers of oxidative stress was shifted, in recent years, towards the search of epigenetic biomarkers of neurodegenerative disorders, following the accumulating evidence of a substantial contribution of epigenetic mechanisms to learning, memory processes, behavioural disorders and neurodegeneration. Increasing evidence is however linking DNA damage and repair with epigenetic phenomena, thereby opening the way to a very attractive and timely research topic in neurodegenerative diseases. We will address those issues in the context of Alzheimer's disease

DNA damage in neurodegenerative diseases

International Nuclear Information System (INIS)

Coppedè, Fabio; Migliore, Lucia

2015-01-01

Highlights: • Oxidative DNA damage is one of the earliest detectable events in the neurodegenerative process. • The mitochondrial DNA is more vulnerable to oxidative attack than the nuclear DNA. • Cytogenetic damage has been largely documented in Alzheimer's disease patients. • The question of whether DNA damage is cause or consequence of neurodegeneration is still open. • Increasing evidence links DNA damage and repair with epigenetic phenomena. - Abstract: Following the observation of increased oxidative DNA damage in nuclear and mitochondrial DNA extracted from post-mortem brain regions of patients affected by neurodegenerative diseases, the last years of the previous century and the first decade of the present one have been largely dedicated to the search of markers of DNA damage in neuronal samples and peripheral tissues of patients in early, intermediate or late stages of neurodegeneration. Those studies allowed to demonstrate that oxidative DNA damage is one of the earliest detectable events in neurodegeneration, but also revealed cytogenetic damage in neurodegenerative conditions, such as for example a tendency towards chromosome 21 malsegregation in Alzheimer's disease. As it happens for many neurodegenerative risk factors the question of whether DNA damage is cause or consequence of the neurodegenerative process is still open, and probably both is true. The research interest in markers of oxidative stress was shifted, in recent years, towards the search of epigenetic biomarkers of neurodegenerative disorders, following the accumulating evidence of a substantial contribution of epigenetic mechanisms to learning, memory processes, behavioural disorders and neurodegeneration. Increasing evidence is however linking DNA damage and repair with epigenetic phenomena, thereby opening the way to a very attractive and timely research topic in neurodegenerative diseases. We will address those issues in the context of Alzheimer's disease

Enterolactone: A novel radiosensitizer for human breast cancer cell lines through impaired DNA repair and increased apoptosis

International Nuclear Information System (INIS)

Bigdeli, Bahareh; Goliaei, Bahram; Masoudi-Khoram, Nastaran; Jooyan, Najmeh; Nikoofar, Alireza; Rouhani, Maryam; Haghparast, Abbas; Mamashli, Fatemeh

2016-01-01

Introduction: Radiotherapy is a potent treatment against breast cancer, which is the most commonly diagnosed cancer among women. However, the emergence of radioresistance due to increased DNA repair leads to radiotherapeutic failure. Applying polyphenols combined with radiation is a more promising method leading to better survival. Enterolactone, a phytoestrogenic polyphenol, has been reported to inhibit an important radioresistance signaling pathway, therefore we conjectured that enterolactone could enhance radiosensitivity in breast cancer. To assess this hypothesis, radiation response of enterolactone treated MDA-MB-231 and T47D cell lines and corresponding cellular mechanisms were investigated. Methods: Cytotoxicity of enterolactone was measured via MTT assay. Cells were treated with enterolactone before X-irradiation, and clonogenic assay was used to evaluate radiosensitivity. Cell cycle distribution and apoptosis were measured by flow cytometric analysis. In addition, DNA damages and corresponding repair, chromosomal damages, and aberrations were assessed by comet, micronucleus, and cytogenetic assays, respectively. Results: Enterolactone decreased the viability of cells in a concentration- and time dependent manner. Enterolactone significantly enhanced radiosensitivity of cells by abrogating G2/M arrest, impairing DNA repair, and increasing radiation-induced apoptosis. Furthermore, increased chromosomal damages and aberrations were detected in cells treated with enterolactone combined with X-rays than X-ray alone. These effects were more prominent in T47D than MDA-MB-231 cells. Discussion: To our knowledge, this is the first report that enterolactone is a novel radiosensitizer for breast cancer irrespective of estrogen receptor status. Authors propose enterolactone as a candidate for combined therapy to decrease the radiation dose delivered to patients and subsequent side effects. - Highlights: • Enterolactone is proposed to be a novel radiosensitizer for

Enterolactone: A novel radiosensitizer for human breast cancer cell lines through impaired DNA repair and increased apoptosis

Energy Technology Data Exchange (ETDEWEB)

Bigdeli, Bahareh, E-mail: bhr.bigdeli@ut.ac.ir [Department of Biophysics, Institute of Biochemistry and Biophysics, University of Tehran, 16th Azar St., Enghelab Sq., Tehran (Iran, Islamic Republic of); Goliaei, Bahram, E-mail: goliaei@ut.ac.ir [Department of Biophysics, Institute of Biochemistry and Biophysics, University of Tehran, 16th Azar St., Enghelab Sq., Tehran (Iran, Islamic Republic of); Masoudi-Khoram, Nastaran, E-mail: n.masoudi@alumni.ut.ac.ir [Department of Biophysics, Institute of Biochemistry and Biophysics, University of Tehran, 16th Azar St., Enghelab Sq., Tehran (Iran, Islamic Republic of); Jooyan, Najmeh, E-mail: n.jooyan@ut.ac.ir [Department of Biophysics, Institute of Biochemistry and Biophysics, University of Tehran, 16th Azar St., Enghelab Sq., Tehran (Iran, Islamic Republic of); Nikoofar, Alireza, E-mail: nikoofar@iums.ac.ir [Department of Radiotherapy, Iran University of Medical Sciences (IUMS), Shahid Hemmat Highway, Tehran (Iran, Islamic Republic of); Rouhani, Maryam, E-mail: rouhani@iasbs.ac.ir [Department of Biological Sciences, Institute for Advanced Studies in Basic Sciences (IASBS), Prof. Yousef Sobouti Blvd., Gava Zang, Zanjan (Iran, Islamic Republic of); Haghparast, Abbas, E-mail: Haghparast@sbmu.ac.ir [Neuroscience Research Center, Shahid Beheshti University of Medical Sciences, Daneshjo St., Evin, Tehran (Iran, Islamic Republic of); Mamashli, Fatemeh, E-mail: mamashli@ut.ac.ir [Department of Biophysics, Institute of Biochemistry and Biophysics, University of Tehran, 16th Azar St., Enghelab Sq., Tehran (Iran, Islamic Republic of)

2016-12-15

Introduction: Radiotherapy is a potent treatment against breast cancer, which is the most commonly diagnosed cancer among women. However, the emergence of radioresistance due to increased DNA repair leads to radiotherapeutic failure. Applying polyphenols combined with radiation is a more promising method leading to better survival. Enterolactone, a phytoestrogenic polyphenol, has been reported to inhibit an important radioresistance signaling pathway, therefore we conjectured that enterolactone could enhance radiosensitivity in breast cancer. To assess this hypothesis, radiation response of enterolactone treated MDA-MB-231 and T47D cell lines and corresponding cellular mechanisms were investigated. Methods: Cytotoxicity of enterolactone was measured via MTT assay. Cells were treated with enterolactone before X-irradiation, and clonogenic assay was used to evaluate radiosensitivity. Cell cycle distribution and apoptosis were measured by flow cytometric analysis. In addition, DNA damages and corresponding repair, chromosomal damages, and aberrations were assessed by comet, micronucleus, and cytogenetic assays, respectively. Results: Enterolactone decreased the viability of cells in a concentration- and time dependent manner. Enterolactone significantly enhanced radiosensitivity of cells by abrogating G2/M arrest, impairing DNA repair, and increasing radiation-induced apoptosis. Furthermore, increased chromosomal damages and aberrations were detected in cells treated with enterolactone combined with X-rays than X-ray alone. These effects were more prominent in T47D than MDA-MB-231 cells. Discussion: To our knowledge, this is the first report that enterolactone is a novel radiosensitizer for breast cancer irrespective of estrogen receptor status. Authors propose enterolactone as a candidate for combined therapy to decrease the radiation dose delivered to patients and subsequent side effects. - Highlights: • Enterolactone is proposed to be a novel radiosensitizer for

Damage-induced DNA replication stalling relies on MAPK-activated protein kinase 2 activity

DEFF Research Database (Denmark)

Köpper, Frederik; Bierwirth, Cathrin; Schön, Margarete

2013-01-01

knockdown of the MAP kinase-activated protein kinase 2 (MK2), a kinase currently implicated in p38 stress signaling and G2 arrest. Depletion or inhibition of MK2 also protected cells from DNA damage-induced cell death, and mice deficient for MK2 displayed decreased apoptosis in the skin upon UV irradiation...

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

Directory of Open Access Journals (Sweden)

Qing Li

2013-09-01

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

Dioxin-like (PCB 126) and non dioxin-like (PCB 153) action on DNA damage and apoptosis in granulosa cells. Preliminary data

Energy Technology Data Exchange (ETDEWEB)

Gregoraszczuk, E.L.; Wojtowicz, A. [Lab. of Physiology and Toxicology of Reproduction, Inst. of Zoology, Jagiellonian Univ., Krakow (Poland); Kapiszewska, M.; Magnowska, Z. [Dept. of General Biochemistry, Jagiellonian Univ., Krakow (Poland)

2004-09-15

Introduction Approximately 60-90% of all cancer cases are now generally believed to be due to environmental factors, to which humans are exposed by taking food, and water or inhaling air. These chemicals, including polycyclic aromatic hydrocarbons (PAHs), can be subdivided into different classes. TCDD was classified as group (documented carcinogen in humans) Benzo [a] pyrene and benzo [a] anthracene were included into group 2A (probably carcinogen in humans). PCB is not mentioned in this list. The International Agency for Research on Cancer (IARC) maintains a register of human carcinogens and suspected human carcinogens. The capacity of PCBs to induce DNA strand breaks has been studied to a lesser degree and is quite controversial. DNA strand breaks are the potentially mutagenic lesions that have been proposed as a genotoxicity biomarker for the biomonitoring of environmental pollutants. The mutations induced by these lesions in pollutant-exposed populations are believed to be induced by chemical carcinogens. The formation of PCB adducts has been demonstrated and measured in vitro and in vivo 3. The capacity of PCBs to form DNA adducts depends on their metabolic rate, which is determined by the degree of chlorination. The aim of the presented preliminary data was to evaluate the follicular stage-specific action of PCB 126 and PCB 153 on DNA damage and apoptosis in granulosa cells.

Patulin causes DNA damage leading to cell cycle arrest and apoptosis through modulation of Bax, p53 and p21/WAF1 proteins in skin of mice

International Nuclear Information System (INIS)

Saxena, Neha; Ansari, Kausar M.; Kumar, Rahul; Dhawan, Alok; Dwivedi, Premendra D.; Das, Mukul

2009-01-01

Patulin (PAT), a mycotoxin found in apples, grapes, oranges, pear and peaches, is a potent genotoxic compound. WHO has highlighted the need for the study of cutaneous toxicity of PAT as manual labour is employed during pre and post harvest stages, thereby causing direct exposure to skin. In the present study cutaneous toxicity of PAT was evaluated following topical application to Swiss Albino mice. Dermal exposure of PAT, to mice for 4 h resulted in a dose (40-160 μg/animal) and time (up to 6 h) dependent enhancement of ornithine decarboxylase (ODC), a marker enzyme of cell proliferation. The ODC activity was found to be normal after 12 and 24 h treatment of patulin. Topical application of PAT (160 μg/100 μl acetone) for 24-72 h caused (a) DNA damage in skin cells showing significant increase (34-63%) in olive tail moment, a parameter of Comet assay (b) significant G 1 and S-phase arrest along with induction of apoptosis (2.8-10 folds) as shown by annexin V and PI staining assay through flow cytometer. Moreover PAT leads to over expression of p 21/WAF1 (3.6-3.9 fold), pro apoptotic protein Bax (1.3-2.6) and tumor suppressor wild type p 53 (2.8-3.9 fold) protein. It was also shown that PAT induced apoptosis was mediated through mitochondrial intrinsic pathway as revealed through the release of cytochrome C protein in cytosol leading to enhancement of caspase-3 activity in skin cells of mice. These results suggest that PAT has a potential to induce DNA damage leading to p 53 mediated cell cycle arrest along with intrinsic pathway mediated apoptosis that may also be correlated with enhanced polyamine production as evident by induction of ODC activity, which may have dermal toxicological implications

DNA damage mediated transcription arrest: Step back to go forward.

Science.gov (United States)

Mullenders, Leon

2015-12-01

The disturbance of DNA helix conformation by bulky DNA damage poses hindrance to transcription elongating due to stalling of RNA polymerase at transcription blocking lesions. Stalling of RNA polymerase provokes the formation of R-loops, i.e. the formation of a DNA-RNA hybrid and a displaced single stranded DNA strand as well as displacement of spliceosomes. R-loops are processed into DNA single and double strand breaks by NER factors depending on TC-NER factors leading to genome instability. Moreover, stalling of RNA polymerase induces a strong signal for cell cycle arrest and apoptosis. These toxic and mutagenic effects are counteracted by a rapid recruitment of DNA repair proteins to perform transcription coupled nucleotide excision repair (TC-NER) to remove the blocking DNA lesions and to restore transcription. Recent studies have highlighted the role of backtracking of RNA polymerase to facilitate TC-NER and identified novel factors that play key roles in TC-NER and in restoration of transcription. On the molecular level these factors facilitate stability of the repair complex by promotion and regulation of various post-translational modifications of NER factors and chromatin substrate. In addition, the continuous flow of new factors that emerge from screening assays hints to several regulatory levels to safeguard the integrity of transcription elongation after disturbance by DNA damage that have yet to be explored. Copyright © 2015 Elsevier B.V. All rights reserved.

The cyclin-dependent kinase inhibitor 5, 6-dichloro-1-beta-D-ribofuranosylbenzimidazole induces nongenotoxic, DNA replication-independent apoptosis of normal and leukemic cells, regardless of their p53 status

International Nuclear Information System (INIS)

Turinetto, Valentina; Porcedda, Paola; Orlando, Luca; De Marchi, Mario; Amoroso, Antonio; Giachino, Claudia

2009-01-01

Current chemotherapy of human cancers focuses on the DNA damage pathway to induce a p53-mediated cellular response leading to either G1 arrest or apoptosis. However, genotoxic treatments may induce mutations and translocations that result in secondary malignancies or recurrent disease. In addition, about 50% of human cancers are associated with mutations in the p53 gene. Nongenotoxic activation of apoptosis by targeting specific molecular pathways thus provides an attractive therapeutic approach. Normal and leukemic cells were evaluated for their sensitivity to 5, 6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) through cell viability and caspase activation tests. The apoptotic pathway induced by DRB was analysed by immunfluorescence and immunoblot analysis. H2AX phosphorylation and cell cycle analysis were performed to study the dependance of apoptosis on DNA damage and DNA replication, respectively. To investigate the role of p53 in DRB-induced apoptosis, specific p53 inhibitors were used. Statistical analysis on cell survival was performed with the test of independence. Here we report that DRB, an inhibitor of the transcriptional cyclin-dependent kinases (CDKs) 7 and 9, triggers DNA replication-independent apoptosis in normal and leukemic human cells regardless of their p53 status and without inducing DNA damage. Our data indicate that (i) in p53-competent cells, apoptosis induced by DRB relies on a cytosolic accumulation of p53 and subsequent Bax activation, (ii) in the absence of p53, it may rely on p73, and (iii) it is independent of ATM and NBS1 proteins. Notably, even apoptosis-resistant leukemic cells such as Raji were sensitive to DRB. Our results indicate that DRB represents a potentially useful cancer chemotherapeutic strategy that employs both the p53-dependent and -independent apoptotic pathways without inducing genotoxic stress, thereby decreasing the risk of secondary malignancies

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

Directory of Open Access Journals (Sweden)

Razmik Mirzayans

2016-05-01

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

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

Science.gov (United States)

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

2017-08-01

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

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

Science.gov (United States)

Fox, Candace R; Parks, Griffith D

2018-04-01

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

Polychlorinated biphenyl quinone induces oxidative DNA damage and repair responses: The activations of NHEJ, BER and NER via ATM-p53 signaling axis

Energy Technology Data Exchange (ETDEWEB)

Dong, Hui; Shi, Qiong; Song, Xiufang; Fu, Juanli; Hu, Lihua; Xu, Demei; Su, Chuanyang; Xia, Xiaomin; Song, Erqun; Song, Yang, E-mail: songyangwenrong@hotmail.com

2015-07-01

Our previous studies demonstrated that polychlorinated biphenyl (PCB) quinone induced oxidative DNA damage in HepG2 cells. To promote genomic integrity, DNA damage response (DDR) coordinates cell-cycle transitions, DNA repair and apoptosis. PCB quinone-induced cell cycle arrest and apoptosis have been documented, however, whether PCB quinone insult induce DNA repair signaling is still unknown. In this study, we identified the activation of DDR and corresponding signaling events in HepG2 cells upon the exposure to a synthetic PCB quinone, PCB29-pQ. Our data illustrated that PCB29-pQ induces the phosphorylation of p53, which was mediated by ataxia telangiectasia mutated (ATM) protein kinase. The observed phosphorylated histone H2AX (γ-H2AX) foci and the elevation of 8-hydroxy-2′-deoxyguanosine (8-OHdG) indicated that DDR was stimulated by PCB29-pQ treatment. Additionally, we found PCB29-pQ activates non-homologous end joining (NHEJ), base excision repair (BER) and nucleotide excision repair (NER) signalings. However, these repair pathways are not error-free processes and aberrant repair of DNA damage may cause the potential risk of carcinogenesis and mutagenesis. - Highlights: • Polychlorinated biphenyl quinone induces oxidative DNA damage in HepG2 cells. • The elevation of γ-H2AX and 8-OHdG indicates the activation of DNA damage response. • ATM-p53 signaling acts as the DNA damage sensor and effector. • Polychlorinated biphenyl quinone activates NHEJ, BER and NER signalings.

Membranes as sensitive targets in thymocyte apoptosis

International Nuclear Information System (INIS)

Ramakrishnan, N.; McClain, D.E.; Catravas, G.N.

1993-01-01

The role of cellular membranes in thymocyte apoptosis has been examined. Trolox, a water soluble analogue of vitamin E and inhibitor of membrane damage, inhibits DNA fragmentation in thymocytes exposed to γ-radiation, and is most effective in inhibiting DNA fragmentation when added to cells within 30 min post-irradiation. Exposure to trolox only during irradiation did not prevent DNA fragmentation. Incubation of the irradiated cell suspension with trolox for 2h post-irradiation was sufficient to prevent DNA fragmentation measured at 24 h in irradiated cells, suggesting that trolox irreversibly inhibits a cellular lesion required for apoptosis. The induction of DNA fragmentation appears to be related to a concurrent, pronounced flow of Ca 2+ into the cell. At 3 h post-irradiation the amount of Ca 2+ in irradiated thymocytes was more than twice that of unirradiated thymocytes. Trolox treatment completely blocked the radiation-induced influx of Ca 2+ into the thymocytes. These results suggest that membrane damage is a critical lesion involved in DNA fragmentation in thymocyte apoptosis. (author)

Developmental stage- and DNA damage-specific functions of C. elegans FANCD2

International Nuclear Information System (INIS)

Lee, Kyong Yun; Yang, Insil; Park, Jung-Eun; Baek, Ok-Ryun; Chung, Kee Yang; Koo, Hyeon-Sook

2007-01-01

In this study, we set out to investigate the role of Fanconi anemia complementation group D2 protein (FANCD2) in developmental stage-specific DNA damage responses in Caenorhabditis elegans. A mutant C. elegans strain containing a deletion in the gene encoding the FANCD2 homolog, FCD-2, exhibited egg-laying defects, precocious oogenesis, and partial defects in fertilization. The mutant strain also had a lower hatching rate than the wild-type after γ-irradiation of embryos, but not after the irradiation of pachytene stage germ cells. This mutation sensitized pachytene stage germ cells to the genotoxic effects of photoactivated psoralen, as seen by a greatly reduced hatching rate and increased chromosomal aberrations. This mutation also enhanced physiological M-phase arrest and apoptosis. Taken together, our data reveal that the C. elegans FANCD2 homolog participates in the repair of spontaneous DNA damage and DNA crosslinks, not only in proliferating cells but also in pachytene stage cells, and it may have an additional role in double-stranded DNA break repair during embryogenesis

DNA damage by Auger emitters

International Nuclear Information System (INIS)

Martin, R.F.; d'Cunha, Glenn; Gibbs, Richard; Murray, Vincent; Pardee, Marshall; Allen, B.J.

1988-01-01

125 I atoms can be introduced at specific locations along a defined DNA target molecule, either by site-directed incorporation of an 125 I-labelled deoxynucleotide or by binding of an 125 I-labelled sequence-selective DNA ligand. After allowing accumulation of 125 I decay-induced damage to the DNA, application of DNA sequencing techniques enables positions of strand breaks to be located relative to the site of decay, at a resolution corresponding to the distance between adjacent nucleotides [0.34 nm]. Thus, DNA provides a molecular framework to analyse the extent of damage following [averaged] individual decay events. Results can be compared with energy deposition data generated by computer-simulation methods developed by Charlton et al. The DNA sequencing technique also provides information about the chemical nature of the termini of the DNA chains produced following Auger decay-induced damage. In addition to reviewing the application of this approach to the analysis of 125 I decay induced DNA damage, some more recent results obtained by using 67 Ga are also presented. (author)

DNA Damage, Repair, and Cancer Metabolism

Science.gov (United States)

Turgeon, Marc-Olivier; Perry, Nicholas J. S.; Poulogiannis, George

2018-01-01

Although there has been a renewed interest in the field of cancer metabolism in the last decade, the link between metabolism and DNA damage/DNA repair in cancer has yet to be appreciably explored. In this review, we examine the evidence connecting DNA damage and repair mechanisms with cell metabolism through three principal links. (1) Regulation of methyl- and acetyl-group donors through different metabolic pathways can impact DNA folding and remodeling, an essential part of accurate double strand break repair. (2) Glutamine, aspartate, and other nutrients are essential for de novo nucleotide synthesis, which dictates the availability of the nucleotide pool, and thereby influences DNA repair and replication. (3) Reactive oxygen species, which can increase oxidative DNA damage and hence the load of the DNA-repair machinery, are regulated through different metabolic pathways. Interestingly, while metabolism affects DNA repair, DNA damage can also induce metabolic rewiring. Activation of the DNA damage response (DDR) triggers an increase in nucleotide synthesis and anabolic glucose metabolism, while also reducing glutamine anaplerosis. Furthermore, mutations in genes involved in the DDR and DNA repair also lead to metabolic rewiring. Links between cancer metabolism and DNA damage/DNA repair are increasingly apparent, yielding opportunities to investigate the mechanistic basis behind potential metabolic vulnerabilities of a substantial fraction of tumors. PMID:29459886

Comparative investigations of sodium arsenite, arsenic trioxide and cadmium sulphate in combination with gamma-radiation on apoptosis, micronuclei induction and DNA damage in a human lymphoblastoid cell line

International Nuclear Information System (INIS)

Hornhardt, Sabine; Gomolka, Maria; Walsh, Linda; Jung, Thomas

2006-01-01

In the field of radiation protection the combined exposure to radiation and other toxic agents is recognised as an important research area. To elucidate the basic mechanisms of simultaneous exposure, the interaction of the carcinogens and environmental toxicants cadmium and two arsenic compounds, arsenite and arsenic trioxide, in combination with gamma-radiation in human lymphoblastoid cells (TK6) were investigated. Gamma-radiation induced significant genotoxic effects such as micronuclei formation, DNA damage and apoptosis, whereas arsenic and cadmium had no significant effect on these indicators of cellular damage at non-toxic concentrations. However, in combination with gamma-radiation arsenic trioxide induced a more than additive apoptotic rate compared to the sum of the single effects. Here, the level of apoptotic cells was increased, in a dose-dependent way, up to two-fold compared to the irradiated control cells. Arsenite did not induce a significant additive effect at any of the concentrations or radiation doses tested. On the other hand, arsenic trioxide was less effective than arsenite in the induction of DNA protein cross-links. These data indicate that the two arsenic compounds interact through different pathways in the cell. Cadmium sulphate, like arsenite, had no significant effect on apoptosis in combination with gamma-radiation at low concentrations and, at high concentrations, even reduced the radiation-induced apoptosis. An additive effect on micronuclei induction was observed with 1 μM cadmium sulphate with an increase of up to 80% compared to the irradiated control cells. Toxic concentrations of cadmium and arsenic trioxide seemed to reduce micronuclei induction. The results presented here indicate that relatively low concentrations of arsenic and cadmium, close to those occurring in nature, may interfere with radiation effects. Differences in action of the two arsenic compounds were identified

Radiation-induced DNA damage as a function of DNA hydration

International Nuclear Information System (INIS)

Swarts, S.G.; Miao, L.; Wheeler, K.T.; Sevilla, M.D.; Becker, D.

1995-01-01

Radiation-induced DNA damage is produced from the sum of the radicals generated by the direct ionization of the DNA (direct effect) and by the reactions of the DNA with free radicals formed in the surrounding environment (indirect effect). The indirect effect has been believed to be the predominant contributor to radiation-induced intracellular DNA damage, mainly as the result of reactions of bulk water radicals (e.g., OH·) with DNA. However, recent evidence suggests that DNA damage, derived from the irradiation of water molecules that are tightly bound in the hydration layer, may occur as the result of the transfer of electron-loss centers (e.g. holes) and electrons from these water molecules to the DNA. Since this mechanism for damaging DNA more closely parallels that of the direct effect, the irradiation of these tightly bound water molecules may contribute to a quasi-direct effect. These water molecules comprise a large fraction of the water surrounding intracellular DNA and could account for a significant proportion of intracellular radiation-induced DNA damage. Consequently, the authors have attempted to characterize this quasi-direct effect to determine: (1) the extent of the DNA hydration layer that is involved with this effect, and (2) what influence this effect has on the types and quantities of radiation-induced DNA damage

Chk2 mediates RITA-induced apoptosis.

Science.gov (United States)

de Lange, J; Verlaan-de Vries, M; Teunisse, A F A S; Jochemsen, A G

2012-06-01

Reactivation of the p53 tumor-suppressor protein by small molecules like Nutlin-3 and RITA (reactivation of p53 and induction of tumor cell apoptosis) is a promising strategy for cancer therapy. The molecular mechanisms involved in the responses to RITA remain enigmatic. Several groups reported the induction of a p53-dependent DNA damage response. Furthermore, the existence of a p53-dependent S-phase checkpoint has been suggested, involving the checkpoint kinase Chk1. We have recently shown synergistic induction of apoptosis by RITA in combination with Nutlin-3, and we observed concomitant Chk2 phosphorylation. Therefore, we investigated whether Chk2 contributes to the cellular responses to RITA. Strikingly, the induction of apoptosis seemed entirely Chk2 dependent. Transcriptional activity of p53 in response to RITA required the presence of Chk2. A partial rescue of apoptosis observed in Noxa knockdown cells emphasized the relevance of p53 transcriptional activity for RITA-induced apoptosis. In addition, we observed an early p53- and Chk2-dependent block of DNA replication upon RITA treatment. Replicating cells seemed more prone to entering RITA-induced apoptosis. Furthermore, the RITA-induced DNA damage response, which was not a secondary effect of apoptosis induction, was strongly attenuated in cells lacking p53 or Chk2. In conclusion, we identified Chk2 as an essential mediator of the cellular responses to RITA.

