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PubMed2002

Growth arrest in G1 protects against oxygen-induced DNA damage and cell death.

Although oxygen is required for normal aerobic respiration, hyperoxia (95% O(2)/5% CO(2)) damages DNA, inhibits proliferation in G1, S and G2 phases of the cell cycle, and induces necrosis. The current study examines whether growth arrest in G1 protects pulmonary epithelial cells from oxidative DNA damage and cell death. Mv1Lu pulmonary adenocarcinoma cells were chosen for studies because hyperoxia inhibits their proliferation in S and G2 phase, while they can be induced to arrest in G1 by altering culture conditions. Hyperoxia inhibited proliferation, increased intracellular redox, and rapidly reduced clonogenic survival. In contrast, Mv1Lu cells treated with transforming growth factor (TGF)-beta1, deprived of serum or grown to confluency, arrested and remained predominantly in G1 even during exposure. Growth arrest in G1 significantly enhanced clonogenic survival by 10-50-fold. Enhanced survival was not due to reduction in the intracellular redox-state of the cells, but instead was associated with reduced DNA strand breaks and p53 expression. Our findings suggest that the protective effects of G1 is mediated not simply by a reduction in intracellular ROS, but rather through an enhanced ability to limit or rapidly recognize and repair damaged DNA.

Adenocarcinoma↗

PubMed2006

Soil fungi reduce the iron content and the DNA damaging effects of asbestos fibers.

Some soil fungi growing on asbestos fibers release chelators and antioxidants. The bioweathering potential of fungi has thus been envisaged as a possible route for bioremediation of asbestos rich soils, where no inactivation procedures have been established so far. The present study reports fungal-mediated modification of the surface reactivity of the fibers and of their potential to damage DNA in vitro. Verticillium sp. and Paecilomyces sp. were selected among the fungi isolated from fragments of chrysotile bearing rocks, as the most potent in iron extraction, and studied in parallel with F. oxysporum, previously reported to modify the surface reactivity of asbestos fibers. One sample of chrysotile from the Western Alps and a sample of UICC (Union Internationale Contre le Cancer) crocidolite were incubated with or without fungi. All fungi extracted iron from both fibers (7.3% from crocidolite and 33.6% from chrysotile by Verticillium sp.), releasing it into the medium. F. oxysporum and Paecilomyces sp. suppressed the potential of the fibers to release hydroxyl radical, while Verticillium sp. suppressed it on crocidolite but enhanced it on chrysotile, a hallmark of ongoing mobilization of reactive iron. Fibers incubated in the growth medium, but in the absence of fungi, exhibited a remarkable potential to damage DNA in vitro, measured by the generation of 8-oxo-7,8-dihydro-2'-deoxyguanosine, while all the fungi reduced such effect. Fungi may thus be regarded as appropriate candidates for bioremediation of asbestos rich soils whereby the reactive iron ions responsible for DNA damage are progressively removed from the fibers.

Asbestos, Crocidolite↗

PubMed2006

New evidence of premature oxidative DNA damage: mitochondrial DNA deletion in gingival tissue of patients with periodontitis.

BACKGROUND: Overproduction of reactive oxygen species (ROS) causes increased oxidative stress in gingival tissue. It has been generally accepted that increased oxidative stress might contribute to additional damage of lipids, proteins, and DNA molecules. The mitochondrial DNA (mtDNA) mutation is a superb biomarker of oxidative damage. The aim of the present study was to investigate the mtDNA deletions in the gingival tissue of patients with periodontitis and to explain the correlations between mtDNA deletion in gingival tissue and clinical parameters of periodontitis and age. METHODS: Gingival tissue and blood samples were collected from 30 patients with chronic periodontitis (CP group) and 30 healthy control subjects (H group). To determine the clinical condition of each subject, the plaque index, gingival index, clinical attachment level, and probing depth were measured. Using the polymerase chain reaction (PCR) method, we examined the 7.4- and 5-kbp mtDNA deletions in tissue and blood samples. Three different pairs of PCR primers were used in this study. RESULTS: In this study, we did not detect any deletions in blood DNA samples in either the CP or H group. Also, the 7.4-kbp mtDNA deletion was not detected in gingival tissues of subjects. However, the 5-kbp mtDNA deletion was detected in 24 of the 30 subjects (80%) in the CP group and was not detected in the H group (0%). Significant correlations were found between the occurrence of the 5-kbp mtDNA deletion and all clinical parameters (P <0.01). A similar correlation was found between the occurrence of the 5-kbp mtDNA deletion and age (P <0.05). CONCLUSIONS: The overproduction of ROS by activated polymorphonuclear leukocytes in chronic inflammation may lead to premature oxidative damage of the mtDNA. In this study, the occurrence of the 5-kbp mtDNA deletion in 24 periodontitis subjects may be evidence of premature oxidative DNA damage.

Adult↗

PubMed2002

UV stimulation of nucleophosmin/B23 expression is an immediate-early gene response induced by damaged DNA.

Nucleophosmin/B23 (NPM/B23), a nucleolar protein, was rapidly up-regulated after UV irradiation (at 254 nm; 30 J/m(2)) in NIH 3T3 cells and HeLa/S3 cells. Levels of NPM/B23 mRNA peaked 45-60 min after UV treatment and returned to baseline by 12 h. Transcription inhibitor actinomycin D (5 microg/ml) prevented the UV-induced increase of NPM/B23 mRNA, suggesting that UV induction of NPM/B23 was mediated at the transcriptional level. Moreover, UV-induced NPM/B23 expression was super-induced by cycloheximide (20 microg/ml), which was characteristic of immediate-early gene response. The transcriptional activation of NPM/B23 by UV was also confirmed by NPM/B23 promoter activity assay. Thymine dinucleotide, mimicking the effects of UV-induced DNA damage, was able to trigger NPM/B23 expression in the absence of genomic DNA damage. UV-induced activation of NPM/B23 promoter could not be blocked by UV-inducible pathway inhibitors, such as those of growth factor tyrosine kinase, mitogen-activated protein kinase, AP-1, NF-kappaB, and DNA-dependent kinase. Our results indicate that UV stimulation of NPM/B23 expression may be mediated through a novel UV-inducible pathway and is an immediate-early gene response induced by damaged DNA. Induction of immediate-early gene is an initial step in the regulation of cellular and genomic responses to external stimuli. Our results thus provide important evidence for an involvement of NPM/B23 in the acute response of mammalian cells to environmental stress.

3T3 Cells↗

PubMed1994

Xeroderma pigmentosum group E binding factor recognizes a broad spectrum of DNA damage.

Xeroderma pigmentosum complementation group E binding factor (XPE