ATM-deficient human fibroblast cells are resistant to low levels of DNA double-strand break induced apoptosis and subsequently undergo drug-induced premature senescence

International Nuclear Information System (INIS)

Park, Jun; Jo, Yong Hwa; Cho, Chang Hoon; Choe, Wonchae; Kang, Insug; Baik, Hyung Hwan; Yoon, Kyung-Sik

2013-01-01

Highlights: ► A-T cells were not hypersensitive to low levels of DNA DSBs. ► A-T cells have enhanced Akt but defect in activation of p53 and apoptotic proteins. ► A-T cells underwent premature senescence after DNA damage accumulated. ► Chemotherapeutic effect in cancer therapy may be associated with premature senescence. -- Abstract: DNA DSBs are induced by IR or radiomimetic drugs such as doxorubicin. It has been indicated that cells from ataxia-telangiectasia patients are highly sensitive to radiation due to defects in DNA repair, but whether they have impairment in apoptosis has not been fully elucidated. A-T cells showed increased sensitivity to high levels of DNA damage, however, they were more resistant to low doses. Normal cells treated with combination of KU55933, a specific ATM kinase inhibitor, and doxorubicin showed increased resistance as they do in a similar manner to A-T cells. A-T cells have higher viability but more DNA breaks, in addition, the activations of p53 and apoptotic proteins (Bax and caspase-3) were deficient, but Akt expression was enhanced. A-T cells subsequently underwent premature senescence after treatment with a low dose of doxorubicin, which was confirmed by G2 accumulation, senescent morphology, and SA-β-gal positive until 15 days repair incubation. Finally, A-T cells are radio-resistant at low doses due to its defectiveness in detecting DNA damage and apoptosis, but the accumulation of DNA damage leads cells to premature senescence.

DNA Damage, Mutagenesis and Cancer

Directory of Open Access Journals (Sweden)

Ashis K. Basu

2018-03-01

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

DNA damage in plant herbarium tissue.

Science.gov (United States)

Staats, Martijn; Cuenca, Argelia; Richardson, James E; Vrielink-van Ginkel, Ria; Petersen, Gitte; Seberg, Ole; Bakker, Freek T

2011-01-01

Dried plant herbarium specimens are potentially a valuable source of DNA. Efforts to obtain genetic information from this source are often hindered by an inability to obtain amplifiable DNA as herbarium DNA is typically highly degraded. DNA post-mortem damage may not only reduce the number of amplifiable template molecules, but may also lead to the generation of erroneous sequence information. A qualitative and quantitative assessment of DNA post-mortem damage is essential to determine the accuracy of molecular data from herbarium specimens. In this study we present an assessment of DNA damage as miscoding lesions in herbarium specimens using 454-sequencing of amplicons derived from plastid, mitochondrial, and nuclear DNA. In addition, we assess DNA degradation as a result of strand breaks and other types of polymerase non-bypassable damage by quantitative real-time PCR. Comparing four pairs of fresh and herbarium specimens of the same individuals we quantitatively assess post-mortem DNA damage, directly after specimen preparation, as well as after long-term herbarium storage. After specimen preparation we estimate the proportion of gene copy numbers of plastid, mitochondrial, and nuclear DNA to be 2.4-3.8% of fresh control DNA and 1.0-1.3% after long-term herbarium storage, indicating that nearly all DNA damage occurs on specimen preparation. In addition, there is no evidence of preferential degradation of organelle versus nuclear genomes. Increased levels of C→T/G→A transitions were observed in old herbarium plastid DNA, representing 21.8% of observed miscoding lesions. We interpret this type of post-mortem DNA damage-derived modification to have arisen from the hydrolytic deamination of cytosine during long-term herbarium storage. Our results suggest that reliable sequence data can be obtained from herbarium specimens.

Nur77 forms novel nuclear structures upon DNA damage that cause transcriptional arrest

International Nuclear Information System (INIS)

Leseleuc, Louis de; Denis, Francois

2006-01-01

The orphan nuclear receptor Nur77 has been implicated in both growth and apoptosis, and its function and activity can be modulated by cellular redistribution. Green fluorescent protein-tagged Nur77 was used to evaluate the role of Nur77 intracellular redistribution in response to genotoxic stress. Selected DNA damaging agents and transcription inhibition lead to rapid redistribution of Nur77 into nuclear structures distinct from conventional nuclear bodies. These nuclear bodies formed transiently were tightly bound to the nuclear matrix and conditions that lead to their appearance were associated with Nur77 transcriptional inhibition. The formation of Nur77 nuclear bodies might be involved in programmed cell death modulation upon exposure to DNA damaging agents that inhibit transcription by sequestrating this proapoptotic factor in dense nuclear structures

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

Energy Technology Data Exchange (ETDEWEB)

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

2016-02-01

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

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

International Nuclear Information System (INIS)

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

2016-01-01

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

Aberrant DNA damage response pathways may predict the outcome of platinum chemotherapy in ovarian cancer.

Directory of Open Access Journals (Sweden)

Dimitra T Stefanou

Full Text Available Ovarian carcinoma (OC is the most lethal gynecological malignancy. Despite the advances in the treatment of OC with combinatorial regimens, including surgery and platinum-based chemotherapy, patients generally exhibit poor prognosis due to high chemotherapy resistance. Herein, we tested the hypothesis that DNA damage response (DDR pathways are involved in resistance of OC patients to platinum chemotherapy. Selected DDR signals were evaluated in two human ovarian carcinoma cell lines, one sensitive (A2780 and one resistant (A2780/C30 to platinum treatment as well as in peripheral blood mononuclear cells (PBMCs from OC patients, sensitive (n = 7 or resistant (n = 4 to subsequent chemotherapy. PBMCs from healthy volunteers (n = 9 were studied in parallel. DNA damage was evaluated by immunofluorescence γH2AX staining and comet assay. Higher levels of intrinsic DNA damage were found in A2780 than in A2780/C30 cells. Moreover, the intrinsic DNA damage levels were significantly higher in OC patients relative to healthy volunteers, as well as in platinum-sensitive patients relative to platinum-resistant ones (all P<0.05. Following carboplatin treatment, A2780 cells showed lower DNA repair efficiency than A2780/C30 cells. Also, following carboplatin treatment of PBMCs ex vivo, the DNA repair efficiency was significantly higher in healthy volunteers than in platinum-resistant patients and lowest in platinum-sensitive ones (t1/2 for loss of γH2AX foci: 2.7±0.5h, 8.8±1.9h and 15.4±3.2h, respectively; using comet assay, t1/2 of platinum-induced damage repair: 4.8±1.4h, 12.9±1.9h and 21.4±2.6h, respectively; all P<0.03. Additionally, the carboplatin-induced apoptosis rate was higher in A2780 than in A2780/C30 cells. In PBMCs, apoptosis rates were inversely correlated with DNA repair efficiencies of these cells, being significantly higher in platinum-sensitive than in platinum-resistant patients and lowest in healthy volunteers (all P<0.05. We conclude

DNA damage and repair in plants

International Nuclear Information System (INIS)

Britt, A.B.

1996-01-01

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

Immunoassay of DNA damage

International Nuclear Information System (INIS)

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

1988-01-01

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

Immunoassay of DNA damage

Energy Technology Data Exchange (ETDEWEB)

Gasparro, F P; Santella, R M

1988-09-01

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

Mechanisms of mutagenesis: DNA replication in the presence of DNA damage.

Science.gov (United States)

Liu, Binyan; Xue, Qizhen; Tang, Yong; Cao, Jia; Guengerich, F Peter; Zhang, Huidong

2016-01-01

Environmental mutagens cause DNA damage that disturbs replication and produces mutations, leading to cancer and other diseases. We discuss mechanisms of mutagenesis resulting from DNA damage, from the level of DNA replication by a single polymerase to the complex DNA replisome of some typical model organisms (including bacteriophage T7, T4, Sulfolobus solfataricus, Escherichia coli, yeast and human). For a single DNA polymerase, DNA damage can affect replication in three major ways: reducing replication fidelity, causing frameshift mutations, and blocking replication. For the DNA replisome, protein interactions and the functions of accessory proteins can yield rather different results even with a single DNA polymerase. The mechanism of mutation during replication performed by the DNA replisome is a long-standing question. Using new methods and techniques, the replisomes of certain organisms and human cell extracts can now be investigated with regard to the bypass of DNA damage. In this review, we consider the molecular mechanism of mutagenesis resulting from DNA damage in replication at the levels of single DNA polymerases and complex DNA replisomes, including translesion DNA synthesis. Copyright © 2016 Elsevier B.V. All rights reserved.

Protection of vanillin derivative VND3207 on genome damage and apoptosis of human lymphoblastoid cells induced by γ-ray irradiation

International Nuclear Information System (INIS)

Huang Rui; Huang Bo; He Xingpeng; Xu Qinzhi; Wang Yu; Zhou Pingkun

2009-01-01

To determine the protective effect of vanillin derivative VND3207 on the genome damage and apoptosis of human lymphoblastoid AHH-1 cells induced by γ-ray irradiation, the techniques of single-cell gel electrophoresis, micronucleus test, Annexin V-FACS assay, and the double-fluorescein staining and fluorescent microscope observation were used. Neutral single-cell gel electrophoresis showed that the initial DNA double-strand breaks caused by 2 Gy 60 Co γ-ray was significantly decreased by VND3207 in the range of 540 μmol/L. This significant phenomenonwas demonstrated by the fact that the comet tail-moment was significantly shortened and the DNA content in the comet tail was reduced when the cells were protected with VND3207, and the radio-protective effect increases along with the increasing of drug concentration. Similarly, the yield of micronucleus was reduced by 540 μmol/L of VND3207 in a concentration-dependency in AHH-1 cells irradiated with 0.5 Gy, 1.0 Gy and 2.0 Gy 60 Co γ-rays. 40 μmol/L VND3207 resulted in 40% reduction in the yield of micronucleus induced by 2.0 Gy. The occurrence of apoptosis enhanced along with the time from 8 h to 48 h post 4 Gy irradiation, and 40 μmol/L of VND3207 significantly decreased the induction of apoptosis. This work has further demonstrated a good protection of VND3207 on γ-ray-induced cell genome damage and apoptosis. (authors)

Soluble histone H2AX is induced by DNA replication stress and sensitizes cells to undergo apoptosis

Directory of Open Access Journals (Sweden)

Duensing Stefan

2008-07-01

Full Text Available Abstract Background Chromatin-associated histone H2AX is a key regulator of the cellular responses to DNA damage. However, non-nucleosomal functions of histone H2AX are poorly characterized. We have recently shown that soluble H2AX can trigger apoptosis but the mechanisms leading to non-chromatin-associated H2AX are unclear. Here, we tested whether stalling of DNA replication, a common event in cancer cells and the underlying mechanism of various chemotherapeutic agents, can trigger increased soluble H2AX. Results Transient overexpression of H2AX was found to lead to a detectable fraction of soluble H2AX and was associated with increased apoptosis. This effect was enhanced by the induction of DNA replication stress using the DNA polymerase α inhibitor aphidicolin. Cells manipulated to stably express H2AX did not contain soluble H2AX, however, short-term treatment with aphidicolin (1 h resulted in detectable amounts of H2AX in the soluble nuclear fraction and enhanced apoptosis. Similarly, soluble endogenous H2AX was detected under these conditions. We found that excessive soluble H2AX causes chromatin aggregation and inhibition of ongoing gene transcription as evidenced by the redistribution and/or loss of active RNA polymerase II as well as the transcriptional co-activators CBP and p300. Conclusion Taken together, these results show that DNA replication stress rapidly leads to increased soluble H2AX and that non-chromatin-associated H2AX can sensitize cells to undergo apoptosis. Our findings encourage further studies to explore H2AX and the cellular pathways that control its expression as anti-cancer drug targets.

Nitrosative stress induces DNA strand breaks but not caspase mediated apoptosis in a lung cancer cell line

Directory of Open Access Journals (Sweden)

Bentz Brandon G

2004-12-01

Full Text Available Abstract Background Key steps crucial to the process of tumor progression are genomic instability and escape from apoptosis. Nitric oxide and its interrelated reactive intermediates (collectively denoted as NOX have been implicated in DNA damage and mutational events leading to cancer development, while also being implicated in the inhibition of apoptosis through S-nitrosation of key apoptotic enzymes. The purpose of this study was to explore the interrelationship between NOX-mediated DNA strand breaks (DSBs and apoptosis in cultured tumor cell lines. Methods Two well-characterized cell lines were exposed to increasing concentrations of exogenous NOX via donor compounds. Production of NOX was quantified by the Greiss reaction and spectrophotometery, and confirmed by nitrotyrosine immunostaining. DSBs were measured by the alkaline single-cell gel electrophoresis assay (the COMET assay, and correlated with cell viability by the MTT assay. Apoptosis was analyzed both by TUNEL staining and Annexin V/propidium iodine FACS. Finally, caspase enzymatic activity was measured using an in-vitro fluorogenic caspase assay. Results Increases in DNA strand breaks in our tumor cells, but not in control fibroblasts, correlated with the concentration as well as rate of release of exogenously administered NOX. This increase in DSBs did not correlate with an increase in cell death or apoptosis in our tumor cell line. Finally, this lack of apoptosis was found to correlate with inhibition of caspase activity upon exposure to thiol- but not NONOate-based NOX donor compounds. Conclusions Genotoxicity appears to be highly interrelated with both the concentration and kinetic delivery of NOX. Moreover, alterations in cell apoptosis can be seen as a consequence of the explicit mechanisms of NOX delivery. These findings lend credence to the hypothesis that NOX may play an important role in tumor progression, and underscores potential pitfalls which should be considered when

Modulation of the DNA repair system and ATR-p53 mediated apoptosis is relevant for tributyltin-induced genotoxic effects in human hepatoma G2 cells.

Science.gov (United States)

Li, Bowen; Sun, Lingbin; Cai, Jiali; Wang, Chonggang; Wang, Mengmeng; Qiu, Huiling; Zuo, Zhenghong

2015-01-01

The toxic effects of tributyltin (TBT) have been extensively documented in several types of cells, but the molecular mechanisms related to the genotoxic effects of TBT have still not been fully elucidated. Our study showed that exposure of human hepatoma G2 cells to 1-4 μmol/L TBT for 3 hr caused severe DNA damage in a concentration-dependent manner. Moreover, the expression levels of key DNA damage sensor genes such as the replication factor C, proliferating cell nuclear antigen and poly (ADP-ribose) polymerase-1 were inhabited in a concentration-dependent manner. We further demonstrated that TBT induced cell apoptosis via the p53-mediated pathway, which was most likely activated by the ataxia telangiectasia mutated and rad-3 related (ATR) protein kinase. The results also showed that cytochrome c, caspase-3, caspase-8, caspase-9, and the B-cell lymphoma 2 were involved in this process. Taken together, we demonstrated for the first time that the inhibition of the DNA repair system might be more responsible for TBT-induced genotoxic effects in cells. Then the generated DNA damage induced by TBT initiated ATR-p53-mediated apoptosis. Copyright © 2014. Published by Elsevier B.V.

JS-K, a GST-activated nitric oxide generator, induces DNA double-strand breaks, activates DNA damage response pathways, and induces apoptosis in vitro and in vivo in human multiple myeloma cells.

Science.gov (United States)

Kiziltepe, Tanyel; Hideshima, Teru; Ishitsuka, Kenji; Ocio, Enrique M; Raje, Noopur; Catley, Laurence; Li, Chun-Qi; Trudel, Laura J; Yasui, Hiroshi; Vallet, Sonia; Kutok, Jeffery L; Chauhan, Dharminder; Mitsiades, Constantine S; Saavedra, Joseph E; Wogan, Gerald N; Keefer, Larry K; Shami, Paul J; Anderson, Kenneth C

2007-07-15

Here we investigated the cytotoxicity of JS-K, a prodrug designed to release nitric oxide (NO(*)) following reaction with glutathione S-transferases, in multiple myeloma (MM). JS-K showed significant cytotoxicity in both conventional therapy-sensitive and -resistant MM cell lines, as well as patient-derived MM cells. JS-K induced apoptosis in MM cells, which was associated with PARP, caspase-8, and caspase-9 cleavage; increased Fas/CD95 expression; Mcl-1 cleavage; and Bcl-2 phosphorylation, as well as cytochrome c, apoptosis-inducing factor (AIF), and endonuclease G (EndoG) release. Moreover, JS-K overcame the survival advantages conferred by interleukin-6 (IL-6) and insulin-like growth factor 1 (IGF-1), or by adherence of MM cells to bone marrow stromal cells. Mechanistic studies revealed that JS-K-induced cytotoxicity was mediated via NO(*) in MM cells. Furthermore, JS-K induced DNA double-strand breaks (DSBs) and activated DNA damage responses, as evidenced by neutral comet assay, as well as H2AX, Chk2 and p53 phosphorylation. JS-K also activated c-Jun NH(2)-terminal kinase (JNK) in MM cells; conversely, inhibition of JNK markedly decreased JS-K-induced cytotoxicity. Importantly, bortezomib significantly enhanced JS-K-induced cytotoxicity. Finally, JS-K is well tolerated, inhibits tumor growth, and prolongs survival in a human MM xenograft mouse model. Taken together, these data provide the preclinical rationale for the clinical evaluation of JS-K to improve patient outcome in MM.

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

Science.gov (United States)

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

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

Directory of Open Access Journals (Sweden)

Kuroda S

2014-08-01

Full Text Available Shinji Kuroda,1 Justina Tam,2 Jack A Roth,1 Konstantin Sokolov,2 Rajagopal Ramesh3–5 1Department of Thoracic and Cardiovascular Surgery, 2Department of Imaging Physics, The University of Texas MD Anderson Cancer Center, Houston, TX, USA; 3Department of Pathology, 4Graduate Program in Biomedical Sciences, 5Stephenson Cancer Center, University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA Background: We have previously demonstrated the epidermal growth factor receptor (EGFR-targeted hybrid plasmonic magnetic nanoparticles (225-NP produce a therapeutic effect in human lung cancer cell lines in vitro. In the present study, we investigated the molecular mechanism of 225-NP-mediated antitumor activity both in vitro and in vivo using the EGFR-mutant HCC827 cell line. Methods: The growth inhibitory effect of 225-NP on lung tumor cells was determined by cell viability and cell-cycle analysis. Protein expression related to autophagy, apoptosis, and DNA-damage were determined by Western blotting and immunofluorescence. An in vivo efficacy study was conducted using a human lung tumor xenograft mouse model. Results: The 225-NP treatment markedly reduced tumor cell viability at 72 hours compared with the cell viability in control treatment groups. Cell-cycle analysis showed the percentage of cells in the G2/M phase was reduced when treated with 225-NP, with a concomitant increase in the number of cells in Sub-G1 phase, indicative of cell death. Western blotting showed LC3B and PARP cleavage, indicating 225-NP-treatment activated both autophagy- and apoptosis-mediated cell death. The 225-NP strongly induced γH2AX and phosphorylated histone H3, markers indicative of DNA damage and mitosis, respectively. Additionally, significant γH2AX foci formation was observed in 225-NP-treated cells compared with control treatment groups, suggesting 225-NP induced cell death by triggering DNA damage. The 225-NP-mediated DNA damage involved abrogation of the

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

International Nuclear Information System (INIS)

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

2015-01-01

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

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

Energy Technology Data Exchange (ETDEWEB)

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

2015-08-01

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

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

Directory of Open Access Journals (Sweden)

Olga Momcilovic

2010-10-01

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

Flow cytometric assessment of DNA damage in the fish Catla catla (Ham.) exposed to gamma radiation

International Nuclear Information System (INIS)

Anbumani, S.; Mohankumar, Mary N.; Selvanayagam, M.

2012-01-01

Environmental mutagens such as ionizing radiation and chemicals induce DNA damage in a wide variety of organisms. The International Commission on Radiological Protection (lCRP) has recently emphasized the need to protect non-human biota from the potential effects of ionizing radiation. Radiation exposures to non-humans can occur as a result of low-level radioactive discharges into the environment. Molecular genetic effects at low-level radiation exposures are largely unexplored and systematic studies using sensitive biomarkers are required to assess DNA damage in representative non-human species. The objective of the study was to detect DNA damage in the fish Catla catla exposed to gamma radiation using flow cytometry at different time intervals. Increases in the coefficient of variation (CV) of the G 0 /G 1 peak, indicating abnormal DNA distributions were observed in fish exposed to gamma radiation than in controls. Significant increase in the CV was observed from day 12-90 and thereafter decreased. This increase in CV might be due to DNA damage in the cell populations at G 0 /G 1 phase or deletions and duplications caused by improper repair of chromosomes in the cell-cycle machinery. Ionizing radiation induced cell-cycle perturbations and apoptosis were also observed after gamma radiation exposure. (author)

Early-stage apoptosis is associated with DNA-damage-independent ATM phosphorylation and chromatin decondensation in NIH3T3 fibroblasts

DEFF Research Database (Denmark)

Schou, Kenneth Bødtker; Schneider, Linda; Christensen, Søren Tvorup

2008-01-01

Chromatin condensation and degradation of DNA into internucleosomal DNA fragments are key hallmarks of apoptosis. The phosphorylation of protein kinase ataxia telangiectasia mutated (ATM) and histone H2A.X was recently shown to occur concurrently with apoptotic DNA fragmentation. We have used...... necrosis factor-alpha mixed with cycloheximide (TNF-alpha/CHX). In extension to previous findings, ATM phosphorylation was associated with chromatin decondensation, i.e., by loss of dense foci of constitutive heterochromatin. These results suggest that chromatin is decondensed and that ATM is activated...

Radiation damage in DNA

International Nuclear Information System (INIS)

Lafleur, V.

1978-01-01

A number of experiments are described with the purpose to obtain a better insight in the chemical nature and the biological significance of radiation-induced damage in DNA, with some emphasis on the significance of alkali-labile sites. It is shown that not only reactions of OH radicals but also of H radicals introduce breaks and other inactivating damage in single-standed phiX174 DNA. It is found that phosphate buffer is very suitable for the study of the reactions of H radicals with DNA, as the H 2 PO 4 - ions convert the hydrated electrons into H radicals. The hydrated electron, which does react with DNA, does not cause a detectable inactivation. (Auth.)

The DNA damage response in mammalian oocytes

Directory of Open Access Journals (Sweden)

John eCarroll

2013-06-01

Full Text Available DNA damage is one of the most common insults that challenge all cells. To cope, an elaborate molecular and cellular response has evolved to sense, respond to and correct the damage. This allows the maintenance of DNA fidelity essential for normal cell viability and the prevention of genomic instability that can lead to tumour formation. In the context of oocytes, the impact of DNA damage is not one of tumour formation but of the maintenance of fertility. Mammalian oocytes are particularly vulnerable to DNA damage because physiologically they may lie dormant in the ovary for many years (>40 in humans until they receive the stimulus to grow and acquire the competence to become fertilized. The implication of this is that in some organisms, such as humans, oocytes face the danger of cumulative genetic damage for decades. Thus, the ability to detect and repair DNA damage is essential to maintain the supply of oocytes necessary for reproduction. Therefore, failure to confront DNA damage in oocytes could cause serious anomalies in the embryo that may be propagated in the form of mutations to the next generation allowing the appearance of hereditary disease. Despite the potential impact of DNA damage on reproductive capacity and genetic fidelity of embryos, the mechanisms available to the oocyte for monitoring and repairing such insults have remained largely unexplored until recently. Here, we review the different aspects of the response to DNA damage in mammalian oocytes. Specifically, we address the oocyte DNA damage response from embryonic life to adulthood and throughout oocyte development.

Oxidative DNA damage & repair: An introduction.

Science.gov (United States)

Cadet, Jean; Davies, Kelvin J A

2017-06-01

This introductory article should be viewed as a prologue to the Free Radical Biology & Medicine Special Issue devoted to the important topic of Oxidatively Damaged DNA and its Repair. This special issue is dedicated to Professor Tomas Lindahl, co-winner of the 2015 Nobel Prize in Chemistry for his seminal discoveries in the area repair of oxidatively damaged DNA. In the past several years it has become abundantly clear that DNA oxidation is a major consequence of life in an oxygen-rich environment. Concomitantly, survival in the presence of oxygen, with the constant threat of deleterious DNA mutations and deletions, has largely been made possible through the evolution of a vast array of DNA repair enzymes. The articles in this Oxidatively Damaged DNA & Repair special issue detail the reactions by which intracellular DNA is oxidatively damaged, and the enzymatic reactions and pathways by which living organisms survive such assaults by repair processes. Copyright © 2017 Elsevier Inc. All rights reserved.

Repair kinetics of DNA double-strand breaks and incidence of apoptosis in mouse neural stem/progenitor cells and their differentiated neurons exposed to ionizing radiation.

Science.gov (United States)

Kashiwagi, Hiroki; Shiraishi, Kazunori; Sakaguchi, Kenta; Nakahama, Tomoya; Kodama, Seiji

2018-05-01

Neuronal loss leads to neurodegenerative disorders, including Alzheimer's disease, Parkinson's disease and Huntington's disease. Because of their long lifespans, neurons are assumed to possess highly efficient DNA repair ability and to be able to protect themselves from deleterious DNA damage such as DNA double-strand breaks (DSBs) produced by intrinsic and extrinsic sources. However, it remains largely unknown whether the DSB repair ability of neurons is more efficient compared with that of other cells. Here, we investigated the repair kinetics of X-ray-induced DSBs in mouse neural cells by scoring the number of phosphorylated 53BP1 foci post irradiation. We found that p53-independent apoptosis was induced time dependently during differentiation from neural stem/progenitor cells (NSPCs) into neurons in culture for 48 h. DSB repair in neurons differentiated from NSPCs in culture was faster than that in mouse embryonic fibroblasts (MEFs), possibly due to the higher DNA-dependent protein kinase activity, but it was similar to that in NSPCs. Further, the incidence of p53-dependent apoptosis induced by X-irradiation in neurons was significantly higher than that in NSPCs. This difference in response of X-ray-induced apoptosis between neurons and NSPCs may reflect a difference in the fidelity of non-homologous end joining or a differential sensitivity to DNA damage other than DSBs.

Persistent DNA damage after high dose in vivo gamma exposure of minipig skin.

Directory of Open Access Journals (Sweden)

Emad A Ahmed

Full Text Available Exposure to high doses of ionizing radiation (IR can lead to localized radiation injury of the skin and exposed cells suffer dsDNA breaks that may elicit cell death or stochastic changes. Little is known about the DNA damage response after high-dose exposure of the skin. Here, we investigate the cellular and DNA damage response in acutely irradiated minipig skin.IR-induced DNA damage, repair and cellular survival were studied in 15 cm(2 of minipig skin exposed in vivo to ~50 Co-60 γ rays. Skin biopsies of control and 4 h up to 96 days post exposure were investigated for radiation-induced foci (RIF formation using γ-H2AX, 53BP1, and active ATM-p immunofluorescence. High-dose IR induced massive γ-H2AX phosphorylation and high 53BP1 RIF numbers 4 h, 20 h after IR. As time progressed RIF numbers dropped to a low of 3-fold elevated at all subsequent time points. Replicating basal cells (Ki67+ were reduced 3 days post IR followed by increased proliferation and recovery of epidermal cellularity after 28 days.Acute high dose irradiation of minipig epidermis impaired stem cell replication and induced elevated apoptosis from 3 days onward. DNA repair cleared the high numbers of DBSs in skin cells, while RIFs that persisted in <1% cells marked complex and potentially lethal DNA damage up to several weeks after exposure. An elevated frequency of keratinocytes with persistent RIFs may thus serve as indicator of previous acute radiation exposure, which may be useful in the follow up of nuclear or radiological accident scenarios.

NSC666715 and Its Analogs Inhibit Strand-Displacement Activity of DNA Polymerase β and Potentiate Temozolomide-Induced DNA Damage, Senescence and Apoptosis in Colorectal Cancer Cells.

Directory of Open Access Journals (Sweden)

Aruna S Jaiswal

Full Text Available Recently approved chemotherapeutic agents to treat colorectal cancer (CRC have made some impact; however, there is an urgent need for newer targeted agents and strategies to circumvent CRC growth and metastasis. CRC frequently exhibits natural resistance to chemotherapy and those who do respond initially later acquire drug resistance. A mechanism to potentially sensitize CRC cells is by blocking the DNA polymerase β (Pol-β activity. Temozolomide (TMZ, an alkylating agent, and other DNA-interacting agents exert DNA damage primarily repaired by a Pol-β-directed base excision repair (BER pathway. In previous studies, we used structure-based molecular docking of Pol-β and identified a potent small molecule inhibitor (NSC666715. In the present study, we have determined the mechanism by which NSC666715 and its analogs block Fen1-induced strand-displacement activity of Pol-β-directed LP-BER, cause apurinic/apyrimidinic (AP site accumulation and induce S-phase cell cycle arrest. Induction of S-phase cell cycle arrest leads to senescence and apoptosis of CRC cells through the p53/p21 pathway. Our initial findings also show a 10-fold reduction of the IC50 of TMZ when combined with NSC666715. These results provide a guide for the development of a target-defined strategy for CRC chemotherapy that will be based on the mechanisms of action of NSC666715 and TMZ. This combination strategy can be used as a framework to further reduce the TMZ dosages and resistance in CRC patients.

Nedd4 family interacting protein 1 (Ndfip1) is required for ubiquitination and nuclear trafficking of BRCA1-associated ATM activator 1 (BRAT1) during the DNA damage response.

Science.gov (United States)

Low, Ley-Hian; Chow, Yuh-Lit; Li, Yijia; Goh, Choo-Peng; Putz, Ulrich; Silke, John; Ouchi, Toru; Howitt, Jason; Tan, Seong-Seng

2015-03-13

During injury, cells are vulnerable to apoptosis from a variety of stress conditions including DNA damage causing double-stranded breaks. Without repair, these breaks lead to aberrations in DNA replication and transcription, leading to apoptosis. A major response to DNA damage is provided by the protein kinase ATM (ataxia telangiectasia mutated) that is capable of commanding a plethora of signaling networks for DNA repair, cell cycle arrest, and even apoptosis. A key element in the DNA damage response is the mobilization of activating proteins into the cell nucleus to repair damaged DNA. BRAT1 is one of these proteins, and it functions as an activator of ATM by maintaining its phosphorylated status while also keeping other phosphatases at bay. However, it is unknown how BRAT1 is trafficked into the cell nucleus to maintain ATM phosphorylation. Here we demonstrate that Ndfip1-mediated ubiquitination of BRAT1 leads to BRAT1 trafficking into the cell nucleus. Without Ndfip1, BRAT1 failed to translocate to the nucleus. Under genotoxic stress, cells showed increased expression of both Ndfip1 and phosphorylated ATM. Following brain injury, neurons show increased expression of Ndfip1 and nuclear translocation of BRAT1. These results point to Ndfip1 as a sensor protein during cell injury and Ndfip1 up-regulation as a cue for BRAT1 ubiquitination by Nedd4 E3 ligases, followed by nuclear translocation of BRAT1. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

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

Science.gov (United States)

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

Inhibition of the epidermal growth factor receptor in bladder cancer cells treated with the DNA-damaging drug etoposide markedly increases apoptosis

DEFF Research Database (Denmark)

Munk, Mathias; Memon, Ashfaque Ahmed; Nexo, Ebba

2007-01-01

: The bladder cancer cell lines RT4 and T24, representing low- and high-malignancy grades respectively, were treated with VP16 (10 or 50 microM) and the level of apoptosis determined using a commercial kit. EGFR receptor activity was determined by western blotting using antibodies against phosphorylated EGFR....... The EGFR was either activated by heparin-binding (HB)-EGF (1 nM) or inhibited with the specific EGFR inhibitor gefitinib (1 or 5 microM). The pan-caspase inhibitor Z-VAD (30 microM) was used to test the involvement of caspase activity. RESULTS: Treatment of T24 bladder cancer cells with VP16 (50 micro...... suggest that activation of the EGFR induced a cell-survival function when bladder cancer cells were treated with the DNA-damaging drug VP16, and that combined treatment with VP16 and the EGFR inhibitor gefitinib might improve the efficacy of treatment. Udgivelsesdato: 2007-Jan...

The DNA damage response during mitosis

NARCIS (Netherlands)

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

2013-01-01

Cells are equipped with a cell-intrinsic signaling network called the DNA damage response (DDR). This signaling network recognizes DNA lesions and initiates various downstream pathways to coordinate a cell cycle arrest with the repair of the damaged DNA. Alternatively, the DDR can mediate clearance

Early models of DNA damage formation

International Nuclear Information System (INIS)

Śmiałek, Małgorzata A

2012-01-01

Quantification of DNA damage, induced by various types of incident radiation as well as chemical agents, has been the subject of many theoretical and experimental studies, supporting the development of modern cancer therapy. The primary observations showed that many factors can lead to damage of DNA molecules. It became clear that the development of experimental techniques for exploring this phenomenon is required. Another problem was simultaneously dealt with, anticipating on how the damage is distributed within the double helix of the DNA molecule and how the single strand break formation and accumulation can influence the lethal double strand break formation. In this work the most important probabilistic models for DNA strand breakage and damage propagation are summarized and compared.

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

Directory of Open Access Journals (Sweden)

Donghui Zhang

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

Induction of apoptosis by plumbagin through reactive oxygen species-mediated inhibition of topoisomerase II

International Nuclear Information System (INIS)

Kawiak, Anna; Piosik, Jacek; Stasilojc, Grzegorz; Gwizdek-Wisniewska, Anna; Marczak, Lukasz; Stobiecki, Maciej; Bigda, Jacek; Lojkowska, Ewa

2007-01-01

Reactive oxygen species (ROS) have been recognized as key molecules, which can selectively modify proteins and therefore regulate cellular signalling including apoptosis. Plumbagin, a naphthoquinone exhibiting antitumor activity, is known to generate ROS and has been found to inhibit the activity of topoisomerase II (Topo II) through the stabilization of the Topo II-DNA cleavable complex. The objective of this research was to clarify the role of ROS and Topo II inhibition in the induction of apoptosis mediated by plumbagin. As determined by the comet assay, plumbagin induced DNA cleavage in HL-60 cells, whereas in a cell line with reduced Topo II activity-HL-60/MX2, the level of DNA damage was significantly decreased. The onset of DNA strand break formation in HL-60 cells was delayed in comparison with the generation of intracellular ROS. In HL-60/MX2 cells, ROS were generated at a similar rate, whereas a significant reduction in the level of DNA damage was detected. The pretreatment of cells with N-acetylcysteine (NAC) attenuated plumbagin-induced DNA damage, pointing out to the involvement of ROS generation in cleavable complex formation. These results suggest that plumbagin-induced ROS does not directly damage DNA but requires the involvement of Topo II. Furthermore, experiments carried out using light spectroscopy indicated no direct interactions between plumbagin and DNA. The induction of apoptosis was significantly delayed in HL-60/MX2 cells indicating the involvement of Topo II inhibition in plumbagin-mediated apoptosis. Thus, these findings strongly suggest ROS-mediated inhibition of Topo II as an important mechanism contributing to the apoptosis-inducing properties of plumbagin

Tissue-selective effects of nucleolar stress and rDNA damage in developmental disorders.

Science.gov (United States)

Calo, Eliezer; Gu, Bo; Bowen, Margot E; Aryan, Fardin; Zalc, Antoine; Liang, Jialiang; Flynn, Ryan A; Swigut, Tomek; Chang, Howard Y; Attardi, Laura D; Wysocka, Joanna

2018-02-01

Many craniofacial disorders are caused by heterozygous mutations in general regulators of housekeeping cellular functions such as transcription or ribosome biogenesis. Although it is understood that many of these malformations are a consequence of defects in cranial neural crest cells, a cell type that gives rise to most of the facial structures during embryogenesis, the mechanism underlying cell-type selectivity of these defects remains largely unknown. By exploring molecular functions of DDX21, a DEAD-box RNA helicase involved in control of both RNA polymerase (Pol) I- and II-dependent transcriptional arms of ribosome biogenesis, we uncovered a previously unappreciated mechanism linking nucleolar dysfunction, ribosomal DNA (rDNA) damage, and craniofacial malformations. Here we demonstrate that genetic perturbations associated with Treacher Collins syndrome, a craniofacial disorder caused by heterozygous mutations in components of the Pol I transcriptional machinery or its cofactor TCOF1 (ref. 1), lead to relocalization of DDX21 from the nucleolus to the nucleoplasm, its loss from the chromatin targets, as well as inhibition of rRNA processing and downregulation of ribosomal protein gene transcription. These effects are cell-type-selective, cell-autonomous, and involve activation of p53 tumour-suppressor protein. We further show that cranial neural crest cells are sensitized to p53-mediated apoptosis, but blocking DDX21 loss from the nucleolus and chromatin rescues both the susceptibility to apoptosis and the craniofacial phenotypes associated with Treacher Collins syndrome. This mechanism is not restricted to cranial neural crest cells, as blood formation is also hypersensitive to loss of DDX21 functions. Accordingly, ribosomal gene perturbations associated with Diamond-Blackfan anaemia disrupt DDX21 localization. At the molecular level, we demonstrate that impaired rRNA synthesis elicits a DNA damage response, and that rDNA damage results in tissue-selective and

Recent Advancements in DNA Damage-Transcription Crosstalk and High-Resolution Mapping of DNA Breaks.

Science.gov (United States)

Vitelli, Valerio; Galbiati, Alessandro; Iannelli, Fabio; Pessina, Fabio; Sharma, Sheetal; d'Adda di Fagagna, Fabrizio

2017-08-31

Until recently, DNA damage arising from physiological DNA metabolism was considered a detrimental by-product for cells. However, an increasing amount of evidence has shown that DNA damage could have a positive role in transcription activation. In particular, DNA damage has been detected in transcriptional elements following different stimuli. These physiological DNA breaks are thought to be instrumental for the correct expression of genomic loci through different mechanisms. In this regard, although a plethora of methods are available to precisely map transcribed regions and transcription start sites, commonly used techniques for mapping DNA breaks lack sufficient resolution and sensitivity to draw a robust correlation between DNA damage generation and transcription. Recently, however, several methods have been developed to map DNA damage at single-nucleotide resolution, thus providing a new set of tools to correlate DNA damage and transcription. Here, we review how DNA damage can positively regulate transcription initiation, the current techniques for mapping DNA breaks at high resolution, and how these techniques can benefit future studies of DNA damage and transcription.

An in vivo analysis of Cr6+ induced biochemical, genotoxicological and transcriptional profiling of genes related to oxidative stress, DNA damage and apoptosis in liver of fish, Channa punctatus (Bloch, 1793).

Science.gov (United States)

Awasthi, Yashika; Ratn, Arun; Prasad, Rajesh; Kumar, Manoj; Trivedi, Sunil P

2018-07-01

Present study was designed to assess the hexavalent chromium (Cr 6+ ) mediated oxidative stress that induces DNA damage and apoptosis in adult fish, Channa punctatus (35 ± 3.0 g; 14.5 ± 1.0 cm; Actinopterygii). Fishes were maintained in three groups for 15, 30 and 45 d of exposure periods. They were treated with 5% (Group T1) and 10% (Group T2) of 96 h-LC 50 of chromium trioxide (Cr 6+ ). Controls were run for the similar duration. A significant (p < 0.05) increment in the activities of antioxidant enzymes, SOD and CAT in liver tissues of the exposed fish evinces the persistence of oxidative stress. A significant (p < 0.05) increase in induction of micronuclei (MN) coupled with transcriptional responses of target genes related to antioxidant enzymes, DNA damage and apoptosis (sod, cat, gsr, nox-1, p53, bax, bcl-2, apaf-1 and casp3a) establishes the impact of oxidative stress due to in vivo, Cr 6+ accumulation in liver as compared to control (0 mg/L), in a dose and exposure-dependent manner. Initially, the increased level of reactive oxygen species (ROS) in liver coincided with that of enhanced mRNA expression of antioxidant enzymes, sod, cat, gsr and nox-1 but, later, the overproduction of ROS, after 45 d of exposure of Cr 6+ , resulted in a significant (p < 0.05) up-regulation of p53. Our findings also unveil that the up-regulation of bax, apaf-1 and casp3a and down-regulation of bcl-2 are associated with Cr 6+ -induced oxidative stress mediated-apoptosis in liver of test fish. Aforesaid molecular markers can, thus, be efficiently utilized for bio-monitoring of aquatic regimes and conservation of fish biodiversity. Copyright © 2018 Elsevier B.V. All rights reserved.

Carcinogen-induced damage to DNA

International Nuclear Information System (INIS)

Strauss, B.; Altamirano, M.; Bose, K.; Sklar, R.; Tatsumi, K.

1979-01-01

Human cells respond to carcinogen-induced damage in their DNA in at least two ways. The first response, excision repair, proceeds by at least three variations, depending on the nature of the damage. Nucleotide excision results in relatively large repair patches but few free DNA breaks, since the endonuclease step is limiting. Apurinic repair is characterized by the appearance of numerous breaks in the DNA and by short repair patches. The pathways behave as though they function independently. Lymphoic cells derived from a xeroderma pigmentosum complementation group C patient are deficient in their ability to perform nucleotide excision and also to excise 6 methoxyguanine adducts, but they are apurinic repair competent. Organisms may bypass damage in their DNA. Lymphoblastoid cells, including those derived from xeroderma pigmentosum treated with 3 H-anti-BPDE, can replicate their DNA at low doses of carcinogen. Unexcised 3 H is found in the light or parental strand of the resulting hybrid DNA when replication occurs in medium with BrdUrd. This observation indicates a bypass reaction occurring by a mechanism involving branch migration at DNA growing points. Branch migration in DNA preparations have been observed, but the evidence is that most occurs in BrdUrd-containing DNA during cell lysis. The measurement of the bifilarly substituted DNA resulting from branch migration is a convenient method of estimating the proportion of new synthesis remaining in the vicinity of the DNA growing point. Treatment with carcinogens or caffeine results in accumulation of DNA growing points accompanied by the synthesis of shortened pieces of daughter DNA

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

Science.gov (United States)

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.

Real-time fluorescence imaging of the DNA damage repair response during mitosis.

Science.gov (United States)

Miwa, Shinji; Yano, Shuya; Yamamoto, Mako; Matsumoto, Yasunori; Uehara, Fuminari; Hiroshima, Yukihiko; Toneri, Makoto; Murakami, Takashi; Kimura, Hiroaki; Hayashi, Katsuhiro; Yamamoto, Norio; Efimova, Elena V; Tsuchiya, Hiroyuki; Hoffman, Robert M

2015-04-01

The response to DNA damage during mitosis was visualized using real-time fluorescence imaging of focus formation by the DNA-damage repair (DDR) response protein 53BP1 linked to green fluorescent protein (GFP) (53BP1-GFP) in the MiaPaCa-2(Tet-On) pancreatic cancer cell line. To observe 53BP1-GFP foci during mitosis, MiaPaCa-2(Tet-On) 53BP1-GFP cells were imaged every 30 min by confocal microscopy. Time-lapse imaging demonstrated that 11.4 ± 2.1% of the mitotic MiaPaCa-2(Tet-On) 53BP1-GFP cells had increased focus formation over time. Non-mitotic cells did not have an increase in 53BP1-GFP focus formation over time. Some of the mitotic MiaPaCa-2(Tet-On) 53BP1-GFP cells with focus formation became apoptotic. The results of the present report suggest that DNA strand breaks occur during mitosis and undergo repair, which may cause some of the mitotic cells to enter apoptosis in a phenomenon possibly related to mitotic catastrophe. © 2014 Wiley Periodicals, Inc.

Repair of DNA damage in Deinococcus radiodurans

International Nuclear Information System (INIS)

Evans, D.M.

1984-01-01

The repair of DNA lesions in Deinococcus radiodurans was examined with particular reference to DNA excision repair of ultraviolet light (UV) induced pyrimidine dimers. The characteristics of excision repair via UV endonucleases α and β in vivo varied with respect to (a) the substrate range of the enzymes, (b) the rate of repair of DNA damage (c) the requirement for a protein synthesised in response to DNA damage to attenuate exonuclease action at repairing regions. UV endonuclease α is postulated to incise DNA in a different manner from UV endonuclease β thus defining the method of subsequent repair. Several DNA damage specific endonuclease activities independent of α and β are described. Mutations of the uvsA, uvsF and uvsG genes resulted in an increase in single-strand breaks in response to DNA damage producing uncontrolled DNA degradation. Evidence is presented that these genes have a role in limiting the access of UV endonuclease β to DNA lesions. uvsF and uvsG are also shown to be linked to the mtoA gene. Mutation of uvsH and reo-1 produces further distinct phenotypes which are discussed. An overall model of excision repair of DNA damage in Deinococcus radiodurans is presented. (author)

Acrolein preferentially damages nucleolus eliciting ribosomal stress and apoptosis in human cancer cells.

Science.gov (United States)

Wang, Hsiang-Tsui; Chen, Tzu-Ying; Weng, Ching-Wen; Yang, Chun-Hsiang; Tang, Moon-Shong

2016-12-06

Acrolein (Acr) is a potent cytotoxic and DNA damaging agent which is ubiquitous in the environment and abundant in tobacco smoke. Acr is also an active cytotoxic metabolite of the anti-cancer drugs cyclophosphamide and ifosfamide. The mechanisms via which Acr exerts its anti-cancer activity and cytotoxicity are not clear. In this study, we found that Acr induces cytotoxicity and cell death in human cancer cells with different activities of p53. Acr preferentially binds nucleolar ribosomal DNA (rDNA) to form Acr-deoxyguanosine adducts, and induces oxidative damage to both rDNA and ribosomal RNA (rRNA). Acr triggers ribosomal stress responses, inhibits rRNA synthesis, reduces RNA polymerase I binding to the promoter of rRNA gene, disrupts nucleolar integrity, and impairs ribosome biogenesis and polysome formation. Acr causes an increase in MDM2 levels and phosphorylation of MDM2 in A549 and HeLa cells which are p53 active and p53 inactive, respectively. It enhances the binding of ribosomal protein RPL11 to MDM2 and reduces the binding of p53 and E2F-1 to MDM2 resulting in stabilization/activation of p53 in A549 cells and degradation of E2F-1 in A549 and HeLa cells. We propose that Acr induces ribosomal stress which leads to activation of MDM2 and RPL11-MDM2 binding, consequently, activates p53 and enhances E2F-1 degradation, and that taken together these two processes induce apoptosis and cell death.

Molecular mechanisms in radiation damage to DNA

International Nuclear Information System (INIS)

Osman, R.

1991-01-01

The objectives of this work are to elucidate the molecular mechanisms that are responsible for radiation-induced DNA damage. The overall goal is to understand the relationship between the chemical and structural changes produced by ionizing radiation in DNA and the resulting impairment of biological function expressed as carcinogenesis or cell death. The studies are based on theoretical explorations of possible mechanisms that link initial radiation damage in the form of base and sugar damage to conformational changes in DNA. These mechanistic explorations should lead to the formulation of testable hypothesis regarding the processes of impairment of regulation of gene expression, alternation in DNA repair, and damage to DNA structure involved in cell death or cancer

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

Directory of Open Access Journals (Sweden)

V. V. Moroz

2014-01-01

 pase3, caspase9, superoxide dismutase, and sAPO1/FAS were measured by enzyme immunoassay using test systems(Bender MedSystems, Austria.Results. There were significantly higher plasma levels of free DNA in the victims than in the controls throughout the followup, which is due to its exit from the cells damaged from tissue injury. In the first two weeks after injury, there were increases in DNA damages and white blood cell alteration processes by a necrotic and apoptotic mechanism in the victims with different types of injury, which may be associated with the active participation of leuko cytes in the processes of cellular breakdown product removal in the tissues damaged during injury and the in those of prevention of infectious complications. In the victims, white blood cell alteration in the necrotic pathway does not depend on BLV and hypoxia degree while that in the apoptotic pathway showed a relationship of leukocyte alteration to hypoxia in these patients. The sum of the values of necrotic DNA comets, apoptotic DNA comets, and single and doublestrand breaks on day 3 postinjury may serve as a predictor of the likely development of infectious complications in victims with injury, blood loss, and marked hypoxia. There were differences in the levels of DNA damages and white blood cell apoptosis and necrosis in the victims with BI and SSI. The injury victims showed a threefold decrease in plasma 8hydroxy2 deoxyguanosine concentrations, which was accompanied and, possibly, caused by an increase in the amount of superoxide dismutase.Conclusion. There was a relationship between the degree of DNA damages, apoptosis, and necrosis in the white blood cells of victims with injury and hypoxiainduced blood loss.

Mechanisms for radiation damage in DNA

International Nuclear Information System (INIS)

Sevilla, M.D.

1993-12-01

In this project the author has proposed several mechanisms for radiation damage to DNA and its constituents, and has detailed a series of experiments utilizing electron spin resonance spectroscopy, HPLC, GC-mass spectroscopy and ab initio molecular orbital calculations to test the proposed mechanisms. In this years work he has completed several experiments on the role of hydration water on DNA radiation damage, continued the investigation of the localization of the initial charges and their reactions on DNA, investigated protonation reactions in DNA base anions, and employed ab initio molecular orbital theory to gain insight into the initial events of radiation damage to DNA. Ab initio calculations have provided an understanding of the energetics evolved in anion and cation formation, ion radical transfer in DNA as well as proton transfer with DNA base pair radical ions. This has been extended in this years work to a consideration of ionization energies of various components of the DNA deoxyribose backbone and resulting neutral sugar radicals. This information has aided the formation of new radiation models for the effect of radiation on DNA. During this fiscal year four articles have been published, four are in press, one is submitted and several more are in preparation. Four papers have been presented at scientific meetings. This years effort will include another review article on the open-quotes Electron Spin Resonance of Radiation Damage to DNAclose quotes

SIRT participates at DNA damage response

Energy Technology Data Exchange (ETDEWEB)

Yun, Mi Yong; Joeng, Jae Min; Lee, Kee Ho [Korea Cancer Center Hospital, Seoul (Korea, Republic of); Park, Gil Hong [College of Medicine, Korea University, Seoul (Korea, Republic of)

2009-05-15

Sir2 maintains genomic stability in multiple ways in yeast. As a NAD{sup +}-dependent histone deacetylase, Sir2 has been reported to control chromatin silencing. In both budding yeast and Drosophila, overexpression of Sir2 extends life span. Previous reports have also demonstrated that Sir2 participate at DNA damage repair. A protein complex containing Sir2 has been reported to translocate to DNA double-strand breaks. Following DNA damage response, SIRT1 deacetylates p53 protein and attenuates its ability as a transcription factor. Consequently, SIRT1 over-expression increases cell survival under DNA damage inducing conditions. These previous observations mean a possibility that signals generated during the process of DNA repair are delivered through SIRT1 to acetylated p53. We present herein functional evidence for the involvement of SIRT1 in DNA repair response to radiation. In addition, this modulation of DNA repair activity may be connected to deacetylation of MRN proteins.

The Intertwined Roles of DNA Damage and Transcription

OpenAIRE

Di Palo, Giacomo

2016-01-01

DNA damage and transcription are two interconnected events. Transcription can induce damage and scheduled DNA damage can be required for transcription. Here, we analyzed genome-wide distribution of 8oxodG-marked oxidative DNA damage obtained by OxiDIP-Seq, and we found a correlation with transcription of protein coding genes.

DNA damage-induced inflammation and nuclear architecture.

Science.gov (United States)

Stratigi, Kalliopi; Chatzidoukaki, Ourania; Garinis, George A

2017-07-01

Nuclear architecture and the chromatin state affect most-if not all- DNA-dependent transactions, including the ability of cells to sense DNA lesions and restore damaged DNA back to its native form. Recent evidence points to functional links between DNA damage sensors, DNA repair mechanisms and the innate immune responses. The latter raises the question of how such seemingly disparate processes operate within the intrinsically complex nuclear landscape and the chromatin environment. Here, we discuss how DNA damage-induced immune responses operate within chromatin and the distinct sub-nuclear compartments highlighting their relevance to chronic inflammation. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Faster DNA Repair of Ultraviolet-Induced Cyclobutane Pyrimidine Dimers and Lower Sensitivity to Apoptosis in Human Corneal Epithelial Cells than in Epidermal Keratinocytes.

Directory of Open Access Journals (Sweden)

Justin D Mallet

Full Text Available Absorption of UV rays by DNA generates the formation of mutagenic cyclobutane pyrimidine dimers (CPD and pyrimidine (6-4 pyrimidone photoproducts (6-4PP. These damages are the major cause of skin cancer because in turn, they can lead to signature UV mutations. The eye is exposed to UV light, but the cornea is orders of magnitude less prone to UV-induced cancer. In an attempt to shed light on this paradox, we compared cells of the corneal epithelium and the epidermis for UVB-induced DNA damage frequency, repair and cell death sensitivity. We found similar CPD levels but a 4-time faster UVB-induced CPD, but not 6-4PP, repair and lower UV-induced apoptosis sensitivity in corneal epithelial cells than epidermal. We then investigated levels of DDB2, a UV-induced DNA damage recognition protein mostly impacting CPD repair, XPC, essential for the repair of both CPD and 6-4PP and p53 a protein upstream of the genotoxic stress response. We found more DDB2, XPC and p53 in corneal epithelial cells than in epidermal cells. According to our results analyzing the protein stability of DDB2 and XPC, the higher level of DDB2 and XPC in corneal epithelial cells is most likely due to an increased stability of the protein. Taken together, our results show that corneal epithelial cells have a better efficiency to repair UV-induced mutagenic CPD. On the other hand, they are less prone to UV-induced apoptosis, which could be related to the fact that since the repair is more efficient in the HCEC, the need to eliminate highly damaged cells by apoptosis is reduced.

Hydroxychavicol, a betel leaf component, inhibits prostate cancer through ROS-driven DNA damage and apoptosis

Science.gov (United States)

Gundala, Sushma Reddy; Yang, Chunhua; Mukkavilli, Rao; Paranjpe, Rutugandha; Brahmbhatt, Meera; Pannu, Vaishali; Cheng, Alice; Reid, Michelle D.; Aneja, Ritu

2015-01-01

Dietary phytochemicals are excellent ROS-modulating agents and have been shown to effectively enhance ROS levels beyond toxic threshold in cancer cells to ensure their selective killing while leaving normal cells unscathed. Here we demonstrate that hydroxychavicol (HC), extracted and purified from Piper betel leaves, significantly inhibits growth and proliferation via ROS generation in human prostate cancer, PC-3 cells. HC perturbed cell-cycle kinetics and progression, reduced clonogenicity and mediated cytotoxicity by ROS-induced DNA damage leading to activation of several pro-apoptotic molecules. In addition, HC treatment elicited a novel autophagic response as evidenced by the appearance of acidic vesicular organelles and increased expression of autophagic markers, LC3-IIb and beclin-1. Interestingly, quenching of ROS with tiron, an antioxidant, offered significant protection against HC-induced inhibition of cell growth and down regulation of caspase-3, suggesting the crucial role of ROS in mediating cell death. The collapse of mitochondrial transmembrane potential by HC further revealed the link between ROS generation and induction of caspase-mediated apoptosis in PC-3 cells. Our data showed remarkable inhibition of prostate tumor xenografts by ~72% upon daily oral administration of 150 mg/kg bw HC by quantitative tumor volume measurements and non-invasive real-time bioluminescent imaging. HC was well-tolerated at this dosing level without any observable toxicity. This is the first report to demonstrate the anti-prostate efficacy of HC in vitro and in vivo, which is perhaps attributable to its selective prooxidant activity to eliminate cancer cells thus providing compelling grounds for future preclinical studies to validate its potential usefulness for prostate cancer management. PMID:25064160

Hydroxychavicol, a betel leaf component, inhibits prostate cancer through ROS-driven DNA damage and apoptosis.

Science.gov (United States)

Gundala, Sushma Reddy; Yang, Chunhua; Mukkavilli, Rao; Paranjpe, Rutugandha; Brahmbhatt, Meera; Pannu, Vaishali; Cheng, Alice; Reid, Michelle D; Aneja, Ritu

2014-10-01

Dietary phytochemicals are excellent ROS-modulating agents and have been shown to effectively enhance ROS levels beyond toxic threshold in cancer cells to ensure their selective killing while leaving normal cells unscathed. Here we demonstrate that hydroxychavicol (HC), extracted and purified from Piper betel leaves, significantly inhibits growth and proliferation via ROS generation in human prostate cancer, PC-3 cells. HC perturbed cell-cycle kinetics and progression, reduced clonogenicity and mediated cytotoxicity by ROS-induced DNA damage leading to activation of several pro-apoptotic molecules. In addition, HC treatment elicited a novel autophagic response as evidenced by the appearance of acidic vesicular organelles and increased expression of autophagic markers, LC3-IIb and beclin-1. Interestingly, quenching of ROS with tiron, an antioxidant, offered significant protection against HC-induced inhibition of cell growth and down regulation of caspase-3, suggesting the crucial role of ROS in mediating cell death. The collapse of mitochondrial transmembrane potential by HC further revealed the link between ROS generation and induction of caspase-mediated apoptosis in PC-3 cells. Our data showed remarkable inhibition of prostate tumor xenografts by ~72% upon daily oral administration of 150mg/kg bw HC by quantitative tumor volume measurements and non-invasive real-time bioluminescent imaging. HC was well-tolerated at this dosing level without any observable toxicity. This is the first report to demonstrate the anti-prostate cancer efficacy of HC in vitro and in vivo, which is perhaps attributable to its selective prooxidant activity to eliminate cancer cells thus providing compelling grounds for future preclinical studies to validate its potential usefulness for prostate cancer management. Copyright © 2014 Elsevier Inc. All rights reserved.

Molecular and sensory mechanisms to mitigate sunlight-induced DNA damage in treefrog tadpoles.

Science.gov (United States)

Schuch, André P; Lipinski, Victor M; Santos, Mauricio B; Santos, Caroline P; Jardim, Sinara S; Cechin, Sonia Z; Loreto, Elgion L S

2015-10-01

The increased incidence of solar ultraviolet B (UVB) radiation has been proposed as an environmental stressor, which may help to explain the enigmatic decline of amphibian populations worldwide. Despite growing knowledge regarding the UV-induced biological effects in several amphibian models, little is known about the efficacy of DNA repair pathways. In addition, little attention has been given to the interplay between these molecular mechanisms with other physiological strategies that avoid the damage induced by sunlight. Here, DNA lesions induced by environmental doses of solar UVB and UVA radiation were detected in genomic DNA samples of treefrog tadpoles (Hypsiboas pulchellus) and their DNA repair activity was evaluated. These data were complemented by monitoring the induction of apoptosis in blood cells and tadpole survival. Furthermore, the tadpoles' ability to perceive and escape from UV wavelengths was evaluated as an additional strategy of photoprotection. The results show that tadpoles are very sensitive to UVB light, which could be explained by the slow DNA repair rates for both cyclobutane pyrimidine dimers (CPDs) and pyrimidine (6,4) pyrimidone photoproducts (6,4PPs). However, they were resistant to UVA, probably as a result of the activation of photolyases during UVA irradiation. Surprisingly, a sensory mechanism that triggers their escape from UVB and UVA light avoids the generation of DNA damage and helps to maintain the genomic integrity. This work demonstrates the genotoxic impact of both UVB and UVA radiation on tadpoles and emphasizes the importance of the interplay between molecular and sensory mechanisms to minimize the damage caused by sunlight. © 2015. Published by The Company of Biologists Ltd.

Role of DNA repair in repair of cytogenetic damages. Slowly repaired DNA injuries involved in cytogenetic damages repair

International Nuclear Information System (INIS)

Zaichkina, S.I.; Rozanova, O.M.; Aptikaev, G.F.; Ganassi, E.Eh.

1989-01-01

Caffeine was used to study the kinetics of cytogenetic damages repair in Chinese hamster fibroblasts. Its half-time (90 min) was shown to correlate with that of repair of slowly repaired DNA damages. The caffeine-induced increase in the number of irreparable DNA damages, attributed to inhibition of double-strand break repair, is in a quantitative correlation with the effect of the cytogenetic damage modification

Evaluation of apoptosis and apoptosis proteins as possible markers of radiation at doses 0.1-2 Gy, in comparison to the micronucleus assay in three cell lines

International Nuclear Information System (INIS)

Jaworska, A.; Angelis, P. de

1997-01-01

In recent years the interest in apoptosis as possible indicator of radiation damage has increased. Studies have been done to examine the induction of apoptosis after ionizing radiation using morphological criteria, characteristic DNA damage pattern(ladders), early DNA damage using flow cytometry and/or expression of the proteins involved in apoptosis. But the picture which emerges from these investigations is unclear. Some researchers suggest that apoptosis studies can be used as potential assays of biological dosimetry, others doubt if apoptosis can be used as a marker of irradiation at all. Most studies have been done using relatively high doses of radiation. In this study we focus on apoptosis induction after relatively small doses (0,1-2 Gy). We detected apoptosis with the in situ terminal deoxynucleotidyl transferase assay by flow cytometry, and measured the expression of proteins that regulate apoptosis (Bcl-2, Bax, P53) with Western blotting. As comparison we used the cytokinesis-block micronucleus assay as a reference. The studies were carried out in three lymphoid cell lines: the mouse lymphoma L5178Y resistant and sensitive cell lines widely used in radiobiological studies, and the human pre-B cell leukemia Reh cells. Our results indicate that we can not consider the examined parameters of apoptosis as markers of radiation in these cell lines. (author)

Role of DNA repair in repair of cytogenetic damages. Contribution of repair of single-strand DNA breaks to cytogenetic damages repair

International Nuclear Information System (INIS)

Rozanova, O.M.; Zaichkina, S.I.; Aptikaev, G.F.; Ganassi, E.Eh.

1989-01-01

The comparison was made between the results of the effect of poly(ADP-ribosylation) ingibitors (e.g. nicotinamide and 3-aminobenzamide) and a chromatin proteinase ingibitor, phenylmethylsulfonylfluoride, on the cytogenetic damages repair, by a micronuclear test, and DNA repair in Chinese hamster fibroblasts. The values of the repair half-periods (5-7 min for the cytogenetic damages and 5 min for the rapidly repaired DNA damages) and a similar modyfying effect with regard to radiation cytogenetic damages and kynetics of DNA damages repair were found to be close. This confirms the contribution of repair of DNA single-strand breaks in the initiation of structural damages to chromosomes

The effect of tributyltin chloride on Caenorhabditis elegans germline is mediated by a conserved DNA damage checkpoint pathway.

Science.gov (United States)

Cheng, Zhe; Tian, Huimin; Chu, Hongran; Wu, Jianjian; Li, Yingying; Wang, Yanhai

2014-03-21

Tributyltin (TBT), one of the environmental pollutants, has been shown to impact the reproduction of animals. However, due to the lack of appropriate animal model, analysis of the affected molecular pathways in germ cells is lagging and has been particularly challenging. In the present study, we investigated the effects of tributyltin chloride (TBTCL) on the nematode Caenorhabditis elegans germline. We show that exposure of C. elegans to TBTCL causes significantly elevated level of sterility and embryonic lethality. TBTCL exposure results in an increased number of meiotic DNA double-strand breaks in germ cells, subsequently leading to activated DNA damage checkpoint. Exposing C. elegans to TBTCL causes dose- and time-dependent germline apoptosis. This apoptotic response was blocked in loss-of-function mutants of hus-1 (op241), mrt-2 (e2663) and p53/cep-1 (gk138), indicating that checkpoints and p53 are essential for mediating TBTCL-induced germ cell apoptosis. Moreover, TBTCL exposure can inhibit germ cell proliferation, which is also mediated by the conserved checkpoint pathway. We thereby propose that TBT exhibits its effects on the germline by inducing DNA damage and impaired maintenance of genomic integrity. Copyright © 2014 The Authors. Published by Elsevier Ireland Ltd.. All rights reserved.

Transcription and DNA Damage: Holding Hands or Crossing Swords?

Science.gov (United States)

D'Alessandro, Giuseppina; d'Adda di Fagagna, Fabrizio

2017-10-27

Transcription has classically been considered a potential threat to genome integrity. Collision between transcription and DNA replication machinery, and retention of DNA:RNA hybrids, may result in genome instability. On the other hand, it has been proposed that active genes repair faster and preferentially via homologous recombination. Moreover, while canonical transcription is inhibited in the proximity of DNA double-strand breaks, a growing body of evidence supports active non-canonical transcription at DNA damage sites. Small non-coding RNAs accumulate at DNA double-strand break sites in mammals and other organisms, and are involved in DNA damage signaling and repair. Furthermore, RNA binding proteins are recruited to DNA damage sites and participate in the DNA damage response. Here, we discuss the impact of transcription on genome stability, the role of RNA binding proteins at DNA damage sites, and the function of small non-coding RNAs generated upon damage in the signaling and repair of DNA lesions. Copyright © 2016 Elsevier Ltd. All rights reserved.

Mechanisms for radiation damage in DNA

International Nuclear Information System (INIS)

Sevilla, M.D.

1985-07-01

Radiation damage to DNA results from the direct interaction of radiation with DNA where positive ions, electrons and excited states are formed in the DNA, and the indirect effect where radical species formed in the surrounding medium by the radiation attack the DNA. The primary mechanism proposed for radiation damage, by the direct effect, is that positive and negative ions formed within the DNA strand migrate through the stacked DNA bases. The ions can then recombine, react with the DNA bases most likely to react by protonation of the anion and deprotonation or hydroxylation of the cation or transfer out of the DNA chain to the surrounding histone protein. This work as aimed at understanding the possible reactions of the DNA base ion radicals, as well as their initial distribution in the DNA strand. 31 refs

Protein kinase Cη activates NF-κB in response to camptothecin-induced DNA damage

International Nuclear Information System (INIS)

Raveh-Amit, Hadas; Hai, Naama; Rotem-Dai, Noa; Shahaf, Galit; Gopas, Jacob; Livneh, Etta

2011-01-01

Highlights: → Protein kinase C-eta (PKCη) is an upstream regulator of the NF-κB signaling pathway. → PKCη activates NF-κB in non-stressed conditions and in response to DNA damage. → PKCη regulates NF-κB by activating IκB kinase (IKK) and inducing IκB degradation. -- Abstract: The nuclear factor κB (NF-κB) family of transcription factors participates in the regulation of genes involved in innate- and adaptive-immune responses, cell death and inflammation. The involvement of the Protein kinase C (PKC) family in the regulation of NF-κB in inflammation and immune-related signaling has been extensively studied. However, not much is known on the role of PKC in NF-κB regulation in response to DNA damage. Here we demonstrate for the first time that PKC-eta (PKCη) regulates NF-κB upstream signaling by activating the IκB kinase (IKK) and the degradation of IκB. Furthermore, PKCη enhances the nuclear translocation and transactivation of NF-κB under non-stressed conditions and in response to the anticancer drug camptothecin. We and others have previously shown that PKCη confers protection against DNA damage-induced apoptosis. Our present study suggests that PKCη is involved in NF-κB signaling leading to drug resistance.

DNA Damage Signals and Space Radiation Risk

Science.gov (United States)

Cucinotta, Francis A.

2011-01-01

Space radiation is comprised of high-energy and charge (HZE) nuclei and protons. The initial DNA damage from HZE nuclei is qualitatively different from X-rays or gamma rays due to the clustering of damage sites which increases their complexity. Clustering of DNA damage occurs on several scales. First there is clustering of single strand breaks (SSB), double strand breaks (DSB), and base damage within a few to several hundred base pairs (bp). A second form of damage clustering occurs on the scale of a few kbp where several DSB?s may be induced by single HZE nuclei. These forms of damage clusters do not occur at low to moderate doses of X-rays or gamma rays thus presenting new challenges to DNA repair systems. We review current knowledge of differences that occur in DNA repair pathways for different types of radiation and possible relationships to mutations, chromosomal aberrations and cancer risks.

Chlorella vulgaris Induces Apoptosis of Human Non-Small Cell Lung Carcinoma (NSCLC) Cells.

Science.gov (United States)

Zhang, Zhi-Dong; Liang, Kai; Li, Kun; Wang, Guo-Quan; Zhang, Ke-Wei; Cai, Lei; Zhai, Shui-Ting; Chou, Kuo-Chen

2017-01-01

Chlorella vulgaris (C. vulgaris), a unicellular green microalga, has been widely used as a food supplement and reported to have antioxidant and anticancer properties. The current study was designed to assess the cytotoxic, apoptotic, and DNA-damaging effects of C. vulgaris growth factor (CGF), hot water C. vulgaris extracts, inlung tumor A549 and NCI-H460 cell lines. A549 cells, NCI-H460 cells, and normal human fibroblasts were treated with CGF at various concentrations (0-300 μg/ml) for 24 hr. The comet assay and γH2AX assay showed DNA damage in A549 and NCI-H460 cells upon CGF exposure. Evaluation of apoptosis by the TUNEL assay and DNA fragmentation analysis by agarose gel electrophoresis showed that CGF induced apoptosis in A549 and NCI-H460 cells. Chlorella vulgaris hot water extract induced apoptosis and DNA damage in human lung carcinoma cells. CGF can thus be considered a potential cytotoxic or genotoxic drug for treatment of lung carcinoma. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Involvement of DNA-PK and ATM in radiation- and heat-induced DNA damage recognition and apoptotic cell death

International Nuclear Information System (INIS)

Tomita, Masanori

2010-01-01

Exposure to ionizing radiation and hyperthermia results in important biological consequences, e.g. cell death, chromosomal aberrations, mutations, and DNA strand breaks. There is good evidence that the nucleus, specifically cellular DNA, is the principal target for radiation-induced cell lethality. DNA double-strand breaks (DSBs) are considered to be the most serious type of DNA damage induced by ionizing radiation. On the other hand, verifiable mechanisms which can lead to heat-induced cell death are damage to the plasma membrane and/or inactivation of heat-labile proteins caused by protein denaturation and subsequent aggregation. Recently, several reports have suggested that DSBs can be induced after hyperthermia because heat-induced phosphorylated histone H2AX (γ-H2AX) foci formation can be observed in several mammalian cell lines. In mammalian cells, DSBs are repaired primarily through two distinct and complementary mechanisms: non-homologous end joining (NHEJ), and homologous recombination (HR) or homology-directed repair (HDR). DNA-dependent protein kinase (DNA-PK) and ataxia-telangiectasia mutated (ATM) are key players in the initiation of DSB repair and phosphorylate and/or activate many substrates, including themselves. These phosphorylated substrates have important roles in the functioning of cell cycle checkpoints and in cell death, as well as in DSB repair. Apoptotic cell death is a crucial cell suicide mechanism during development and in the defense of homeostasis. If DSBs are unrepaired or misrepaired, apoptosis is a very important system which can protect an organism against carcinogenesis. This paper reviews recently obtained results and current topics concerning the role of DNA-PK and ATM in heat- or radiation-induced apoptotic cell death. (author)

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

International Nuclear Information System (INIS)

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

2014-01-01

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

Repair of Clustered Damage and DNA Polymerase Iota.

Science.gov (United States)

Belousova, E A; Lavrik, O I

2015-08-01

Multiple DNA lesions occurring within one or two turns of the DNA helix known as clustered damage are a source of double-stranded DNA breaks, which represent a serious threat to the cells. Repair of clustered lesions is accomplished in several steps. If a clustered lesion contains oxidized bases, an individual DNA lesion is repaired by the base excision repair (BER) mechanism involving a specialized DNA polymerase after excising DNA damage. Here, we investigated DNA synthesis catalyzed by DNA polymerase iota using damaged DNA templates. Two types of DNA substrates were used as model DNAs: partial DNA duplexes containing breaks of different length, and DNA duplexes containing 5-formyluracil (5-foU) and uracil as a precursor of apurinic/apyrimidinic sites (AP) in opposite DNA strands. For the first time, we showed that DNA polymerase iota is able to catalyze DNA synthesis using partial DNA duplexes having breaks of different length as substrates. In addition, we found that DNA polymerase iota could catalyze DNA synthesis during repair of clustered damage via the BER system by using both undamaged and 5-foU-containing templates. We found that hPCNA (human proliferating cell nuclear antigen) increased efficacy of DNA synthesis catalyzed by DNA polymerase iota.

DNA DAMAGE QUANTITATION BY ALKALINE GEL ELECTROPHORESIS.

Energy Technology Data Exchange (ETDEWEB)

SUTHERLAND,B.M.; BENNETT,P.V.; SUTHERLAND, J.C.

2004-03-24

Physical and chemical agents in the environment, those used in clinical applications, or encountered during recreational exposures to sunlight, induce damages in DNA. Understanding the biological impact of these agents requires quantitation of the levels of such damages in laboratory test systems as well as in field or clinical samples. Alkaline gel electrophoresis provides a sensitive (down to {approx} a few lesions/5Mb), rapid method of direct quantitation of a wide variety of DNA damages in nanogram quantities of non-radioactive DNAs from laboratory, field, or clinical specimens, including higher plants and animals. This method stems from velocity sedimentation studies of DNA populations, and from the simple methods of agarose gel electrophoresis. Our laboratories have developed quantitative agarose gel methods, analytical descriptions of DNA migration during electrophoresis on agarose gels (1-6), and electronic imaging for accurate determinations of DNA mass (7-9). Although all these components improve sensitivity and throughput of large numbers of samples (7,8,10), a simple version using only standard molecular biology equipment allows routine analysis of DNA damages at moderate frequencies. We present here a description of the methods, as well as a brief description of the underlying principles, required for a simplified approach to quantitation of DNA damages by alkaline gel electrophoresis.

Phorate-induced oxidative stress, DNA damage and transcriptional activation of p53 and caspase genes in male Wistar rats

Energy Technology Data Exchange (ETDEWEB)

Saquib, Quaiser [Department of Zoology, College of Science, King Saud University, Riyadh (Saudi Arabia); Attia, Sabry M. [Department of Pharmacology, College of Pharmacy, King Saud University, Riyadh (Saudi Arabia); Siddiqui, Maqsood A. [Department of Zoology, College of Science, King Saud University, Riyadh (Saudi Arabia); Aboul-Soud, Mourad A.M. [Department of Zoology, College of Science, King Saud University, Riyadh (Saudi Arabia); Biochemistry Department, Faculty of Agriculture, Cairo University, 12613 Giza (Egypt); Al-Khedhairy, Abdulaziz A. [Department of Zoology, College of Science, King Saud University, Riyadh (Saudi Arabia); Giesy, John P. [Department of Zoology, College of Science, King Saud University, Riyadh (Saudi Arabia); Department of Biomedical and Veterinary Biosciences and Toxicology Centre, University of Saskatchewan, Saskatoon, Canada S7N 5B3 (Canada); Zoology Department and Center for Integrative Toxicology, Michigan State University, East Lansing 48824 (United States); Musarrat, Javed, E-mail: musarratj1@yahoo.com [Department of Zoology, College of Science, King Saud University, Riyadh (Saudi Arabia); Department of Microbiology, Faculty of Agricultural Sciences, AMU, Aligarh (India)

2012-02-15

Male Wistar rats exposed to a systemic organophosphorus insecticide, phorate [O,O-diethyl S-[(ethylthio) methyl] phosphorothioate] at varying oral doses of 0.046, 0.092 or 0.184 mg phorate/kg bw for 14 days, exhibited substantial oxidative stress, cellular DNA damage and activation of apoptosis-related p53, caspase 3 and 9 genes. The histopathological changes including the pyknotic nuclei, inflammatory leukocyte infiltrations, renal necrosis, and cardiac myofiber degeneration were observed in the liver, kidney and heart tissues. Biochemical analysis of catalase and glutathione revealed significantly lesser activities of antioxidative enzymes and lipid peroxidation in tissues of phorate exposed rats. Furthermore, generation of intracellular reactive oxygen species and reduced mitochondrial membrane potential in bone marrow cells confirmed phorate-induced oxidative stress. Significant DNA damage was measured through comet assay in terms of the Olive tail moment in bone marrow cells of treated animals as compared to control. Cell cycle analysis also demonstrated the G{sub 2}/M arrest and appearance of a distinctive SubG{sub 1} peak, which signified induction of apoptosis. Up-regulation of tumor suppressor p53 and caspase 3 and 9 genes, determined by quantitative real-time PCR and enzyme-linked immunosorbent assay, elucidated the activation of intrinsic apoptotic pathways in response to cellular stress. Overall, the results suggest that phorate induces genetic alterations and cellular toxicity, which can adversely affect the normal cellular functioning in rats. -- Highlights: ► This is the first report on molecular toxicity of phorate in an in vivo test system. ► Phorate induces biochemical and histological changes in liver, kidney and heart. ► Rats treated with phorate exhibited DNA damage in bone marrow cells. ► Phorate induces apoptosis, oxidative stress and alters mitochondrial fluorescence. ► Phorate induces transcriptional changes and enhanced

Phorate-induced oxidative stress, DNA damage and transcriptional activation of p53 and caspase genes in male Wistar rats

International Nuclear Information System (INIS)

Saquib, Quaiser; Attia, Sabry M.; Siddiqui, Maqsood A.; Aboul-Soud, Mourad A.M.; Al-Khedhairy, Abdulaziz A.; Giesy, John P.; Musarrat, Javed

2012-01-01

Male Wistar rats exposed to a systemic organophosphorus insecticide, phorate [O,O-diethyl S-[(ethylthio) methyl] phosphorothioate] at varying oral doses of 0.046, 0.092 or 0.184 mg phorate/kg bw for 14 days, exhibited substantial oxidative stress, cellular DNA damage and activation of apoptosis-related p53, caspase 3 and 9 genes. The histopathological changes including the pyknotic nuclei, inflammatory leukocyte infiltrations, renal necrosis, and cardiac myofiber degeneration were observed in the liver, kidney and heart tissues. Biochemical analysis of catalase and glutathione revealed significantly lesser activities of antioxidative enzymes and lipid peroxidation in tissues of phorate exposed rats. Furthermore, generation of intracellular reactive oxygen species and reduced mitochondrial membrane potential in bone marrow cells confirmed phorate-induced oxidative stress. Significant DNA damage was measured through comet assay in terms of the Olive tail moment in bone marrow cells of treated animals as compared to control. Cell cycle analysis also demonstrated the G 2 /M arrest and appearance of a distinctive SubG 1 peak, which signified induction of apoptosis. Up-regulation of tumor suppressor p53 and caspase 3 and 9 genes, determined by quantitative real-time PCR and enzyme-linked immunosorbent assay, elucidated the activation of intrinsic apoptotic pathways in response to cellular stress. Overall, the results suggest that phorate induces genetic alterations and cellular toxicity, which can adversely affect the normal cellular functioning in rats. -- Highlights: ► This is the first report on molecular toxicity of phorate in an in vivo test system. ► Phorate induces biochemical and histological changes in liver, kidney and heart. ► Rats treated with phorate exhibited DNA damage in bone marrow cells. ► Phorate induces apoptosis, oxidative stress and alters mitochondrial fluorescence. ► Phorate induces transcriptional changes and enhanced activities of

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

Science.gov (United States)

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

2015-07-09

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

Aniline Induces Oxidative Stress and Apoptosis of Primary Cultured Hepatocytes

Directory of Open Access Journals (Sweden)

Yue Wang

2016-11-01

Full Text Available The toxicity and carcinogenicity of aniline in humans and animals have been well documented. However, the molecular mechanism involved in aniline-induced liver toxicity and carcinogenesis remains unclear. In our research, primary cultured hepatocytes were exposed to aniline (0, 1.25, 2.50, 5.0 and 10.0 μg/mL for 24 h in the presence or absence of N-acetyl-l-cysteine (NAC. Levels of reactive oxygen species (ROS, malondialdehyde (MDA, and glutathione (GSH, activities of superoxide dismutase (SOD and catalase (CAT, mitochondrial membrane potential, DNA damage, cell viability, and apoptosis were detected. Levels of ROS and MDA were significantly increased and levels of GSH and CAT, activity of SOD, and mitochondrial membrane potential in hepatocytes were significantly decreased by aniline compared with the negative control group. The tail moment and DNA content of the tail in exposed groups were significantly higher than those in the negative control group. Cell viability was reduced and apoptotic death was induced by aniline in a concentration-dependent manner. The phenomena of ROS generation, oxidative damage, loss of mitochondrial membrane potential, DNA damage and apoptosis could be prevented if ROS inhibitor NAC was added. ROS generation is involved in the loss of mitochondrial membrane potential and DNA injury, which may play a role in aniline-induced apoptosis in hepatocytes. Our study provides insight into the mechanism of aniline-induced toxicity and apoptosis of hepatocytes.

Radiation and non-radiation damage to DNA. Onset of molecular instability and carcinogenesis. Theoretical explorations on DNA damage and repair

International Nuclear Information System (INIS)

Pinak, Miroslay; Bunta, J.K.

2006-01-01

The current work is focused on results of molecular dynamics simulations performed on two DNA damages: 8-oxoguanine as the most significant oxidative damage leading to transversion mutation cytosine-guanine→adenine-thymine', which is common mutation found in human cancer cells; and on the DNA strand break, the type of damage that is considered to be one of the most significant damage leading to genetic instability that may result in enhanced cell proliferation or carcinogenesis. Except the structural changes induced by these two lesions the role and importance of electrostatic energy in recognition process in which a respective repair enzyme recognizes damaged DNA site is also described. Among the significant results can be included the fact, that most of the damages on DNA alternate locally electronic state by modifying chemical and electron orbital configuration. This modified configuration may be represented outside DNA molecule as an enhanced electrostatic interaction with surrounding environment, that may signal the presence of the damaged site toward the repair enzyme. Work on the DNA strand break shows that open valences at broken strand ends are quickly filled by the electrons generated during radiolysis. Results of simulation indicate a local instability of hydrogen bonds between complementary bases. (author)

Cellular radiosensitivity and DNA damage in primary human fibroblasts

International Nuclear Information System (INIS)

Wurm, R.; Burnet, N.G.; Duggal, N.

1994-01-01

To evaluate the relationship between radiation-induced cell survival and DNA damage in primary human fibroblasts to decide whether the initial or residual DNA damage levels are more predictive of normal tissue cellular radiosensitivity. Five primary human nonsyndromic and two primary ataxia telangiectasia fibroblast strains grown in monolayer were studied. Cell survival was assessed by clonogenic assay. Irradiation was given at high dose rate (HDR) 1-2 Gy/min. DNA damage was measured in stationary phase cells and expressed as fraction released from the well by pulsed-field gel electrophoresis (PFGE). For initial damage, cells were embedded in agarose and irradiated at HDR on ice. Residual DNA damage was measured in monolayer by allowing a 4-h repair period after HDR irradiation. Following HDR irradiation, cell survival varied between SF 2 0.025 to 0.23. Measurement of initial DNA damage demonstrated linear induction up to 30 Gy, with small differences in the slope of the dose-response curve between strains. No correlation between cell survival and initial damage was found. Residual damage increased linearly up to 80 Gy with a variation in slope by a factor of 3.2. Cell survival correlated with the slope of the dose-response curves for residual damage of the different strains (p = 0.003). The relationship between radiation-induced cell survival and DNA damage in primary human fibroblasts of differing radiosensitivity is closest with the amount of DNA damage remaining after repair. If assays of DNA damage are to be used as predictors of normal tissue response to radiation, residual DNA damage provides the most likely correlation with cell survival. 52 refs., 5 figs., 2 tabs

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

Science.gov (United States)

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

2007-01-01

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

Processing of free radical damaged DNA bases

International Nuclear Information System (INIS)

Wallace, S.

2003-01-01

Free radicals produced during the radiolysis of water gives rise to a plethora of DNA damages including single strand breaks, sites of base loss and a wide variety of purine and pyrimidine base lesions. All these damages are processed in cells by base excision repair. The oxidative DNA glycosylases which catalyze the first step in the removal of a base damage during base excision repair evolved primarily to protect the cells from the deleterious mutagenic effects of single free radical-induced DNA lesions arising during oxidative metabolism. This is evidenced by the high spontaneous mutation rate in bacterial mutants lacking the oxidative DNA glycosylases. However, when a low LET photon transverses the DNA molecule, a burst of free radicals is produced during the radiolysis of water that leads to the formation of clustered damages in the DNA molecule, that are recognized by the oxidative DNA glycosylases. When substrates containing two closely opposed sugar damages or base and sugar damages are incubated with the oxidative DNA glycosylases in vitro, one strand is readily incised by the lyase activity of the DNA glycosylase. Whether or not the second strand is incised depends on the distance between the strand break resulting from the incised first strand and the remaining DNA lesion on the other strand. If the lesions are more than two or three base pairs apart, the second strand is readily cleaved by the DNA glycosylase, giving rise to a double strand break. Even if the entire base excision repair system is reconstituted in vitro, whether or not a double strand break ensues depends solely upon the ability of the DNA glycosylase to cleave the second strand. These data predicted that cells deficient in the oxidative DNA glycosylases would be radioresistant while those that overproduce an oxidative DNA glycosylase would be radiosensitive. This prediction was indeed borne in Escherichia coli that is, mutants lacking the oxidative DNA glycosylases are radioresistant

DNA damage and defence gene expression after oxidative stress induced by x-rays and diesel exhaust particles

Energy Technology Data Exchange (ETDEWEB)

Risom, Lotte

2004-07-01

Particulate air pollution is one the most important environmental health factors for people living in cities. Especially the exhaust particles from traffic are possible causes for cancer and cardiopulmonary diseases. The aim of this thesis was to characterize the health effects of diesel exhaust particles (DEP) by inducing oxidative stress and analyse the underlying mechanisms. Methods for determining oxidative stress, DNA damage, and gene expression were validated and calibrated in lung tissue by studying the dose response relations after ionizing radiation. The study showed the feasibility of partial-body x-ray irradiation as an in vivo model for induction and repair of oxidative DNA damage, of DNA repair enzymes expression, and antioxidant defense genes. A 'nose-only' mouse model for inhalation of ultra-fine particles showed that particles induce oxidative DNA damage in lung tissue and in bronchoalveolar lavage cells. The exposure increased the expression of HO-1 mRNA and oxoguanine DNA glycosylase OGG1 mRNA. The levels of 8-oxodG and OGG1 mRNA were mirror images. Colon and liver were analysed after administration of DEP in the diet with or without increasing doses of sucrose. This study indicated that DEP induces DNA adducts and oxidative stress through formation of DNA strand breaks, DNA repair enzyme expression, apoptosis, and protein oxidisation in colon and liver at relatively low exposure doses. The thesis is based on four published journal articles. (ln)

DNA damage and defence gene expression after oxidative stress induced by x-rays and diesel exhaust particles

International Nuclear Information System (INIS)

Risom, Lotte

2004-01-01

Particulate air pollution is one the most important environmental health factors for people living in cities. Especially the exhaust particles from traffic are possible causes for cancer and cardiopulmonary diseases. The aim of this thesis was to characterize the health effects of diesel exhaust particles (DEP) by inducing oxidative stress and analyse the underlying mechanisms. Methods for determining oxidative stress, DNA damage, and gene expression were validated and calibrated in lung tissue by studying the dose response relations after ionizing radiation. The study showed the feasibility of partial-body x-ray irradiation as an in vivo model for induction and repair of oxidative DNA damage, of DNA repair enzymes expression, and antioxidant defense genes. A 'nose-only' mouse model for inhalation of ultra-fine particles showed that particles induce oxidative DNA damage in lung tissue and in bronchoalveolar lavage cells. The exposure increased the expression of HO-1 mRNA and oxoguanine DNA glycosylase OGG1 mRNA. The levels of 8-oxodG and OGG1 mRNA were mirror images. Colon and liver were analysed after administration of DEP in the diet with or without increasing doses of sucrose. This study indicated that DEP induces DNA adducts and oxidative stress through formation of DNA strand breaks, DNA repair enzyme expression, apoptosis, and protein oxidisation in colon and liver at relatively low exposure doses. The thesis is based on four published journal articles. (ln)

Radiation damage to DNA-binding proteins

International Nuclear Information System (INIS)

Culard, G.; Eon, S.; DeVuyst, G.; Charlier, M.; Spotheim-Maurizot, M.

2003-01-01

The DNA-binding properties of proteins are strongly affected upon irradiation. The tetrameric lactose repressor (a dimer of dimers) losses its ability to bind operator DNA as soon as at least two damages per protomer of each dimer occur. The monomeric MC1 protein losses its ability to bind DNA in two steps : i) at low doses only the specific binding is abolished, whereas the non-specific one is still possible; ii) at high doses all binding vanishes. Moreover, the DNA bending induced by MC1 binding is less pronounced for a protein that underwent the low dose irradiation. When the entire DNA-protein complexes are irradiated, the observed disruption of the complexes is mainly due to the damage of the proteins and not to that of DNA. The doses necessary for complex disruption are higher than those inactivating the free protein. This difference, larger for MC1 than for lactose repressor, is due to the protection of the protein by the bound DNA. The oxidation of the protein side chains that are accessible to the radiation-induced hydroxyl radicals seems to represent the inactivating damage

DNA Damage Response and Immune Defence: Links and Mechanisms

Directory of Open Access Journals (Sweden)

Björn Schumacher

2016-08-01

Full Text Available DNA damage plays a causal role in numerous human pathologies including cancer, premature aging and chronic inflammatory conditions. In response to genotoxic insults, the DNA damage response (DDR orchestrates DNA damage checkpoint activation and facilitates the removal of DNA lesions. The DDR can also arouse the immune system by for example inducing the expression of antimicrobial peptides as well as ligands for receptors found on immune cells. The activation of immune signalling is triggered by different components of the DDR including DNA damage sensors, transducer kinases, and effectors. In this review, we describe recent advances on the understanding of the role of DDR in activating immune signalling. We highlight evidence gained into (i which molecular and cellular pathways of DDR activate immune signalling, (ii how DNA damage drives chronic inflammation, and (iii how chronic inflammation causes DNA damage and pathology in humans.

Analysis of damaged DNA / proteins interactions: Methodological optimizations and applications to DNA lesions induced by platinum anticancer drugs

International Nuclear Information System (INIS)

Bounaix Morand du Puch, Ch

2010-10-01

DNA lesions contribute to the alteration of DNA structure, thereby inhibiting essential cellular processes. Such alterations may be beneficial for chemotherapies, for example in the case of platinum anticancer agents. They generate bulky adducts that, if not repaired, ultimately cause apoptosis. A better understanding of the biological response to such molecules can be obtained through the study of proteins that directly interact with the damages. These proteins constitute the DNA lesions interactome. This thesis presents the development of tools aiming at increasing the list of platinum adduct-associated proteins. Firstly, we designed a ligand fishing system made of damaged plasmids immobilized onto magnetic beads. Three platinum drugs were selected for our study: cisplatin, oxali-platin and satra-platin. Following exposure of the trap to nuclear extracts from HeLa cancer cells and identification of retained proteins by proteomics, we obtained already known candidates (HMGB1, hUBF, FACT complex) but also 29 new members of the platinated-DNA interactome. Among them, we noted the presence of PNUTS, TOX4 and WDR82, which associate to form the recently-discovered PTW/PP complex. Their capture was then confirmed with a second model, namely breast cancer cell line MDA MB 231, and the biological consequences of such an interaction now need to be elucidated. Secondly, we adapted a SPRi bio-chip to the study of platinum-damaged DNA/proteins interactions. Affinity of HMGB1 and newly characterized TOX4 for adducts generated by our three platinum drugs could be validated thanks to the bio-chip. Finally, we used our tools, as well as analytical chemistry and biochemistry methods, to evaluate the role of DDB2 (a factor involved in the recognition of UV-induced lesions) in the repair of cisplatin adducts. Our experiments using MDA MB 231 cells differentially expressing DDB2 showed that this protein is not responsible for the repair of platinum damages. Instead, it appears to act

MDM2 Antagonists Counteract Drug-Induced DNA Damage

Directory of Open Access Journals (Sweden)

Anna E. Vilgelm

2017-10-01

Full Text Available Antagonists of MDM2-p53 interaction are emerging anti-cancer drugs utilized in clinical trials for malignancies that rarely mutate p53, including melanoma. We discovered that MDM2-p53 antagonists protect DNA from drug-induced damage in melanoma cells and patient-derived xenografts. Among the tested DNA damaging drugs were various inhibitors of Aurora and Polo-like mitotic kinases, as well as traditional chemotherapy. Mitotic kinase inhibition causes mitotic slippage, DNA re-replication, and polyploidy. Here we show that re-replication of the polyploid genome generates replicative stress which leads to DNA damage. MDM2-p53 antagonists relieve replicative stress via the p53-dependent activation of p21 which inhibits DNA replication. Loss of p21 promoted drug-induced DNA damage in melanoma cells and enhanced anti-tumor activity of therapy combining MDM2 antagonist with mitotic kinase inhibitor in mice. In summary, MDM2 antagonists may reduce DNA damaging effects of anti-cancer drugs if they are administered together, while targeting p21 can improve the efficacy of such combinations.

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

Directory of Open Access Journals (Sweden)

Ana Belén Herrero

2017-05-01

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

Preterm newborns show slower repair of oxidative damage and paternal smoking associated DNA damage.

Science.gov (United States)

Vande Loock, Kim; Ciardelli, Roberta; Decordier, Ilse; Plas, Gina; Haumont, Dominique; Kirsch-Volders, Micheline

2012-09-01

Newborns have to cope with hypoxia during delivery and a sudden increase in oxygen at birth. Oxygen will partly be released as reactive oxygen species having the potential to cause damage to DNA and proteins. In utero, increase of most (non)-enzymatic antioxidants occurs during last weeks of gestation, making preterm neonates probably more sensitive to oxidative stress. Moreover, it has been hypothesized that oxidative stress might be the common etiological factor for certain neonatal diseases in preterm infants. The aim of this study was to assess background DNA damage; in vitro H(2)O(2) induced oxidative DNA damage and repair capacity (residual DNA damage) in peripheral blood mononucleated cells from 25 preterm newborns and their mothers. In addition, demographic data were taken into account and repair capacity of preterm was compared with full-term newborns. Multivariate linear regression analysis revealed that preterm infants from smoking fathers have higher background DNA damage levels than those from non-smoking fathers, emphasizing the risk of paternal smoking behaviour for the progeny. Significantly higher residual DNA damage found after 15-min repair in preterm children compared to their mothers and higher residual DNA damage after 2 h compared to full-term newborns suggest a slower DNA repair capacity in preterm children. In comparison with preterm infants born by caesarean delivery, preterm infants born by vaginal delivery do repair more slowly the in vitro induced oxidative DNA damage. Final impact of passive smoking and of the slower DNA repair activity of preterm infants need to be confirmed in a larger study population combining transgenerational genetic and/or epigenetic effects, antioxidant levels, genotypes, repair enzyme efficiency/levels and infant morbidity.

Bortezomib-induced sensitization of malignant human glioma cells to vorinostat-induced apoptosis depends on reactive oxygen species production, mitochondrial dysfunction, Noxa upregulation, Mcl-1 cleavage, and DNA damage.

Science.gov (United States)

Premkumar, Daniel R; Jane, Esther P; Agostino, Naomi R; DiDomenico, Joseph D; Pollack, Ian F

2013-02-01

Glioblastomas are invasive tumors with poor prognosis despite current therapies. Histone deacetylase inhibitors (HDACIs) represent a class of agents that can modulate gene expression to reduce tumor growth, and we and others have noted some antiglioma activity from HDACIs, such as vorinostat, although insufficient to warrant use as monotherapy. We have recently demonstrated that proteasome inhibitors, such as bortezomib, dramatically sensitized highly resistant glioma cells to apoptosis induction, suggesting that proteasomal inhibition may be a promising combination strategy for glioma therapeutics. In this study, we examined whether bortezomib could enhance response to HDAC inhibition in glioma cells. Although primary cells from glioblastoma multiforme (GBM) patients and established glioma cell lines did not show significant induction of apoptosis with vorinostat treatment alone, the combination of vorinostat plus bortezomib significantly enhanced apoptosis. The enhanced efficacy was due to proapoptotic mitochondrial injury and increased generation of reactive oxygen species. Our results also revealed that combination of bortezomib with vorinostat enhanced apoptosis by increasing Mcl-1 cleavage, Noxa upregulation, Bak and Bax activation, and cytochrome c release. Further downregulation of Mcl-1 using shRNA enhanced cell killing by the bortezomib/vorinostat combination. Vorinostat induced a rapid and sustained phosphorylation of histone H2AX in primary GBM and T98G cells, and this effect was significantly enhanced by co-administration of bortezomib. Vorinostat/bortezomib combination also induced Rad51 downregulation, which plays an important role in the synergistic enhancement of DNA damage and apoptosis. The significantly enhanced antitumor activity that results from the combination of bortezomib and HDACIs offers promise as a novel treatment for glioma patients. Copyright © 2011 Wiley Periodicals, Inc.

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

Energy Technology Data Exchange (ETDEWEB)

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

2016-08-05

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

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

Directory of Open Access Journals (Sweden)

Adcock Ian M

2002-11-01

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

Chromosomal damage and apoptosis analysis in exfoliated oral epithelial cells from mouthwash and alcohol users

Science.gov (United States)

Rocha, Rodrigo dos Santos; Meireles, José Roberto Cardoso; de Moraes Marcílio Cerqueira, Eneida

2014-01-01

Chromosomal damage and apoptosis were analyzed in users of mouthwash and/or alcoholic beverages, using the micronucleus test on exfoliated oral mucosa cells. Samples from four groups of 20 individuals each were analyzed: three exposed groups (EG1, EG2 and EG3) and a control group (CG). EG1 comprised mouthwash users; EG2 comprised drinkers, and EG3 users of both mouthwashes and alcoholic beverages. Cell material was collected by gently scraping the insides of the cheeks. Then the cells were fixed in a methanol/acetic acid (3:1) solution and stained and counterstained, respectively, with Schiff reactive and fast green. Endpoints were computed on 2,000 cells in a blind test. Statistical analysis showed that chromosomal damage and apoptosis were significantly higher in individuals of groups EG1 and EG3 than in controls (p < 0.005 and p < 0.001, respectively). No significant difference in chromosomal damage and apoptosis was observed between the exposed groups. In EG2, only the occurrence of apoptosis was significantly higher than in the controls. These results suggest that mouthwashes alone or in association with alcoholic drinks induce genotoxic effects, manifested as chromosomal damage and apoptosis. They also suggest that alcoholic drinks are effective for stimulating the process of apoptosis. However, these data need to be confirmed in larger samples. PMID:25505845

Vitamin C for DNA damage prevention

International Nuclear Information System (INIS)

Sram, Radim J.; Binkova, Blanka; Rossner, Pavel

2012-01-01

The ability of vitamin C to affect genetic damage was reviewed in human studies that used molecular epidemiology methods, including analysis of DNA adducts, DNA strand breakage (using the Comet assay), oxidative damage measured as levels of 8-oxo-7,8-dihydroxy-2′-deoxyguanosine (8-oxodG), cytogenetic analysis of chromosomal aberrations and micronuclei, and the induction of DNA repair proteins. The protective effect of vitamin C was observed at plasma levels > 50 μmol/l. Vitamin C supplementation decreased the frequency of chromosomal aberrations in groups with insufficient dietary intake who were occupationally exposed to mutagens, and also decreased the sensitivity to mutagens as assessed using the bleomycin assay. High vitamin C levels in plasma decreased the frequency of genomic translocations in groups exposed to ionizing radiation or c-PAHs in polluted air. The frequency of micronuclei was decreased by vitamin C supplementation in smokers challenged with γ-irradiation, and higher vitamin C levels in plasma counteracted the damage induced by air pollution. The prevalence of DNA adducts inversely correlated with vitamin C levels in groups environmentally exposed to high concentrations of c-PAHs. Increased vitamin C levels decreased DNA strand breakage induced by air pollution. Oxidative damage (8-oxodG levels) was decreased by vitamin C supplementation in groups with plasma levels > 50 μmol/l exposed to PM2.5 and c-PAHs. Modulation of DNA repair by vitamin C supplementation was observed both in poorly nourished subjects and in groups with vitamin C plasma levels > 50 μmol/l exposed to higher concentrations of c-PAHs. It is possible that the impact of vitamin C on DNA damage depends both on background values of vitamin C in the individual as well as on the level of exposure to xenobiotics or oxidative stress.

Vitamin C for DNA damage prevention

Energy Technology Data Exchange (ETDEWEB)

Sram, Radim J., E-mail: sram@biomed.cas.cz [Institute of Experimental Medicine, Academy of Sciences of the Czech Republic, 14220 Prague 4 (Czech Republic); Binkova, Blanka; Rossner, Pavel [Institute of Experimental Medicine, Academy of Sciences of the Czech Republic, 14220 Prague 4 (Czech Republic)

2012-05-01

The ability of vitamin C to affect genetic damage was reviewed in human studies that used molecular epidemiology methods, including analysis of DNA adducts, DNA strand breakage (using the Comet assay), oxidative damage measured as levels of 8-oxo-7,8-dihydroxy-2 Prime -deoxyguanosine (8-oxodG), cytogenetic analysis of chromosomal aberrations and micronuclei, and the induction of DNA repair proteins. The protective effect of vitamin C was observed at plasma levels > 50 {mu}mol/l. Vitamin C supplementation decreased the frequency of chromosomal aberrations in groups with insufficient dietary intake who were occupationally exposed to mutagens, and also decreased the sensitivity to mutagens as assessed using the bleomycin assay. High vitamin C levels in plasma decreased the frequency of genomic translocations in groups exposed to ionizing radiation or c-PAHs in polluted air. The frequency of micronuclei was decreased by vitamin C supplementation in smokers challenged with {gamma}-irradiation, and higher vitamin C levels in plasma counteracted the damage induced by air pollution. The prevalence of DNA adducts inversely correlated with vitamin C levels in groups environmentally exposed to high concentrations of c-PAHs. Increased vitamin C levels decreased DNA strand breakage induced by air pollution. Oxidative damage (8-oxodG levels) was decreased by vitamin C supplementation in groups with plasma levels > 50 {mu}mol/l exposed to PM2.5 and c-PAHs. Modulation of DNA repair by vitamin C supplementation was observed both in poorly nourished subjects and in groups with vitamin C plasma levels > 50 {mu}mol/l exposed to higher concentrations of c-PAHs. It is possible that the impact of vitamin C on DNA damage depends both on background values of vitamin C in the individual as well as on the level of exposure to xenobiotics or oxidative stress.

Role of the Checkpoint Clamp in DNA Damage Response

Directory of Open Access Journals (Sweden)

Mihoko Kai

2013-01-01

Full Text Available DNA damage occurs during DNA replication, spontaneous chemical reactions, and assaults by external or metabolism-derived agents. Therefore, all living cells must constantly contend with DNA damage. Cells protect themselves from these genotoxic stresses by activating the DNA damage checkpoint and DNA repair pathways. Coordination of these pathways requires tight regulation in order to prevent genomic instability. The checkpoint clamp complex consists of Rad9, Rad1 and Hus1 proteins, and is often called the 9-1-1 complex. This PCNA (proliferating cell nuclear antigen-like donut-shaped protein complex is a checkpoint sensor protein that is recruited to DNA damage sites during the early stage of the response, and is required for checkpoint activation. As PCNA is required for multiple pathways of DNA metabolism, the checkpoint clamp has also been implicated in direct roles in DNA repair, as well as in coordination of the pathways. Here we discuss roles of the checkpoint clamp in DNA damage response (DDR.

DNA damage-inducible transcripts in mammalian cells

International Nuclear Information System (INIS)

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

1988-01-01

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

Cells Lacking mtDNA Display Increased dNTP Pools upon DNA Damage

DEFF Research Database (Denmark)

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

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

Imaging the DNA damage response with PET and SPECT

Energy Technology Data Exchange (ETDEWEB)

Knight, James C.; Koustoulidou, Sofia; Cornelissen, Bart [University of Oxford, CR-UK/MRC Oxford Institute for Radiation Oncology, Department of Oncology, Oxford (United Kingdom)

2017-06-15

DNA integrity is constantly challenged by endogenous and exogenous factors that can alter the DNA sequence, leading to mutagenesis, aberrant transcriptional activity, and cytotoxicity. Left unrepaired, damaged DNA can ultimately lead to the development of cancer. To overcome this threat, a series of complex mechanisms collectively known as the DNA damage response (DDR) are able to detect the various types of DNA damage that can occur and stimulate the appropriate repair process. Each DNA damage repair pathway leads to the recruitment, upregulation, or activation of specific proteins within the nucleus, which, in some cases, can represent attractive targets for molecular imaging. Given the well-established involvement of DDR during tumorigenesis and cancer therapy, the ability to monitor these repair processes non-invasively using nuclear imaging techniques may facilitate the earlier detection of cancer and may also assist in monitoring response to DNA damaging treatment. This review article aims to provide an overview of recent efforts to develop PET and SPECT radiotracers for imaging of DNA damage repair proteins. (orig.)

A decrease in cyclin B1 levels leads to polyploidization in DNA damage-induced senescence.

Science.gov (United States)

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.

Quantitative analysis of gene-specific DNA damage in human spermatozoa

International Nuclear Information System (INIS)

Sawyer, Dennis E.; Mercer, Belinda G.; Wiklendt, Agnieszka M.; Aitken, R. John

2003-01-01

Recent studies have suggested that human spermatozoa are highly susceptible to DNA damage induced by oxidative stress. However, a detailed analysis of the precise nature of this damage and the extent to which it affects the mitochondrial and nuclear genomes has not been reported. To induce DNA damage, human spermatozoa were treated in vitro with hydrogen peroxide (H 2 O 2 ; 0-5 mM) or iron (as Fe(II)SO 4 , 0-500 μM). Quantitative PCR (QPCR) was used to measure DNA damage in individual nuclear genes (hprt, β-pol and β-globin) and mitochondrial DNA. Single strand breaks were also assessed by alkaline gel electrophoresis. H 2 O 2 was found to be genotoxic toward spermatozoa at concentrations as high as 1.25 mM, but DNA damage was not detected in these cells with lower concentrations of H 2 O 2 . The mitochondrial genome of human spermatozoa was significantly (P 2 O 2 -induced DNA damage than the nuclear genome. However, both nDNA and mtDNA in human spermatozoa were significantly (P<0.001) more resistant to damage than DNA from a variety of cell lines of germ cell and myoblastoid origin. Interestingly, significant DNA damage was also not detected in human spermatozoa treated with iron. These studies report, for the first time, quantitative measurements of DNA damage in specific genes of male germ cells, and challenge the commonly held belief that human spermatozoa are particularly vulnerable to DNA damage

A p53-independent role for the MDM2 antagonist Nutlin-3 in DNA damage response initiation

Directory of Open Access Journals (Sweden)

Kumar Sonia

2011-02-01

Full Text Available Abstract Background The mammalian DNA-damage response (DDR has evolved to protect genome stability and maximize cell survival following DNA-damage. One of the key regulators of the DDR is p53, itself tightly regulated by MDM2. Following double-strand DNA breaks (DSBs, mediators including ATM are recruited to the site of DNA-damage. Subsequent phosphorylation of p53 by ATM and ATM-induced CHK2 results in p53 stabilization, ultimately intensifying transcription of p53-responsive genes involved in DNA repair, cell-cycle checkpoint control and apoptosis. Methods In the current study, we investigated the stabilization and activation of p53 and associated DDR proteins in response to treatment of human colorectal cancer cells (HCT116p53+/+ with the MDM2 antagonist, Nutlin-3. Results Using immunoblotting, Nutlin-3 was observed to stabilize p53, and activate p53 target proteins. Unexpectedly, Nutlin-3 also mediated phosphorylation of p53 at key DNA-damage-specific serine residues (Ser15, 20 and 37. Furthermore, Nutlin-3 induced activation of CHK2 and ATM - proteins required for DNA-damage-dependent phosphorylation and activation of p53, and the phosphorylation of BRCA1 and H2AX - proteins known to be activated specifically in response to DNA damage. Indeed, using immunofluorescent labeling, Nutlin-3 was seen to induce formation of γH2AX foci, an early hallmark of the DDR. Moreover, Nutlin-3 induced phosphorylation of key DDR proteins, initiated cell cycle arrest and led to formation of γH2AX foci in cells lacking p53, whilst γH2AX foci were also noted in MDM2-deficient cells. Conclusion To our knowledge, this is the first solid evidence showing a secondary role for Nutlin-3 as a DDR triggering agent, independent of p53 status, and unrelated to its role as an MDM2 antagonist.

Sunlight-induced DNA damage in human mononuclear cells

DEFF Research Database (Denmark)

Møller, Peter; Wallin, Hakan; Holst, Erik

2002-01-01

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

DNA damage induction of ribonucleotide reductase.

OpenAIRE

Elledge, S J; Davis, R W

1989-01-01

RNR2 encodes the small subunit of ribonucleotide reductase, the enzyme that catalyzes the first step in the pathway for the production of deoxyribonucleotides needed for DNA synthesis. RNR2 is a member of a group of genes whose activities are cell cycle regulated and that are transcriptionally induced in response to the stress of DNA damage. An RNR2-lacZ fusion was used to further characterize the regulation of RNR2 and the pathway responsible for its response to DNA damage. beta-Galactosidas...

Mechanisms of free radical-induced damage to DNA.

Science.gov (United States)

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.

The apoptosis of CHO cells induced by X-rays

International Nuclear Information System (INIS)

Lu Zhaohong; Zhao Jingyong; Zhu Mingqing; Shi Xijin; Wang Chunlei

2004-01-01

The work is to study the mechanism of toxic effects on reproductive system and apoptosis of Chinese hamster ovary (CHO) cells induced by X-rays. CHO cell was exposed to X-rays 2 to 20 Gy. Apoptosis and morphological changes of the cells were observed by fluorescent microscopy and flow cytometry analyzer with double staining with Annexin V/PI. The apoptosis could be observed at 24, 48 and 72h after the exposure, but it was more obvious 48 and 72 h after the exposure. Rate of the apoptosis increased along with radiation dose were elevated. Some morphological changes, such as irregular agglomerate of chromatins, pycnosis and periphery distribution of nuclei, crescent-moon-like cells, small apoptosis body, were observed. Radiation results DNA damage in the CHO cells, and the damage cannot be repaired, hence the induced cell apoptosis. (authors)

uv photobiology: DNA damage and repair

International Nuclear Information System (INIS)

Sutherland, B.M.

1978-01-01

The following topics are discussed: targets that determine the fate of the cell when uv light interacts with a cell; comparison of action spectrum for a given biological effect with the absorption spectrum of different biological macromolecules; biological effects of damage to DNA; measurement of mutations; chemical damage to DNA; photoreactivation; role of pyrimidine dimers in induction of skin cancer by uv

DNA damage in plant herbarium tissue.

NARCIS (Netherlands)

Staats, M.; Cuenca, A.; Richardson, J.E.; Ginkel, R.V.; Petersen, G.; Seberg, O.; Bakker, F.T.

2011-01-01

Dried plant herbarium specimens are potentially a valuable source of DNA. Efforts to obtain genetic information from this source are often hindered by an inability to obtain amplifiable DNA as herbarium DNA is typically highly degraded. DNA post-mortem damage may not only reduce the number of

Radiation damage of DNA. Model for direct ionization of DNA

International Nuclear Information System (INIS)

Kobayashi, Kazuo; Tagawa, Seiichi

2004-01-01

Current aspects of radiation damage of DNA, particularly induced by the direct effect of radiation, and author's method of pulse radiolysis are described in relation to behavior of ions formed by radiation and active principles to induce the strand break. In irradiation of DNA solution in water, the direct effect of radiation is derived from ionization of DNA itself and indirect one, from the reaction between DNA and radicals generated from water molecules and the former direct one has been scarcely investigated due to difficulty of experimental approach. Radicals generated in sugar moiety of DNA are shown important in the strand break by recent studies on crystalline DNA irradiated by X-ray, DNA solution by electron and photon beams, hydrated DNA by γ-ray and by high linear energy transfer (LET) ion. Author's pulse radiolysis studies have revealed behaviors of guanine and adenine radical cations in dynamics of DNA oxidation. Since reactions described are the model, the experimental approach is thought necessary for elucidation of the actually occurring DNA damage in living cells. (N.I.)

DNA-damage response during mitosis induces whole-chromosome missegregation.

Science.gov (United States)

Bakhoum, Samuel F; Kabeche, Lilian; Murnane, John P; Zaki, Bassem I; Compton, Duane A

2014-11-01

Many cancers display both structural (s-CIN) and numerical (w-CIN) chromosomal instabilities. Defective chromosome segregation during mitosis has been shown to cause DNA damage that induces structural rearrangements of chromosomes (s-CIN). In contrast, whether DNA damage can disrupt mitotic processes to generate whole chromosomal instability (w-CIN) is unknown. Here, we show that activation of the DNA-damage response (DDR) during mitosis selectively stabilizes kinetochore-microtubule (k-MT) attachments to chromosomes through Aurora-A and PLK1 kinases, thereby increasing the frequency of lagging chromosomes during anaphase. Inhibition of DDR proteins, ATM or CHK2, abolishes the effect of DNA damage on k-MTs and chromosome segregation, whereas activation of the DDR in the absence of DNA damage is sufficient to induce chromosome segregation errors. Finally, inhibiting the DDR during mitosis in cancer cells with persistent DNA damage suppresses inherent chromosome segregation defects. Thus, the DDR during mitosis inappropriately stabilizes k-MTs, creating a link between s-CIN and w-CIN. The genome-protective role of the DDR depends on its ability to delay cell division until damaged DNA can be fully repaired. Here, we show that when DNA damage is induced during mitosis, the DDR unexpectedly induces errors in the segregation of entire chromosomes, thus linking structural and numerical chromosomal instabilities. ©2014 American Association for Cancer Research.

Berberine induces p53-dependent cell cycle arrest and apoptosis of human osteosarcoma cells by inflicting DNA damage

International Nuclear Information System (INIS)

Liu Zhaojian; Liu Qiao; Xu Bing; Wu Jingjing; Guo Chun; Zhu Faliang; Yang Qiaozi; Gao Guimin; Gong Yaoqin; Shao Changshun

2009-01-01

Alkaloid berberine is widely used for the treatment of diarrhea and other diseases. Many laboratory studies showed that it exhibits anti-proliferative activity against a wide spectrum of cancer cells in culture. In this report we studied the mechanisms underlying the inhibitory effects of berberine on human osteosarcoma cells and on normal osteoblasts. The inhibition was largely attributed to cell cycle arrest at G1 and G2/M, and to a less extent, to apoptosis. The G1 arrest was dependent on p53, as G1 arrest was abolished in p53-deficient osteosarcoma cells. The induction of G1 arrest and apoptosis was accompanied by a p53-dependent up-regulation of p21 and pro-apoptotic genes. However, the G2/M arrest could be induced by berberine regardless of the status of p53. Interestingly, DNA double-strand breaks, as measured by the phosphorylation of H2AX, were remarkably accumulated in berberine-treated cells in a dose-dependent manner. Thus, one major mechanism by which berberine exerts its growth-inhibitory effect is to inflict genomic lesions on cells, which in turn trigger the activation of p53 and the p53-dependent cellular responses including cell cycle arrest and apoptosis

Berberine induces p53-dependent cell cycle arrest and apoptosis of human osteosarcoma cells by inflicting DNA damage

Energy Technology Data Exchange (ETDEWEB)

Liu Zhaojian; Liu Qiao; Xu Bing; Wu Jingjing [Key Laboratory of Experimental Teratology of Ministry of Education and Institute of Molecular Medicine and Genetics, Shandong University School of Medicine, Jinan, Shandong 250012 (China); Guo Chun; Zhu Faliang [Institute of Immunology, Shandong University School of Medicine, Jinan, Shandong 250012 (China); Yang Qiaozi [Department of Genetics, Rutgers University, Piscataway, NJ 08854 (United States); Gao Guimin [Key Laboratory of Experimental Teratology of Ministry of Education and Institute of Molecular Medicine and Genetics, Shandong University School of Medicine, Jinan, Shandong 250012 (China); Gong Yaoqin [Key Laboratory of Experimental Teratology of Ministry of Education and Institute of Molecular Medicine and Genetics, Shandong University School of Medicine, Jinan, Shandong 250012 (China)], E-mail: yxg8@sdu.edu.cn; Shao Changshun [Key Laboratory of Experimental Teratology of Ministry of Education and Institute of Molecular Medicine and Genetics, Shandong University School of Medicine, Jinan, Shandong 250012 (China); Department of Genetics, Rutgers University, Piscataway, NJ 08854 (United States)], E-mail: shao@biology.rutgers.edu

2009-03-09

Alkaloid berberine is widely used for the treatment of diarrhea and other diseases. Many laboratory studies showed that it exhibits anti-proliferative activity against a wide spectrum of cancer cells in culture. In this report we studied the mechanisms underlying the inhibitory effects of berberine on human osteosarcoma cells and on normal osteoblasts. The inhibition was largely attributed to cell cycle arrest at G1 and G2/M, and to a less extent, to apoptosis. The G1 arrest was dependent on p53, as G1 arrest was abolished in p53-deficient osteosarcoma cells. The induction of G1 arrest and apoptosis was accompanied by a p53-dependent up-regulation of p21 and pro-apoptotic genes. However, the G2/M arrest could be induced by berberine regardless of the status of p53. Interestingly, DNA double-strand breaks, as measured by the phosphorylation of H2AX, were remarkably accumulated in berberine-treated cells in a dose-dependent manner. Thus, one major mechanism by which berberine exerts its growth-inhibitory effect is to inflict genomic lesions on cells, which in turn trigger the activation of p53 and the p53-dependent cellular responses including cell cycle arrest and apoptosis.

Antioxidative Dietary Compounds Modulate Gene Expression Associated with Apoptosis, DNA Repair, Inhibition of Cell Proliferation and Migration

Directory of Open Access Journals (Sweden)

Likui Wang

2014-09-01

Full Text Available Many dietary compounds are known to have health benefits owing to their antioxidative and anti-inflammatory properties. To determine the molecular mechanism of these food-derived compounds, we analyzed their effect on various genes related to cell apoptosis, DNA damage and repair, oxidation and inflammation using in vitro cell culture assays. This review further tests the hypothesis proposed previously that downstream products of COX-2 (cyclooxygenase-2 called electrophilic oxo-derivatives induce antioxidant responsive elements (ARE, which leads to cell proliferation under antioxidative conditions. Our findings support this hypothesis and show that cell proliferation was inhibited when COX-2 was down-regulated by polyphenols and polysaccharides. Flattened macrophage morphology was also observed following the induction of cytokine production by polysaccharides extracted from viili, a traditional Nordic fermented dairy product. Coix lacryma-jobi (coix polysaccharides were found to reduce mitochondrial membrane potential and induce caspase-3- and 9-mediated apoptosis. In contrast, polyphenols from blueberries were involved in the ultraviolet-activated p53/Gadd45/MDM2 DNA repair system by restoring the cell membrane potential. Inhibition of hypoxia-inducible factor-1 by saponin extracts of ginsenoside (Ginsen and Gynostemma and inhibition of S100A4 by coix polysaccharides inhibited cancer cell migration and invasion. These observations suggest that antioxidants and changes in cell membrane potential are the major driving forces that transfer signals through the cell membrane into the cytosol and nucleus, triggering gene expression, changes in cell proliferation and the induction of apoptosis or DNA repair.

Association of sperm apoptosis and DNA ploidy with sperm chromatin quality in human spermatozoa.

Science.gov (United States)

Mahfouz, Reda Z; Sharma, Rakesh K; Said, Tamer M; Erenpreiss, Juris; Agarwal, Ashok

2009-04-01

To examine the relationship among sperm apoptosis, sperm chromatin status, and DNA ploidy in different sperm fractions. Prospective study. Reproductive research center in a tertiary care hospital. Sperm prepared by density gradient were evaluated for sperm count, motility, apoptosis, and sperm chromatin assessment. Sperm count, sperm motility, toluidine blue (TB) results, DNA fragmentation index (%DFI), high DNA stainability, DNA cytometry, and early and late apoptosis. Sperm motility was related to late apoptotic and subhaploid apoptotic sperm (r = -0.56 and -0.53, respectively). The sperm %DFI showed significant correlation with late apoptotic and subhaploid sperm (r = 0.62 and 0.68). TB-stained sperm were significantly correlated with late apoptotic sperm (r = 0.51). Significantly higher proportions of haploid sperm and light blue TB-stained sperm were seen in mature compared with immature fractions. Even in semen samples with low %DFI, semen processing results in a lower incidence of nuclear immaturity and subhaploidy, but the incidence of late apoptotic sperm remains unchanged. Therefore, simultaneous evaluation of apoptosis and sperm chromatin status is important for processing sperm in assisted reproductive procedures.

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

Science.gov (United States)

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

2015-01-01

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

Hydroxytyrosol Protects against Oxidative DNA Damage in Human Breast Cells

Directory of Open Access Journals (Sweden)

José J. Gaforio

2011-10-01

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

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

Science.gov (United States)

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

2018-04-16

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

DNA damage caused by UV- and near UV-irradiation

International Nuclear Information System (INIS)

Ohnishi, Takeo

1986-01-01

Much work with mutants deficient in DNA repair has been performed concerning UV-induced DNA damage under the condition where there is no artificial stimulation. In an attempt to infer the effects of solar wavelengths, the outcome of the work is discussed in terms of cellular radiation sensitivity, unscheduled DNA synthesis, and mutation induction, leading to the conclusion that some DNA damage occurs even by irradiation of the shorter wavelength light (270 - 315 nm) and is repaired by excision repair. It has been thought to date that pyrimidine dimer (PD) plays the most important role in UV-induced DNA damage, followed by (6 - 4) photoproducts. As for DNA damage induced by near UV irradiation, the yield of DNA single-strand breaks and of DNA-protein crosslinking, other than PD, is considered. The DNA-protein crosslinking has proved to be induced by irradiation at any wavelength of UV ranging from 260 to 425 nm. Near UV irradiation causes the inhibition of cell proliferation to take place. (Namekawa, K.)

DNA damage induced by radionuclide internal irradiation

International Nuclear Information System (INIS)

Cui Fengmei; Zhao Jingyong; Hong Chengjiao; Lao Qinhua; Wang Liuyi; Yang Shuqin

2004-01-01

Objective: To study the DNA damage of peripheral blood mononuclear cell (PBMC) in rats exposed to radionuclide internal irradiation. Methods: The radionuclides were injected into the rats and single cell get electrophoresis (SCGE) was performed to detect the length of DNA migration in the rat PBMC. Results: DNA migration in the rat PBMC increased with accumulative dose or dose-rate. It showed good relationship of dose vs. response and of dose-rate vs. response, both relationship could be described as linear models. Conclusion: Radionuclide internal irradiation could cause DNA damage in rat PBMC. (authors)

Damages to DNA that result in neoplastic transformation

International Nuclear Information System (INIS)

Setlow, R.B.

1975-01-01

Some topics discussed are: correlation between carcinogens and mutagens; defective DNA repair in uv-damaged xeroderma pigmentosum cells; analysis of nucleotide damage to DNA following exposure to chemicals or radiations; photoreactivation in uv-irradiated Escherichia coli; tumor development in fish; excision repair as an aid in identifying damage; detection of excision repair; role of endonucleases in repair of uv damage; and alkylation products and tumors

Molecular models for DNA damaged by photoreaction

International Nuclear Information System (INIS)

Pearlman, D.A.; Holbrook, S.R.; Pirkle, D.H.; Kim, S.H.

1985-01-01

Structural models of a DNA molecule containing a radiation-induced psoralen cross-link and of a DNA containing a thymine photodimer were constructed by applying energy-minimization techniques and model-building procedures to data from x-ray crystallographic studies. The helical axes of the models show substantial kinking and unwinding at the sites of the damage, which may have long-range as well as local effects arising from the concomitant changes in the supercoiling and overall structure of the DNA. The damaged areas may also serve as recognition sites for repair enzymes. These results should help in understanding the biologic effects of radiation-induced damage on cells

Parvovirus infection-induced DNA damage response

Science.gov (United States)

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

Oxidative phosphorylation-dependent regulation of cancer cell apoptosis in response to anticancer agents.

Science.gov (United States)

Yadav, N; Kumar, S; Marlowe, T; Chaudhary, A K; Kumar, R; Wang, J; O'Malley, J; Boland, P M; Jayanthi, S; Kumar, T K S; Yadava, N; Chandra, D

2015-11-05

Cancer cells tend to develop resistance to various types of anticancer agents, whether they adopt similar or distinct mechanisms to evade cell death in response to a broad spectrum of cancer therapeutics is not fully defined. Current study concludes that DNA-damaging agents (etoposide and doxorubicin), ER stressor (thapsigargin), and histone deacetylase inhibitor (apicidin) target oxidative phosphorylation (OXPHOS) for apoptosis induction, whereas other anticancer agents including staurosporine, taxol, and sorafenib induce apoptosis in an OXPHOS-independent manner. DNA-damaging agents promoted mitochondrial biogenesis accompanied by increased accumulation of cellular and mitochondrial ROS, mitochondrial protein-folding machinery, and mitochondrial unfolded protein response. Induction of mitochondrial biogenesis occurred in a caspase activation-independent mechanism but was reduced by autophagy inhibition and p53-deficiency. Abrogation of complex-I blocked DNA-damage-induced caspase activation and apoptosis, whereas inhibition of complex-II or a combined deficiency of OXPHOS complexes I, III, IV, and V due to impaired mitochondrial protein synthesis did not modulate caspase activity. Mechanistic analysis revealed that inhibition of caspase activation in response to anticancer agents associates with decreased release of mitochondrial cytochrome c in complex-I-deficient cells compared with wild type (WT) cells. Gross OXPHOS deficiencies promoted increased release of apoptosis-inducing factor from mitochondria compared with WT or complex-I-deficient cells, suggesting that cells harboring defective OXPHOS trigger caspase-dependent as well as caspase-independent apoptosis in response to anticancer agents. Interestingly, DNA-damaging agent doxorubicin showed strong binding to mitochondria, which was disrupted by complex-I-deficiency but not by complex-II-deficiency. Thapsigargin-induced caspase activation was reduced upon abrogation of complex-I or gross OXPHOS deficiency

Molecular mechanisms in radiation damage to DNA. Progress report

International Nuclear Information System (INIS)

Osman, R.

1994-01-01

The objectives of this work are to elucidate the molecular mechanisms that are responsible for radiation-induced DNA damage. The overall goal is to understand the relationship between the chemical and structural changes produced by ionizing radiation in DNA and the resulting impairment of biological function expressed as carcinogenesis or cell death. The studies are based on theoretical explorations of possible mechanisms that link initial radiation damage in the form of base and sugar damage to conformational changes in DNA. These mechanistic explorations should lead to the formulation of testable hypotheses regarding the processes of impairment of regulation of gene expression, alteration in DNA repair, and damage to DNA structure involved in cell death or cancer

Quantification of DNA damage by single-cell electrophoresis

International Nuclear Information System (INIS)

Ikushima, Takaji

1990-01-01

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

Mouse Rad9b is essential for embryonic development and promotes resistance to DNA damage

Science.gov (United States)

Leloup, Corinne; Hopkins, Kevin M.; Wang, Xiangyuan; Zhu, Aiping; Wolgemuth, Debra J.; Lieberman, Howard B.

2010-01-01

RAD9 participates in promoting resistance to DNA damage, cell cycle checkpoint control, DNA repair, apoptosis, embryogenesis, and regulation of transcription. A paralogue of RAD9 (named RAD9B) has been identified. To define the function of mouse Rad9b (Mrad9b), embryonic stem (ES) cells with a targeted gene deletion were constructed and used to generate Mrad9b mutant mice. Mrad9b−/− embryos are resorbed after E7.5 while some of the heterozygotes die between E12.5 and a few days after birth. Mrad9b is expressed in embryonic brain and Mrad9b+/− embryos exhibit abnormal neural tube closure. Mrad9b−/− mouse embryonic fibroblasts are not viable. Mrad9b−/− ES cells are more sensitive to gamma rays and mitomycin C than Mrad9b+/+ controls, but show normal gamma-ray-induced G2/M checkpoint control. There is no evidence of spontaneous genomic instability in Mrad9b−/− cells. Our findings thus indicate that Mrad9b is essential for embryonic development and mediates resistance to certain DNA damaging agents. PMID:20842695

ATP-dependent chromatin remodeling in the DNA-damage response

Directory of Open Access Journals (Sweden)

Lans Hannes

2012-01-01

Full Text Available Abstract The integrity of DNA is continuously challenged by metabolism-derived and environmental genotoxic agents that cause a variety of DNA lesions, including base alterations and breaks. DNA damage interferes with vital processes such as transcription and replication, and if not repaired properly, can ultimately lead to premature aging and cancer. Multiple DNA pathways signaling for DNA repair and DNA damage collectively safeguard the integrity of DNA. Chromatin plays a pivotal role in regulating DNA-associated processes, and is itself subject to regulation by the DNA-damage response. Chromatin influences access to DNA, and often serves as a docking or signaling site for repair and signaling proteins. Its structure can be adapted by post-translational histone modifications and nucleosome remodeling, catalyzed by the activity of ATP-dependent chromatin-remodeling complexes. In recent years, accumulating evidence has suggested that ATP-dependent chromatin-remodeling complexes play important, although poorly characterized, roles in facilitating the effectiveness of the DNA-damage response. In this review, we summarize the current knowledge on the involvement of ATP-dependent chromatin remodeling in three major DNA repair pathways: nucleotide excision repair, homologous recombination, and non-homologous end-joining. This shows that a surprisingly large number of different remodeling complexes display pleiotropic functions during different stages of the DNA-damage response. Moreover, several complexes seem to have multiple functions, and are implicated in various mechanistically distinct repair pathways.

Study on DNA damages induced by UV radiation

International Nuclear Information System (INIS)

Doan Hong Van; Dinh Ba Tuan; Tran Tuan Anh; Nguyen Thuy Ngan; Ta Bich Thuan; Vo Thi Thuong Lan; Tran Minh Quynh; Nguyen Thi Thom

2015-01-01

DNA damages in Escherichia coli (E. coli) exposed to UV radiation have been investigated. After 30 min of exposure to UV radiation of 5 mJ/cm"2, the growth of E. coli in LB broth medium was about only 10% in compared with non-irradiated one. This results suggested that the UV radiation caused the damages for E. coli genome resulted in reduction in its growth and survival, and those lesions can be somewhat recovered. For both solutions of plasmid DNAs and E. coli cells containing plasmid DNA, this dose also caused the breakage on single and double strands of DNA, shifted the morphology of DNA plasmid from supercoiled to circular and linear forms. The formation of pyrimidine dimers upon UV radiation significantly reduced when the DNA was irradiated in the presence of Ganoderma lucidum extract. Thus, studies on UV-induced DNA damage at molecular level are very essential to determine the UV radiation doses corresponding to the DNA damages, especially for creation and selection of useful radiation-induced mutants, as well as elucidation the protective effects of the specific compounds against UV light. (author)

Aging of hematopoietic stem cells: DNA damage and mutations?

Science.gov (United States)

Moehrle, Bettina M; Geiger, Hartmut

2016-10-01

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

Epigenetic telomere protection by Drosophila DNA damage response pathways.

Science.gov (United States)

Oikemus, Sarah R; Queiroz-Machado, Joana; Lai, KuanJu; McGinnis, Nadine; Sunkel, Claudio; Brodsky, Michael H

2006-05-01

Analysis of terminal deletion chromosomes indicates that a sequence-independent mechanism regulates protection of Drosophila telomeres. Mutations in Drosophila DNA damage response genes such as atm/tefu, mre11, or rad50 disrupt telomere protection and localization of the telomere-associated proteins HP1 and HOAP, suggesting that recognition of chromosome ends contributes to telomere protection. However, the partial telomere protection phenotype of these mutations limits the ability to test if they act in the epigenetic telomere protection mechanism. We examined the roles of the Drosophila atm and atr-atrip DNA damage response pathways and the nbs homolog in DNA damage responses and telomere protection. As in other organisms, the atm and atr-atrip pathways act in parallel to promote telomere protection. Cells lacking both pathways exhibit severe defects in telomere protection and fail to localize the protection protein HOAP to telomeres. Drosophila nbs is required for both atm- and atr-dependent DNA damage responses and acts in these pathways during DNA repair. The telomere fusion phenotype of nbs is consistent with defects in each of these activities. Cells defective in both the atm and atr pathways were used to examine if DNA damage response pathways regulate telomere protection without affecting telomere specific sequences. In these cells, chromosome fusion sites retain telomere-specific sequences, demonstrating that loss of these sequences is not responsible for loss of protection. Furthermore, terminally deleted chromosomes also fuse in these cells, directly implicating DNA damage response pathways in the epigenetic protection of telomeres. We propose that recognition of chromosome ends and recruitment of HP1 and HOAP by DNA damage response proteins is essential for the epigenetic protection of Drosophila telomeres. Given the conserved roles of DNA damage response proteins in telomere function, related mechanisms may act at the telomeres of other organisms.

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

Science.gov (United States)

Solozobova, Valeriya; Rolletschek, Alexandra; Blattner, Christine

2009-06-17

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

E2F1 induces p19INK4d, a protein involved in the DNA damage response, following UV irradiation.

Science.gov (United States)

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.

Infrared A radiation promotes survival of human melanocytes carrying ultraviolet radiation-induced DNA damage.

Science.gov (United States)

Kimeswenger, Susanne; Schwarz, Agatha; Födinger, Dagmar; Müller, Susanne; Pehamberger, Hubert; Schwarz, Thomas; Jantschitsch, Christian

2016-06-01

The link between solar radiation and melanoma is still elusive. Although infrared radiation (IR) accounts for over 50% of terrestrial solar energy, its influence on human skin is not well explored. There is increasing evidence that IR influences the expression patterns of several molecules independently of heat. A previous in vivo study revealed that pretreatment with IR might promote the development of UVR-induced non-epithelial skin cancer and possibly of melanoma in mice. To expand on this, the aim of the present study was to evaluate the impact of IR on UVR-induced apoptosis and DNA repair in normal human epidermal melanocytes. The balance between these two effects is a key factor of malignant transformation. Human melanocytes were exposed to physiologic doses of IR and UVR. Compared to cells irradiated with UVR only, simultaneous exposure to IR significantly reduced the apoptotic rate. However, IR did not influence the repair of UVR-induced DNA damage. IR partly reversed the pro-apoptotic effects of UVR via modification of the expression and activity of proteins mainly of the extrinsic apoptotic pathway. In conclusion, IR enhances the survival of melanocytes carrying UVR-induced DNA damage and thereby might contribute to melanomagenesis. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Aging and oxidatively damaged nuclear DNA in animal organs

DEFF Research Database (Denmark)

Møller, Peter; Løhr, Mille; Folkmann, Janne K

2010-01-01

Oxidative stress is considered to contribute to aging and is associated with the generation of oxidatively damaged DNA, including 8-oxo-7,8-dihydroguanine. We have identified 69 studies that have measured the level of oxidatively damaged DNA in organs of animals at various ages. In general, organs...... with limited cell proliferation, i.e., liver, kidney, brain, heart, pancreas, and muscle, tended to show accumulation of DNA damage with age, whereas organs with highly proliferating cells, such as intestine, spleen, and testis, showed more equivocal or no effect of age. A restricted analysis of studies...... evidence for aging-associated accumulation of oxidatively damaged DNA in organs with limited cell proliferation....

DNA Tetrahedron Delivery Enhances Doxorubicin-Induced Apoptosis of HT-29 Colon Cancer Cells

Science.gov (United States)

Zhang, Guiyu; Zhang, Zhiyong; Yang, Junen

2017-08-01

As a nano-sized drug carrier with the advantage of modifiability and proper biocompatibility, DNA tetrahedron (DNA tetra) delivery is hopeful to enhance the inhibitory efficiency of nontargeted anticancer drugs. In this investigation, doxorubicin (Dox) was assembled to a folic acid-modified DNA tetra via click chemistry to prepare a targeted antitumor agent. Cellular uptake efficiency was measured via fluorescent imaging. Cytotoxicity, inhibition efficiency, and corresponding mechanism on colon cancer cell line HT-29 were evaluated by MTT assay, cell proliferation curve, western blot, and flow cytometry. No cytotoxicity was induced by DNA tetra, but the cellular uptake ratio increased obviously resulting from the DNA tetra-facilitated penetration through cellular membrane. Accordingly, folic acid-DNA tetra-Dox markedly increased the antitumor efficiency with increased apoptosis levels. In details, 100 μM was the effective concentration and a 6-h incubation period was needed for apoptosis induction. In conclusion, nano-sized DNA tetrahedron was a safe and effective delivery system for Dox and correspondingly enhanced the anticancer efficiency.

Mitochondrial DNA repair and aging

Energy Technology Data Exchange (ETDEWEB)

Mandavilli, Bhaskar S.; Santos, Janine H.; Van Houten, Bennett

2002-11-30

The mitochondrial electron transport chain plays an important role in energy production in aerobic organisms and is also a significant source of reactive oxygen species that damage DNA, RNA and proteins in the cell. Oxidative damage to the mitochondrial DNA is implicated in various degenerative diseases, cancer and aging. The importance of mitochondrial ROS in age-related degenerative diseases is further strengthened by studies using animal models, Caenorhabditis elegans, Drosophila and yeast. Research in the last several years shows that mitochondrial DNA is more susceptible to various carcinogens and ROS when compared to nuclear DNA. DNA damage in mammalian mitochondria is repaired by base excision repair (BER). Studies have shown that mitochondria contain all the enzymes required for BER. Mitochondrial DNA damage, if not repaired, leads to disruption of electron transport chain and production of more ROS. This vicious cycle of ROS production and mtDNA damage ultimately leads to energy depletion in the cell and apoptosis.

Mitochondrial DNA repair and aging

International Nuclear Information System (INIS)

Mandavilli, Bhaskar S.; Santos, Janine H.; Van Houten, Bennett

2002-01-01

The mitochondrial electron transport chain plays an important role in energy production in aerobic organisms and is also a significant source of reactive oxygen species that damage DNA, RNA and proteins in the cell. Oxidative damage to the mitochondrial DNA is implicated in various degenerative diseases, cancer and aging. The importance of mitochondrial ROS in age-related degenerative diseases is further strengthened by studies using animal models, Caenorhabditis elegans, Drosophila and yeast. Research in the last several years shows that mitochondrial DNA is more susceptible to various carcinogens and ROS when compared to nuclear DNA. DNA damage in mammalian mitochondria is repaired by base excision repair (BER). Studies have shown that mitochondria contain all the enzymes required for BER. Mitochondrial DNA damage, if not repaired, leads to disruption of electron transport chain and production of more ROS. This vicious cycle of ROS production and mtDNA damage ultimately leads to energy depletion in the cell and apoptosis

Cellular responses to environmental DNA damage

Energy Technology Data Exchange (ETDEWEB)

1994-08-01

This volume contains the proceedings of the conference entitled Cellular Responses to Environmental DNA Damage held in Banff,Alberta December 1--6, 1991. The conference addresses various aspects of DNA repair in sessions titled DNA repair; Basic Mechanisms; Lesions; Systems; Inducible Responses; Mutagenesis; Human Population Response Heterogeneity; Intragenomic DNA Repair Heterogeneity; DNA Repair Gene Cloning; Aging; Human Genetic Disease; and Carcinogenesis. Individual papers are represented as abstracts of about one page in length.

Tumour-cell apoptosis after cisplatin treatment is not telomere dependent.

Science.gov (United States)

Jeyapalan, Jessie C; Saretzki, Gabriele; Leake, Alan; Tilby, Michael J; von Zglinicki, Thomas

2006-06-01

Cisplatin is a major chemotherapeutic agent, especially for the treatment of neuroblastoma. Telomeres with their sequence (TTAGGG)n are probable targets for cisplatin intrastrand cross-linking, but the role of telomeres in mediating cisplatin cytotoxicity is not clear. After exposure to cisplatin as single dose or continuous treatment, we found no loss of telomeres in either SHSY5Y neuroblastoma cells (telomere length, approximately 4 kbp), HeLa 229 cells (telomere length, 20 kbp) or in the acute lymphoblastic T cell line 1301 (telomere length, approximately 80 kbp). There was no induction of telomeric single strand breaks, telomeric overhangs were not degraded and telomerase activity was down-regulated only after massive onset of apoptosis. In contrast, cisplatin induced a delayed formation of DNA strand breaks and induced DNA damage foci containing gamma-H2A.X at nontelomeric sites. Interstitial DNA damage appears to be more important than telomere loss or telomeric damage as inducer of the signal pathway towards apoptosis and/or growth arrest in cisplatin-treated tumour cells.

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

Science.gov (United States)

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

2017-11-22

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

Colorimetric detection of DNA damage by using hemin-graphene nanocomposites

Science.gov (United States)

Wei, W.; Zhang, D. M.; Yin, L. H.; Pu, Y. P.; Liu, S. Q.

2013-04-01

A colorimetric method for detection of DNA damage was developed by using hemin-graphene nanosheets (H-GNs). H-GNs were skillfully synthesized by adsorping of hemin on graphene through π-π interactions. The as-prepared H-GNs possessed both the ability of graphene to differentiate the damage DNA from intact DNA and the catalytic action of hemin. The damaged DNA made H-GNs coagulated to different degrees from the intact DNA because there were different amount of negative charge exposed on their surface, which made a great impact on the solubility of H-GNs. As a result, the corresponding centrifugal supernatant of H-GNs solution showed different color in the presence of 3,3',5,5'-tetramethylbenzidine (TMB) and H2O2, which could be discriminated by naked eyes or by ultraviolet (UV)-visible spectrometer. Based on this, the damaged effects of styrene oxide (SO), NaAsO2 and UV radiation on DNA were studied. Results showed that SO exerted most serious damage effect on DNA although all of them damaged DNA seriously. The new method for detection of DNA damage showed good prospect in the evaluation of genotoxicity of new compounds, the maximum limit of pesticide residue, food additives, and so on, which is important in the fields of food science, pharmaceutical science and pesticide science.

Viral interference with DNA repair by targeting of the single-stranded DNA binding protein RPA.

Science.gov (United States)

Banerjee, Pubali; DeJesus, Rowena; Gjoerup, Ole; Schaffhausen, Brian S

2013-10-01

Correct repair of damaged DNA is critical for genomic integrity. Deficiencies in DNA repair are linked with human cancer. Here we report a novel mechanism by which a virus manipulates DNA damage responses. Infection with murine polyomavirus sensitizes cells to DNA damage by UV and etoposide. Polyomavirus large T antigen (LT) alone is sufficient to sensitize cells 100 fold to UV and other kinds of DNA damage. This results in activated stress responses and apoptosis. Genetic analysis shows that LT sensitizes via the binding of its origin-binding domain (OBD) to the single-stranded DNA binding protein replication protein A (RPA). Overexpression of RPA protects cells expressing OBD from damage, and knockdown of RPA mimics the LT phenotype. LT prevents recruitment of RPA to nuclear foci after DNA damage. This leads to failure to recruit repair proteins such as Rad51 or Rad9, explaining why LT prevents repair of double strand DNA breaks by homologous recombination. A targeted intervention directed at RPA based on this viral mechanism could be useful in circumventing the resistance of cancer cells to therapy.

The co-repressor SMRT delays DNA damage-induced caspase activation by repressing pro-apoptotic genes and modulating the dynamics of checkpoint kinase 2 activation.

Directory of Open Access Journals (Sweden)

Claudio Scafoglio

Full Text Available Checkpoint kinase 2 (Chk2 is a major regulator of DNA damage response and can induce alternative cellular responses: cell cycle arrest and DNA repair or programmed cell death. Here, we report the identification of a new role of Chk2 in transcriptional regulation that also contributes to modulating the balance between survival and apoptosis following DNA damage. We found that Chk2 interacts with members of the NCoR/SMRT transcriptional co-regulator complexes and serves as a functional component of the repressor complex, being required for recruitment of SMRT on the promoter of pro-apoptotic genes upon DNA damage. Thus, the co-repressor SMRT exerts a critical protective action against genotoxic stress-induced caspase activation, repressing a functionally important cohort of pro-apoptotic genes. Amongst them, SMRT is responsible for basal repression of Wip1, a phosphatase that de-phosphorylates and inactivates Chk2, thus affecting a feedback loop responsible for licensing the correct timing of Chk2 activation and the proper execution of the DNA repair process.

Primary DNA Damage in Dry Cleaners with Perchlorethylene Exposure

Directory of Open Access Journals (Sweden)

Mohammad Azimi

2017-10-01

Full Text Available Background: Perchloroethylene is a halogenated solvent widely used in dry cleaning. International agency of research on cancer classified this chemical as a probable human carcinogen. Objective: To evaluate the extent of primary DNA damage in dry cleaner workers who were exposed to perchloroethylene as compared to non-exposed subjects. The effect of exposure modifying factors such as use of personal protective equipment, perceived risk, and reported safe behaviors on observed DNA damage were also studied. Methods: 59 exposed and non-exposed workers were selected from Yazd, Iran. All the 33 exposed workers had work history at least 3 months in the dry cleaning shops. Peripheral blood sampling was performed. Microscope examination was performed under fluorescent microscope (400×. Open comet software was used for image analysis. All biological analysis was performed in one laboratory. Results: Primary DNA damage to leukocytes in dry cleaners was relatively high. The median tail length, %DNA in tail, and tail moment in exposed group were significantly higher than those in non-exposed group. There was no significant difference between smokers and nonsmokers in terms of tail length, tail moment, and %DNA in tail. There was no significant correlation between duration of employment in dry cleaning and observed DNA damage in terms of tail length, tail moment and %DNA in tail. Stratified analysis based on exposed and nonexposed category showed no significant relationship between age and observed DNA damage. Conclusion: Occupationally exposure to perchloroethylene can cause early DNA damage in dry cleaners.

Chemical determination of free radical-induced damage to DNA.

Science.gov (United States)

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.

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

International Nuclear Information System (INIS)

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

2015-01-01

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

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

Science.gov (United States)

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

2015-12-22

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

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

Directory of Open Access Journals (Sweden)

Natalia Bailon-Moscoso

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

Measurement of oxidative damage to DNA in nanomaterial exposed cells and animals

DEFF Research Database (Denmark)

Møller, Peter; Jensen, Ditte Marie; Christophersen, Daniel Vest

2015-01-01

-reactivity with other molecules in cells. This review provides an overview of efforts to reliably detect oxidatively damaged DNA and a critical assessment of the published studies on DNA damage levels. Animal studies with high baseline levels of oxidatively damaged DNA are more likely to show positive associations...... of oxidatively damaged DNA in lung tissue. Oral exposure to nanosized carbon black, TiO2 , carbon nanotubes and ZnO is associated with elevated levels of oxidatively damaged DNA in tissues. These observations are supported by cell culture studies showing concentration-dependent associations between ENM exposure...... and oxidatively damaged DNA measured by the comet assay. Cell culture studies show relatively high variation in the ability of ENMs to oxidatively damage DNA; hence, it is currently impossible to group ENMs according to their DNA damaging potential. Environ. Mol. Mutagen., 2014. © 2014 Wiley Periodicals, Inc....

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

Directory of Open Access Journals (Sweden)

Nadine Schuler

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

Molecular mechanisms in radiation damage to DNA: Final report

International Nuclear Information System (INIS)

Osman, R.

1996-01-01

The objectives of this work were to elucidate the molecular mechanisms that were responsible for radiation-induced DNA damage. The studies were based on theoretical explorations of possible mechanisms that link initial radiation damage in the form of base and sugar damage to conformational changes in DNA

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

International Nuclear Information System (INIS)

Roper, Katherine; Coverley, Dawn

2012-01-01

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

Damage of DNA by radiation and it's recovery, 3

International Nuclear Information System (INIS)

Narita, Noboru; Matsuura, Tomio; Sato, Hiroyuki.

1974-01-01

The damage and recovery of DNA was investigated by the incorporation of thymine derivatives (DHT, I trans, II trans, cis and glycol) into exponentially growing Tetrahymena cells. The strain employed was Tetrahymena pyriformis, Variety I, mating type IV. It is well known that these thymine derivatives are induced in vivo by radiation. The in vivo damage of DNA induced by radiation, and its recovery, were confirmed experimentally by means of gradient separation of sucrose density and by analytical ultra centrifugation (UVC). The recovery of DNA, its excision repair and its recombinational repair were compared with the recovery of Bacillus subtilis whose recovery kinetics were already known. 1) The damage of DNA was more sensitive to glycol than to II trans and cis. On the other hand, DHT is not sensitive for breaking DNA strand. 2) In its recovery damaged DNA was no more sensitive to glycol than to hhp as was true for Bacillus subtilis. (author)

Global chromatin fibre compaction in response to DNA damage

International Nuclear Information System (INIS)

Hamilton, Charlotte; Hayward, Richard L.; Gilbert, Nick

2011-01-01

Highlights: ► Robust KAP1 phosphorylation in response to DNA damage in HCT116 cells. ► DNA repair foci are found in soluble chromatin. ► Biophysical analysis reveals global chromatin fibre compaction after DNA damage. ► DNA damage is accompanied by rapid linker histone dephosphorylation. -- Abstract: DNA is protected by packaging it into higher order chromatin fibres, but this can impede nuclear processes like DNA repair. Despite considerable research into the factors required for signalling and repairing DNA damage, it is unclear if there are concomitant changes in global chromatin fibre structure. In human cells DNA double strand break (DSB) formation triggers a signalling cascade resulting in H2AX phosphorylation (γH2AX), the rapid recruitment of chromatin associated proteins and the subsequent repair of damaged sites. KAP1 is a transcriptional corepressor and in HCT116 cells we found that after DSB formation by chemicals or ionising radiation there was a wave of, predominantly ATM dependent, KAP1 phosphorylation. Both KAP1 and phosphorylated KAP1 were readily extracted from cells indicating they do not have a structural role and γH2AX was extracted in soluble chromatin indicating that sites of damage are not attached to an underlying structural matrix. After DSB formation we did not find a concomitant change in the sensitivity of chromatin fibres to micrococcal nuclease digestion. Therefore to directly investigate higher order chromatin fibre structures we used a biophysical sedimentation technique based on sucrose gradient centrifugation to compare the conformation of chromatin fibres isolated from cells before and after DNA DSB formation. After damage we found global chromatin fibre compaction, accompanied by rapid linker histone dephosphorylation, consistent with fibres being more regularly folded or fibre deformation being stabilized by linker histones. We suggest that following DSB formation, although there is localised chromatin unfolding to

Mechanistic Studies with DNA Polymerases Reveal Complex Outcomes following Bypass of DNA Damage

Directory of Open Access Journals (Sweden)

Robert L. Eoff

2010-01-01

Full Text Available DNA is a chemically reactive molecule that is subject to many different covalent modifications from sources that are both endogenous and exogenous in origin. The inherent instability of DNA is a major obstacle to genomic maintenance and contributes in varying degrees to cellular dysfunction and disease in multi-cellular organisms. Investigations into the chemical and biological aspects of DNA damage have identified multi-tiered and overlapping cellular systems that have evolved as a means of stabilizing the genome. One of these pathways supports DNA replication events by in a sense adopting the mantra that one must “make the best of a bad situation” and tolerating covalent modification to DNA through less accurate copying of the damaged region. Part of this so-called DNA damage tolerance pathway involves the recruitment of specialized DNA polymerases to sites of stalled or collapsed replication forks. These enzymes have unique structural and functional attributes that often allow bypass of adducted template DNA and successful completion of genomic replication. What follows is a selective description of the salient structural features and bypass properties of specialized DNA polymerases with an emphasis on Y-family members.

The circadian clock controls sunburn apoptosis and erythema in mouse skin.

Science.gov (United States)

Gaddameedhi, Shobhan; Selby, Christopher P; Kemp, Michael G; Ye, Rui; Sancar, Aziz

2015-04-01

Epidemiological studies of humans and experimental studies with mouse models suggest that sunburn resulting from exposure to excessive UV light and damage to DNA confers an increased risk for melanoma and non-melanoma skin cancer. Previous reports have shown that both nucleotide excision repair, which is the sole pathway in humans for removing UV photoproducts, and DNA replication are regulated by the circadian clock in mouse skin. Furthermore, the timing of UV exposure during the circadian cycle has been shown to affect skin carcinogenesis in mice. Because sunburn and skin cancer are causally related, we investigated UV-induced sunburn apoptosis and erythema in mouse skin as a function of circadian time. Interestingly, we observed that sunburn apoptosis, inflammatory cytokine induction, and erythema were maximal following an acute early-morning exposure to UV and minimal following an afternoon exposure. Early-morning exposure to UV also produced maximal activation of ataxia telangiectasia mutated and Rad3-related (Atr)-mediated DNA damage checkpoint signaling, including activation of the tumor suppressor p53, which is known to control the process of sunburn apoptosis. These data provide early evidence that the circadian clock has an important role in the erythemal response in UV-irradiated skin. The early morning is when DNA repair is at a minimum, and thus the acute responses likely are associated with unrepaired DNA damage. The prior report that mice are more susceptible to skin cancer induction following chronic irradiation in the AM, when p53 levels are maximally induced, is discussed in terms of the mutational inactivation of p53 during chronic irradiation.

DNA Repair Mechanisms and the Bypass of DNA Damage in Saccharomyces cerevisiae

Science.gov (United States)

Boiteux, Serge; Jinks-Robertson, Sue

2013-01-01

DNA repair mechanisms are critical for maintaining the integrity of genomic DNA, and their loss is associated with cancer predisposition syndromes. Studies in Saccharomyces cerevisiae have played a central role in elucidating the highly conserved mechanisms that promote eukaryotic genome stability. This review will focus on repair mechanisms that involve excision of a single strand from duplex DNA with the intact, complementary strand serving as a template to fill the resulting gap. These mechanisms are of two general types: those that remove damage from DNA and those that repair errors made during DNA synthesis. The major DNA-damage repair pathways are base excision repair and nucleotide excision repair, which, in the most simple terms, are distinguished by the extent of single-strand DNA removed together with the lesion. Mistakes made by DNA polymerases are corrected by the mismatch repair pathway, which also corrects mismatches generated when single strands of non-identical duplexes are exchanged during homologous recombination. In addition to the true repair pathways, the postreplication repair pathway allows lesions or structural aberrations that block replicative DNA polymerases to be tolerated. There are two bypass mechanisms: an error-free mechanism that involves a switch to an undamaged template for synthesis past the lesion and an error-prone mechanism that utilizes specialized translesion synthesis DNA polymerases to directly synthesize DNA across the lesion. A high level of functional redundancy exists among the pathways that deal with lesions, which minimizes the detrimental effects of endogenous and exogenous DNA damage. PMID:23547164

Chafuroside B, an Oolong tea polyphenol, ameliorates UVB-induced DNA damage and generation of photo-immunosuppression related mediators in human keratinocytes.

Directory of Open Access Journals (Sweden)

Tatsuya Hasegawa

Full Text Available Chafuroside B was recently isolated as a new polyphenolic constituent of oolong tea leaves. However, the effects of chafuroside B on skin function have not been examined. In this study, we investigated the protective effects of chafuroside B against UVB-induced DNA damage, apoptosis and generation of photo-immunosuppression related mediators in cultured normal human epidermal keratinocytes (NHEK. Chafuroside B at 1 µM attenuated both UVB-induced apoptosis, evaluated in terms of caspase-3/7 activity, and UVB-induced DNA damage, evaluated in terms of formation of cyclobutane pyrimidine dimers (CPD, in NHEK exposed to UVB (20 mJ/cm2. In addition, chafuroside B at 0.3 or 1 µM suppressed the UVB-induced production of interleukin (IL-10, tumor necrosis factor (TNF-α, and prostaglandin E2 (PGE2, as determined by ELISA, and conversely enhanced IL-12 mRNA expression and production, as measured by RT-PCR and ELISA. Further, chafuroside B at 1 µM also suppressed UVB-induced expression of receptor activator of nuclear factor κB ligand (RANKL mRNA. These results indicate that chafuroside B promotes repair of UVB-induced DNA damage and ameliorates the generation of IL-10, TNF-α, PGE2, and RANKL, all of which are UVB-induced immunosuppression related mediators. These effects of chafuroside B may be mediated at least in part through induction of IL-12 synthesis in human keratinocytes. Because chafuroside B might have practical value as a photoprotective agent, a further study of the in vivo effects of chafuroside B seems warranted.

The complexity of DNA damage: relevance to biological consequences

International Nuclear Information System (INIS)

Ward, J.F.

1994-01-01

Ionizing radiation causes both singly and multiply damaged sites in DNA when the range of radical migration is limited by the presence of hydroxyl radical scavengers (e.g. within cells). Multiply damaged sites are considered to be more biologically relevant because of the challenges they present to cellular repair mechanisms. These sites occur in the form of DNA double-strand breaks (dsb) but also as other multiple damages that can be converted to dsb during attempted repair. The presence of a dsb can lead to loss of base sequence information and/or can permit the two ends of a break to separate and rejoin with the wrong partner. (Multiply damaged sites may also be the biologically relevant type of damage caused by other agents, such as UVA, B and/or C light, and some antitumour antibiotics). The quantitative data available from radiation studies of DNA are shown to support the proposed mechanisms for the production of complex damage in cellular DNA, i.e. via scavengable and non-scavengable mechanisms. The yields of complex damages can in turn be used to support the conclusion that cellular mutations are a consequence of the presence of these damages within a gene. (Author)

DNA repair and the evolution of transformation in Bacillus subtilis. 3. Sex with damaged DNA

International Nuclear Information System (INIS)

Hoelzer, M.A.; Michod, R.E.

1991-01-01

Natural genetic transformation in the bacterium Bacillus subtilis provides an experimental system for studying the evolutionary function of sexual recombination. The repair hypothesis proposes that during transformation the exogenous DNA taken up by cells is used as template for recombinational repair of damages in the recipient cell's genome. Earlier results demonstrated that the population density of transformed cells (i.e., sexual cells) increases, relative to nontransformed cells (primarily asexual cells), with increasing dosage of ultraviolet irradiation, provided that the cells are transformed with undamaged homologous DNA after they have become damaged. In nature, however, donor DNA for transformation is likely to come from cells that are as damaged as the recipient cells. In order to better simulate the effects of transformation in natural populations we conducted similar experiments as those just described using damaged donor DNA. The authors document in this report that transformants continue to increase in relative density even if they are transformed with damaged donor DNA. These results suggest that sites of transformation are often damaged sites in the recipient cell's genome

Silver nanoparticles induced heat shock protein 70, oxidative stress and apoptosis in Drosophila melanogaster.

Science.gov (United States)

Ahamed, Maqusood; Posgai, Ryan; Gorey, Timothy J; Nielsen, Mark; Hussain, Saber M; Rowe, John J

2010-02-01

Due to the intensive commercial application of silver nanoparticles (Ag NPs), risk assessment of this nanoparticle is of great importance. Our previous in vitro study demonstrated that Ag NPs caused DNA damage and apoptosis in mouse embryonic stem cells and fibroblasts. However, toxicity of Ag NPs in vivo is largely lacking. This study was undertaken to examine the toxic effects of well-characterized polysaccharide coated 10 nm Ag NPs on heat shock stress, oxidative stress, DNA damage and apoptosis in Drosophila melanogaster. Third instar larvae of D. melanogaster were fed a diet of standard cornmeal media mixed with Ag NPs at the concentrations of 50 and 100 microg/ml for 24 and 48 h. Ag NPs up-regulated the expression of heat shock protein 70 and induced oxidative stress in D. melanogaster. Malondialdehyde level, an end product of lipid peroxidation was significantly higher while antioxidant glutathione content was significantly lower in Ag NPs exposed organisms. Activities of antioxidant enzyme superoxide dismutase and catalase were also significantly higher in the organisms exposed to Ag NPs. Furthermore, Ag NPs up-regulated the cell cycle checkpoint p53 and cell signaling protein p38 that are involved in the DNA damage repair pathway. Moreover, activities of caspase-3 and caspase-9, markers of apoptosis were significantly higher in Ag NPs exposed organisms. The results indicate that Ag NPs in D. melanogaster induce heat shock stress, oxidative stress, DNA damage and apoptosis. This study suggests that the organism is stressed and thus warrants more careful assessment of Ag NPs using in vivo models to determine if chronic exposure presents developmental and reproductive toxicity. Copyright 2009 Elsevier Inc. All rights reserved.

Silver nanoparticles induced heat shock protein 70, oxidative stress and apoptosis in Drosophila melanogaster

International Nuclear Information System (INIS)

Ahamed, Maqusood; Posgai, Ryan; Gorey, Timothy J.; Nielsen, Mark; Hussain, Saber M.; Rowe, John J.

2010-01-01

Due to the intensive commercial application of silver nanoparticles (Ag NPs), risk assessment of this nanoparticle is of great importance. Our previous in vitro study demonstrated that Ag NPs caused DNA damage and apoptosis in mouse embryonic stem cells and fibroblasts. However, toxicity of Ag NPs in vivo is largely lacking. This study was undertaken to examine the toxic effects of well-characterized polysaccharide coated 10 nm Ag NPs on heat shock stress, oxidative stress, DNA damage and apoptosis in Drosophila melanogaster. Third instar larvae of D. melanogaster were fed a diet of standard cornmeal media mixed with Ag NPs at the concentrations of 50 and 100 μg/ml for 24 and 48 h. Ag NPs up-regulated the expression of heat shock protein 70 and induced oxidative stress in D. melanogaster. Malondialdehyde level, an end product of lipid peroxidation was significantly higher while antioxidant glutathione content was significantly lower in Ag NPs exposed organisms. Activities of antioxidant enzyme superoxide dismutase and catalase were also significantly higher in the organisms exposed to Ag NPs. Furthermore, Ag NPs up-regulated the cell cycle checkpoint p53 and cell signaling protein p38 that are involved in the DNA damage repair pathway. Moreover, activities of caspase-3 and caspase-9, markers of apoptosis were significantly higher in Ag NPs exposed organisms. The results indicate that Ag NPs in D. melanogaster induce heat shock stress, oxidative stress, DNA damage and apoptosis. This study suggests that the organism is stressed and thus warrants more careful assessment of Ag NPs using in vivo models to determine if chronic exposure presents developmental and reproductive toxicity.

Chk2 regulates transcription-independent p53-mediated apoptosis in response to DNA damage

International Nuclear Information System (INIS)

Chen Chen; Shimizu, Shigeomi; Tsujimoto, Yoshihide; Motoyama, Noboru

2005-01-01

The tumor suppressor protein p53 plays a central role in the induction of apoptosis in response to genotoxic stress. The protein kinase Chk2 is an important regulator of p53 function in mammalian cells exposed to ionizing radiation (IR). Cells derived from Chk2-deficient mice are resistant to the induction of apoptosis by IR, and this resistance has been thought to be a result of the defective transcriptional activation of p53 target genes. It was recently shown, however, that p53 itself and histone H1.2 translocate to mitochondria and thereby induces apoptosis in a transcription-independent manner in response to IR. We have now examined whether Chk2 also regulates the transcription-independent induction of apoptosis by p53 and histone H1.2. The reduced ability of IR to induce p53 stabilization in Chk2-deficient thymocytes was associated with a marked impairment of p53 and histone H1 translocation to mitochondria. These results suggest that Chk2 regulates the transcription-independent mechanism of p53-mediated apoptosis by inducing stabilization of p53 in response to IR

The sequence specificity of UV-induced DNA damage in a systematically altered DNA sequence.

Science.gov (United States)

Khoe, Clairine V; Chung, Long H; Murray, Vincent

2018-06-01

The sequence specificity of UV-induced DNA damage was investigated in a specifically designed DNA plasmid using two procedures: end-labelling and linear amplification. Absorption of UV photons by DNA leads to dimerisation of pyrimidine bases and produces two major photoproducts, cyclobutane pyrimidine dimers (CPDs) and pyrimidine(6-4)pyrimidone photoproducts (6-4PPs). A previous study had determined that two hexanucleotide sequences, 5'-GCTC*AC and 5'-TATT*AA, were high intensity UV-induced DNA damage sites. The UV clone plasmid was constructed by systematically altering each nucleotide of these two hexanucleotide sequences. One of the main goals of this study was to determine the influence of single nucleotide alterations on the intensity of UV-induced DNA damage. The sequence 5'-GCTC*AC was designed to examine the sequence specificity of 6-4PPs and the highest intensity 6-4PP damage sites were found at 5'-GTTC*CC nucleotides. The sequence 5'-TATT*AA was devised to investigate the sequence specificity of CPDs and the highest intensity CPD damage sites were found at 5'-TTTT*CG nucleotides. It was proposed that the tetranucleotide DNA sequence, 5'-YTC*Y (where Y is T or C), was the consensus sequence for the highest intensity UV-induced 6-4PP adduct sites; while it was 5'-YTT*C for the highest intensity UV-induced CPD damage sites. These consensus tetranucleotides are composed entirely of consecutive pyrimidines and must have a DNA conformation that is highly productive for the absorption of UV photons. Crown Copyright © 2018. Published by Elsevier B.V. All rights reserved.

Anticancer potential of Conium maculatum extract against cancer cells in vitro: Drug-DNA interaction and its ability to induce apoptosis through ROS generation.

Science.gov (United States)

Mondal, Jesmin; Panigrahi, Ashis Kumar; Khuda-Bukhsh, Anisur Rahman

2014-08-01

Conium maculatum extract is used as a traditional medicine for cervix carcinoma including homeopathy. However, no systematic work has so far been carried out to test its anti-cancer potential against cervix cancer cells in vitro. Thus, in this study, we investigated whether ethanolic extract of conium is capable of inducing cytotoxicity in different normal and cancer cell lines including an elaborate study in HeLa cells. Conium's effects on cell cycle, reactive oxygen species (ROS) accumulation, mitochondrial membrane potential (MMP) and apoptosis, if any, were analyzed through flow cytometry. Whether Conium could damage DNA and induce morphological changes were also determined microscopically. Expression of different proteins related to cell death and survival was critically studied by western blotting and ELISA methods. If Conium could interact directly with DNA was also determined by circular dichroism (CD) spectroscopy. Conium treatment reduced cell viability and colony formation at 48 h and inhibited cell proliferation, arresting cell cycle at sub-G stage. Conium treatment lead to increased generation of reactive oxygen species (ROS) at 24 h, increase in MMP depolarization, morphological changes and DNA damage in HeLa cells along with externalization of phosphatidyl serine at 48 hours. While cytochrome c release and caspase-3 activation led HeLa cells toward apoptosis, down-regulation of Akt and NFkB inhibited cellular proliferation, indicating the signaling pathway to be mediated via the mitochondria-mediated caspase-3-dependent pathway. CD-spectroscopy revealed that Conium interacted with DNA molecule. Overall results validate anti-cancer potential of Conium and provide support for its use in traditional systems of medicine.

DNA Mismatch Repair and Oxidative DNA Damage: Implications for Cancer Biology and Treatment

International Nuclear Information System (INIS)

Bridge, Gemma; Rashid, Sukaina; Martin, Sarah A.

2014-01-01

Many components of the cell, including lipids, proteins and both nuclear and mitochondrial DNA, are vulnerable to deleterious modifications caused by reactive oxygen species. If not repaired, oxidative DNA damage can lead to disease-causing mutations, such as in cancer. Base excision repair and nucleotide excision repair are the two DNA repair pathways believed to orchestrate the removal of oxidative lesions. However, recent findings suggest that the mismatch repair pathway may also be important for the response to oxidative DNA damage. This is particularly relevant in cancer where mismatch repair genes are frequently mutated or epigenetically silenced. In this review we explore how the regulation of oxidative DNA damage by mismatch repair proteins may impact on carcinogenesis. We discuss recent studies that identify potential new treatments for mismatch repair deficient tumours, which exploit this non-canonical role of mismatch repair using synthetic lethal targeting

DNA Mismatch Repair and Oxidative DNA Damage: Implications for Cancer Biology and Treatment

Energy Technology Data Exchange (ETDEWEB)

Bridge, Gemma; Rashid, Sukaina; Martin, Sarah A., E-mail: sarah.martin@qmul.ac.uk [Centre for Molecular Oncology, Barts Cancer Institute, Queen Mary University of London, Charterhouse Square, London EC1M 6BQ (United Kingdom)

2014-08-05

Many components of the cell, including lipids, proteins and both nuclear and mitochondrial DNA, are vulnerable to deleterious modifications caused by reactive oxygen species. If not repaired, oxidative DNA damage can lead to disease-causing mutations, such as in cancer. Base excision repair and nucleotide excision repair are the two DNA repair pathways believed to orchestrate the removal of oxidative lesions. However, recent findings suggest that the mismatch repair pathway may also be important for the response to oxidative DNA damage. This is particularly relevant in cancer where mismatch repair genes are frequently mutated or epigenetically silenced. In this review we explore how the regulation of oxidative DNA damage by mismatch repair proteins may impact on carcinogenesis. We discuss recent studies that identify potential new treatments for mismatch repair deficient tumours, which exploit this non-canonical role of mismatch repair using synthetic lethal targeting.

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

Directory of Open Access Journals (Sweden)

Rolletschek Alexandra

2009-06-01

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

The effect of higher order chromatin structure on DNA damage and repair

International Nuclear Information System (INIS)

Yasui, L.S.; Warters, R.L.; Higashikubo, R.

1985-01-01

Alterations in chromatin structure are thought to play an important role in various radiobiological end points, i.e., DNA damage, DNA damage repair and cell survival. The authors use here the isoleucine deprivation technique to decondense higher order chromatin structure and asses X-ray induced DNA damage, DNA damage repair and cell survival on cells with decondensed chromatin as compared to controls. This chromatin decondensation manifests itself as a 30 fold decrease in nuclear area occupied by heterochromatin, an increased rate of Micrococcal nuclease digestion, 15% increased ethidium bromide intercalation and an altered binding capacity of Hl histone. These chromatin/nuclear changes do not affect X-ray induced DNA damage as measured by the alkaline elution technique or cell survival but slows DNA damage repair by 2 fold. Therefore, even though the chromatin appears more accessible to DNA damage and repair processes, these particular nuclear changes do not affect the DNA damaging effects of X-rays and in addition, repair is not enhanced by the ''relaxed'' state of chromatin. It is proposed that the altered metabolic state of isoleucine deprived cells provides a less efficient system for the repair of X-ray induced DNA damage

Visualization of complex DNA damage along accelerated ions tracks

Science.gov (United States)

Kulikova, Elena; Boreyko, Alla; Bulanova, Tatiana; Ježková, Lucie; Zadneprianetc, Mariia; Smirnova, Elena

2018-04-01

The most deleterious DNA lesions induced by ionizing radiation are clustered DNA double-strand breaks (DSB). Clustered or complex DNA damage is a combination of a few simple lesions (single-strand breaks, base damage etc.) within one or two DNA helix turns. It is known that yield of complex DNA lesions increases with increasing linear energy transfer (LET) of radiation. For investigation of the induction and repair of complex DNA lesions, human fibroblasts were irradiated with high-LET 15N ions (LET = 183.3 keV/μm, E = 13MeV/n) and low-LET 60Co γ-rays (LET ≈ 0.3 keV/μm) radiation. DNA DSBs (γH2AX and 53BP1) and base damage (OGG1) markers were visualized by immunofluorecence staining and high-resolution microscopy. The obtained results showed slower repair kinetics of induced DSBs in cells irradiated with accelerated ions compared to 60Co γ-rays, indicating induction of more complex DNA damage. Confirming previous assumptions, detailed 3D analysis of γH2AX/53BP1 foci in 15N ions tracks revealed more complicated structure of the foci in contrast to γ-rays. It was shown that proteins 53BP1 and OGG1 involved in repair of DNA DSBs and modified bases, respectively, were colocalized in tracks of 15N ions and thus represented clustered DNA DSBs.

Plasmid DNA damage induced by helium atmospheric pressure plasma jet

Science.gov (United States)

Han, Xu; Cantrell, William A.; Escobar, Erika E.; Ptasinska, Sylwia

2014-03-01

A helium atmospheric pressure plasma jet (APPJ) is applied to induce damage to aqueous plasmid DNA. The resulting fractions of the DNA conformers, which indicate intact molecules or DNA with single- or double-strand breaks, are determined using agarose gel electrophoresis. The DNA strand breaks increase with a decrease in the distance between the APPJ and DNA samples under two working conditions of the plasma source with different parameters of applied electric pulses. The damage level induced in the plasmid DNA is also enhanced with increased plasma irradiation time. The reactive species generated in the APPJ are characterized by optical emission spectra, and their roles in possible DNA damage processes occurring in an aqueous environment are also discussed.

Chromosomal Damage and Apoptosis in Exfoliated Buccal Cells from Individuals with Oral Cancer

Science.gov (United States)

Dórea, Lavínia Tércia Magalhães; Meireles, José Roberto Cardoso; Lessa, Júlia Paula Ramos; Oliveira, Márcio Campos; de Bragança Pereira, Carlos Alberto; Polpo de Campos, Adriano; Cerqueira, Eneida de Moraes Macílio

2012-01-01

This study aimed to investigate cytological abnormalities indicative of chromosome damage (micronuclei) and apoptosis (karyorrhexis, pyknosis, and condensed chromatin) in exfoliated cells from the buccal mucosa of patients with oral cancer and control subjects. The sample included twenty individuals with oral cancer and forty individuals with normal buccal mucosa. Material was collected from the cheek epithelium in areas with lesions and areas without abnormalities. A minimum of one thousand cells was analyzed. Micronuclei were found significantly more frequently in cells collected from lesions than in cells from normal areas, independent of the presence/absence of cancer (P < 0.0001). They were also significantly more frequent in smokers and in mouthwash users (P < 0.0001). Apoptosis occurred significantly less frequently in individuals with oral cancer (P < 0.0001). These results show that oral cancer is associated with higher frequency of chromosomal damage and suggest that apoptosis is compromised in the buccal cells of individuals with this kind of neoplasia. PMID:22315605

Interactions and Localization of Escherichia coli Error-Prone DNA Polymerase IV after DNA Damage.

Science.gov (United States)

Mallik, Sarita; Popodi, Ellen M; Hanson, Andrew J; Foster, Patricia L

2015-09-01

Escherichia coli's DNA polymerase IV (Pol IV/DinB), a member of the Y family of error-prone polymerases, is induced during the SOS response to DNA damage and is responsible for translesion bypass and adaptive (stress-induced) mutation. In this study, the localization of Pol IV after DNA damage was followed using fluorescent fusions. After exposure of E. coli to DNA-damaging agents, fluorescently tagged Pol IV localized to the nucleoid as foci. Stepwise photobleaching indicated ∼60% of the foci consisted of three Pol IV molecules, while ∼40% consisted of six Pol IV molecules. Fluorescently tagged Rep, a replication accessory DNA helicase, was recruited to the Pol IV foci after DNA damage, suggesting that the in vitro interaction between Rep and Pol IV reported previously also occurs in vivo. Fluorescently tagged RecA also formed foci after DNA damage, and Pol IV localized to them. To investigate if Pol IV localizes to double-strand breaks (DSBs), an I-SceI endonuclease-mediated DSB was introduced close to a fluorescently labeled LacO array on the chromosome. After DSB induction, Pol IV localized to the DSB site in ∼70% of SOS-induced cells. RecA also formed foci at the DSB sites, and Pol IV localized to the RecA foci. These results suggest that Pol IV interacts with RecA in vivo and is recruited to sites of DSBs to aid in the restoration of DNA replication. DNA polymerase IV (Pol IV/DinB) is an error-prone DNA polymerase capable of bypassing DNA lesions and aiding in the restart of stalled replication forks. In this work, we demonstrate in vivo localization of fluorescently tagged Pol IV to the nucleoid after DNA damage and to DNA double-strand breaks. We show colocalization of Pol IV with two proteins: Rep DNA helicase, which participates in replication, and RecA, which catalyzes recombinational repair of stalled replication forks. Time course experiments suggest that Pol IV recruits Rep and that RecA recruits Pol IV. These findings provide in vivo evidence

Chromatin modifications and the DNA damage response to ionizing radiation

International Nuclear Information System (INIS)

Kumar, Rakesh; Horikoshi, Nobuo; Singh, Mayank; Gupta, Arun; Misra, Hari S.; Albuquerque, Kevin; Hunt, Clayton R.; Pandita, Tej K.

2013-01-01

In order to survive, cells have evolved highly effective repair mechanisms to deal with the potentially lethal DNA damage produced by exposure to endogenous as well as exogenous agents. Ionizing radiation exposure induces highly lethal DNA damage, especially DNA double-strand breaks (DSBs), that is sensed by the cellular machinery and then subsequently repaired by either of two different DSB repair mechanisms: (1) non-homologous end joining, which re-ligates the broken ends of the DNA and (2) homologous recombination, that employs an undamaged identical DNA sequence as a template, to maintain the fidelity of DNA repair. Repair of DSBs must occur within the natural context of the cellular DNA which, along with specific proteins, is organized to form chromatin, the overall structure of which can impede DNA damage site access by repair proteins. The chromatin complex is a dynamic structure and is known to change as required for ongoing cellular processes such as gene transcription or DNA replication. Similarly, during the process of DNA damage sensing and repair, chromatin needs to undergo several changes in order to facilitate accessibility of the repair machinery. Cells utilize several factors to modify the chromatin in order to locally open up the structure to reveal the underlying DNA sequence but post-translational modification of the histone components is one of the primary mechanisms. In this review, we will summarize chromatin modifications by the respective chromatin modifying factors that occur during the DNA damage response.

The nucleosome: orchestrating DNA damage signaling and repair within chromatin.

Science.gov (United States)

Agarwal, Poonam; Miller, Kyle M

2016-10-01

DNA damage occurs within the chromatin environment, which ultimately participates in regulating DNA damage response (DDR) pathways and repair of the lesion. DNA damage activates a cascade of signaling events that extensively modulates chromatin structure and organization to coordinate DDR factor recruitment to the break and repair, whilst also promoting the maintenance of normal chromatin functions within the damaged region. For example, DDR pathways must avoid conflicts between other DNA-based processes that function within the context of chromatin, including transcription and replication. The molecular mechanisms governing the recognition, target specificity, and recruitment of DDR factors and enzymes to the fundamental repeating unit of chromatin, i.e., the nucleosome, are poorly understood. Here we present our current view of how chromatin recognition by DDR factors is achieved at the level of the nucleosome. Emerging evidence suggests that the nucleosome surface, including the nucleosome acidic patch, promotes the binding and activity of several DNA damage factors on chromatin. Thus, in addition to interactions with damaged DNA and histone modifications, nucleosome recognition by DDR factors plays a key role in orchestrating the requisite chromatin response to maintain both genome and epigenome integrity.

RRR-alpha-tocopheryl succinate inhibits EL4 thymic lymphoma cell growth by inducing apoptosis and DNA synthesis arrest.

Science.gov (United States)

Yu, W; Sanders, B G; Kline, K

1997-01-01

RRR-alpha-tocopheryl succinate (vitamin E succinate, VES) treatment of murine EL4 T lymphoma cells induced the cells to undergo apoptosis. After 48 hours of VES treatment at 20 micrograms/ml, 95% of cells were apoptotic. Evidence for the induction of apoptosis by VES treatments is based on staining of DNA for detection of chromatin condensation/fragmentation, two-color flow-cytometric analyses of DNA content, and end-labeled DNA and electrophoretic analyses for detection of DNA ladder formation. VES-treated EL4 cells were blocked in the G1 cell cycle phase; however, apoptotic cells came from all cell cycle phases. Analyses of mRNA expression of genes involved in apoptosis revealed decreased c-myc and increased bcl-2, c-fos, and c-jun mRNAs within three to six hours after treatment. Western analyses showed increased c-Jun, c-Fos, and Bcl-2 protein levels. Electrophoretic mobility shift assays showed increased AP-1 binding at 6, 12, and 24 hours after treatment and decreased c-Myc binding after 12 and 24 hours of VES treatment. Treatments of EL4 cells with VES+RRR-alpha-to-copherol reduced apoptosis without effecting DNA synthesis arrest. Treatments of EL4 cells with VES+rac-6-hydroxyl-2, 5,7,8-tetramethyl-chroman-2-carboxylic acid, butylated hydroxytoluene, or butylated hydroxyanisole had no effect on apoptosis or DNA synthesis arrest caused by VES treatments. Analyses of bcl-2, c-myc, c-jun, and c-fos mRNA levels in cells receiving VES + RRR-alpha-tocopherol treatments showed no change from cells receiving VES treatments alone, implying that these changes are correlated with VES treatments but are not causal for apoptosis. However, treatments with VES + RRR-alpha-tocopherol decreased AP-1 binding to consensus DNA oligomer, suggesting AP-1 involvement in apoptosis induced by VES treatments.

UV and ionizing radiations induced DNA damage, differences and similarities

Science.gov (United States)

Ravanat, Jean-Luc; Douki, Thierry

2016-11-01

Both UV and ionizing radiations damage DNA. Two main mechanisms, so-called direct and indirect pathways, are involved in the degradation of DNA induced by ionizing radiations. The direct effect of radiation corresponds to direct ionization of DNA (one electron ejection) whereas indirect effects are produced by reactive oxygen species generated through water radiolysis, including the highly reactive hydroxyl radicals, which damage DNA. UV (and visible) light damages DNA by again two distinct mechanisms. UVC and to a lesser extend UVB photons are directly absorbed by DNA bases, generating their excited states that are at the origin of the formation of pyrimidine dimers. UVA (and visible) light by interaction with endogenous or exogenous photosensitizers induce the formation of DNA damage through photosensitization reactions. The excited photosensitizer is able to induce either a one-electron oxidation of DNA (type I) or to produce singlet oxygen (type II) that reacts with DNA. In addition, through an energy transfer from the excited photosensitizer to DNA bases (sometime called type III mechanism) formation of pyrimidine dimers could be produced. Interestingly it has been shown recently that pyrimidine dimers are also produced by direct absorption of UVA light by DNA, even if absorption of DNA bases at these wavelengths is very low. It should be stressed that some excited photosensitizers (such as psoralens) could add directly to DNA bases to generate adducts. The review will described the differences and similarities in terms of damage formation (structure and mechanisms) between these two physical genotoxic agents.

Reduction of arsenite-enhanced ultraviolet radiation-induced DNA damage by supplemental zinc

Energy Technology Data Exchange (ETDEWEB)

Cooper, Karen L.; King, Brenee S.; Sandoval, Monica M.; Liu, Ke Jian; Hudson, Laurie G., E-mail: lhudson@salud.unm.edu

2013-06-01

Arsenic is a recognized human carcinogen and there is evidence that arsenic augments the carcinogenicity of DNA damaging agents such as ultraviolet radiation (UVR) thereby acting as a co-carcinogen. Inhibition of DNA repair is one proposed mechanism to account for the co-carcinogenic actions of arsenic. We and others find that arsenite interferes with the function of certain zinc finger DNA repair proteins. Furthermore, we reported that zinc reverses the effects of arsenite in cultured cells and a DNA repair target protein, poly (ADP-ribose) polymerase-1. In order to determine whether zinc ameliorates the effects of arsenite on UVR-induced DNA damage in human keratinocytes and in an in vivo model, normal human epidermal keratinocytes and SKH-1 hairless mice were exposed to arsenite, zinc or both before solar-simulated (ss) UVR exposure. Poly (ADP-ribose) polymerase activity, DNA damage and mutation frequencies at the Hprt locus were measured in each treatment group in normal human keratinocytes. DNA damage was assessed in vivo by immunohistochemical staining of skin sections isolated from SKH-1 hairless mice. Cell-based findings demonstrate that ssUVR-induced DNA damage and mutagenesis are enhanced by arsenite, and supplemental zinc partially reverses the arsenite effect. In vivo studies confirm that zinc supplementation decreases arsenite-enhanced DNA damage in response to ssUVR exposure. From these data we can conclude that zinc offsets the impact of arsenic on ssUVR-stimulated DNA damage in cells and in vivo suggesting that zinc supplementation may provide a strategy to improve DNA repair capacity in arsenic exposed human populations. - Highlights: • Low levels of arsenite enhance UV-induced DNA damage in human keratinocytes. • UV-initiated HPRT mutation frequency is enhanced by arsenite. • Zinc supplementation offsets DNA damage and mutation frequency enhanced by arsenite. • Zinc-dependent reduction of arsenite enhanced DNA damage is confirmed in vivo.

Overexpression of DNA damage-induced 45 α gene contributes to esophageal squamous cell cancer by promoter hypomethylation

Directory of Open Access Journals (Sweden)

Wang Bao xiang

2012-02-01

Full Text Available Abstract Background Environmental factors-induced dysfunction of esophageal squamous epithelium, including genomic DNA impairment and apoptosis, play an important role in the pathogenesis of esophageal squamous cell cancer. DNA damage-induced 45α (GADD45α has been found promoting DNA repair and removing methylation marker, Therefore, in this study we will investigate whether GADD45α expression is induced and its mechanism in esophageal squamous cell cancer. Methods Two human esophageal squamous cell lines (ESCC, ECA109 and KYSE510 were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS. Lipofectamine 2000 was used to transfect cells. mRNA level of GADD45α was measured by reverse transcription-quantitive PCR (RT-qPCR, protein level of GADD45α was detected by western blot and Immunohistochemistry. Global DNA methylation of tissue sample was measured using the Methylamp Global DNA Methylation Quantification Ultra kit (Epigentek Group and promoter methylation was measured by bisulfite sequencing. Results GADD45a mRNA and protein levels were increased significantly in tumor tissue than that in adjacent normal tissue. Hypomethylation of global genomic DNA and GADD45α promoter were found in ESCC. The cell sensitivity to Cisplatin DDP was decreased significantly in Eca109 and Kyse510 cells, in which GADD45α expression was down-regulated by RNA interference (RNAi. In addition, silence of GADD45a expression in ESCC cells inhibited proliferation and promoted apoptosis. Conclusion Overexpression of GADD45α gene is due to DNA hypomethylation in ESCC. GADD45α may be a protective factor in DDP chemotherapy for esophageal squamous cell carcinoma.

The cAMP signaling system inhibits the repair of {gamma}-ray-induced DNA damage by promoting Epac1-mediated proteasomal degradation of XRCC1 protein in human lung cancer cells

Energy Technology Data Exchange (ETDEWEB)

Cho, Eun-Ah [Department of Biochemistry and Molecular Biology, Cancer Research Center, Seoul National University College of Medicine, Seoul 110-799 (Korea, Republic of); Juhnn, Yong-Sung, E-mail: juhnn@snu.ac.kr [Department of Biochemistry and Molecular Biology, Cancer Research Center, Seoul National University College of Medicine, Seoul 110-799 (Korea, Republic of)

2012-06-01

Highlights: Black-Right-Pointing-Pointer cAMP signaling system inhibits repair of {gamma}-ray-induced DNA damage. Black-Right-Pointing-Pointer cAMP signaling system inhibits DNA damage repair by decreasing XRCC1 expression. Black-Right-Pointing-Pointer cAMP signaling system decreases XRCC1 expression by promoting its proteasomal degradation. Black-Right-Pointing-Pointer The promotion of XRCC1 degradation by cAMP signaling system is mediated by Epac1. -- Abstract: Cyclic AMP is involved in the regulation of metabolism, gene expression, cellular growth and proliferation. Recently, the cAMP signaling system was found to modulate DNA-damaging agent-induced apoptosis by regulating the expression of Bcl-2 family proteins and inhibitors of apoptosis. Thus, we hypothesized that the cAMP signaling may modulate DNA repair activity, and we investigated the effects of the cAMP signaling system on {gamma}-ray-induced DNA damage repair in lung cancer cells. Transient expression of a constitutively active mutant of stimulatory G protein (G{alpha}sQL) or treatment with forskolin, an adenylyl cyclase activator, augmented radiation-induced DNA damage and inhibited repair of the damage in H1299 lung cancer cells. Expression of G{alpha}sQL or treatment with forskolin or isoproterenol inhibited the radiation-induced expression of the XRCC1 protein, and exogenous expression of XRCC1 abolished the DNA repair-inhibiting effect of forskolin. Forskolin treatment promoted the ubiquitin and proteasome-dependent degradation of the XRCC1 protein, resulting in a significant decrease in the half-life of the protein after {gamma}-ray irradiation. The effect of forskolin on XRCC1 expression was not inhibited by PKA inhibitor, but 8-pCPT-2 Prime -O-Me-cAMP, an Epac-selective cAMP analog, increased ubiquitination of XRCC1 protein and decreased XRCC1 expression. Knockdown of Epac1 abolished the effect of 8-pCPT-2 Prime -O-Me-cAMP and restored XRCC1 protein level following {gamma}-ray irradiation. From

UV-B induces DNA damage and DNA synthesis delay in the marine diatom Cyclotella sp

NARCIS (Netherlands)

Buma, A.G.J.; Van Hannen, E.J.; Veldhuis, M.; Gieskes, W.W.C.

1996-01-01

The effect of UV-B on the occurrence of DNA damage and consequences for the cell cycle were studied in the marine diatom Cyclotella sp. DNA damage was quantified by immunofluorescent detection of thymine dimers in nuclear DNA of single cells using flow cytometry. A total UV-B dose (biologically

UV-B induces DNA damage and DNA synthesis delay in the marine diatom Cyclotella sp.

NARCIS (Netherlands)

Buma, A.G.J.; van Hannen, E.J; Veldhuis, M.J W; Gieskes, W.W C

The effect of UV-B on the occurrence of DNA damage and consequences for the cell cycle were studied in the marine diatom Cyclotella sp. DNA damage was quantified by immunofluorescent detection of thymine dimers in nuclear DNA of single cells using flow cytometry. A total UV-B dose (biologically

Chimeric proteins for detection and quantitation of DNA mutations, DNA sequence variations, DNA damage and DNA mismatches

Science.gov (United States)

McCutchen-Maloney, Sandra L.

2002-01-01

Chimeric proteins having both DNA mutation binding activity and nuclease activity are synthesized by recombinant technology. The proteins are of the general formula A-L-B and B-L-A where A is a peptide having DNA mutation binding activity, L is a linker and B is a peptide having nuclease activity. The chimeric proteins are useful for detection and identification of DNA sequence variations including DNA mutations (including DNA damage and mismatches) by binding to the DNA mutation and cutting the DNA once the DNA mutation is detected.

Oxidative DNA damage during sleep periods among nightshift workers.

Science.gov (United States)

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/

DNA Damage among Wood Workers Assessed with the Comet Assay

Science.gov (United States)

Bruschweiler, Evin Danisman; Wild, Pascal; Huynh, Cong Khanh; Savova-Bianchi, Dessislava; Danuser, Brigitta; Hopf, Nancy B.

2016-01-01

Exposure to wood dust, a human carcinogen, is common in wood-related industries, and millions of workers are occupationally exposed to wood dust worldwide. The comet assay is a rapid, simple, and sensitive method for determining DNA damage. The objective of this study was to investigate the DNA damage associated with occupational exposure to wood dust using the comet assay (peripheral blood samples) among nonsmoking wood workers (n = 31, furniture and construction workers) and controls (n = 19). DNA damage was greater in the group exposed to composite wood products compared to the group exposed to natural woods and controls (P < 0.001). No difference in DNA damage was observed between workers exposed to natural woods and controls (P = 0.13). Duration of exposure and current dust concentrations had no effect on DNA damage. In future studies, workers’ exposures should include cumulative dust concentrations and exposures originating from the binders used in composite wood products. PMID:27398027

Apoptosis-promoted tumorigenesis: γ-irradiation-induced thymic lymphomagenesis requires Puma-driven leukocyte death

OpenAIRE

Michalak, Ewa M.; Vandenberg, Cassandra J.; Delbridge, Alex R.D.; Wu, Li; Scott, Clare L.; Adams, Jerry M.; Strasser, Andreas

2010-01-01

Although tumor development requires impaired apoptosis, we describe a novel paradigm of apoptosis-dependent tumorigenesis. Because DNA damage triggers apoptosis through p53-mediated induction of BH3-only proteins Puma and Noxa, we explored their roles in γ-radiation-induced thymic lymphomagenesis. Surprisingly, whereas Noxa loss accelerated it, Puma loss ablated tumorigenesis. Tumor suppression by Puma deficiency reflected its protection of leukocytes from γ-irradiation-induced death, because...

Phosphorylation of human INO80 is involved in DNA damage tolerance

International Nuclear Information System (INIS)

Kato, Dai; Waki, Mayumi; Umezawa, Masaki; Aoki, Yuka; Utsugi, Takahiko; Ohtsu, Masaya; Murakami, Yasufumi

2012-01-01

Highlights: ► Depletion of hINO80 significantly reduced PCNA ubiquitination. ► Depletion of hINO80 significantly reduced nuclear dots intensity of RAD18 after UV irradiation. ► Western blot analyses showed phosphorylated hINO80 C-terminus. ► Overexpression of phosphorylation mutant hINO80 reduced PCNA ubiquitination. -- Abstract: Double strand breaks (DSBs) are the most serious type of DNA damage. DSBs can be generated directly by exposure to ionizing radiation or indirectly by replication fork collapse. The DNA damage tolerance pathway, which is conserved from bacteria to humans, prevents this collapse by overcoming replication blockages. The INO80 chromatin remodeling complex plays an important role in the DNA damage response. The yeast INO80 complex participates in the DNA damage tolerance pathway. The mechanisms regulating yINO80 complex are not fully understood, but yeast INO80 complex are necessary for efficient proliferating cell nuclear antigen (PCNA) ubiquitination and for recruitment of Rad18 to replication forks. In contrast, the function of the mammalian INO80 complex in DNA damage tolerance is less clear. Here, we show that human INO80 was necessary for PCNA ubiquitination and recruitment of Rad18 to DNA damage sites. Moreover, the C-terminal region of human INO80 was phosphorylated, and overexpression of a phosphorylation-deficient mutant of human INO80 resulted in decreased ubiquitination of PCNA during DNA replication. These results suggest that the human INO80 complex, like the yeast complex, was involved in the DNA damage tolerance pathway and that phosphorylation of human INO80 was involved in the DNA damage tolerance pathway. These findings provide new insights into the DNA damage tolerance pathway in mammalian cells.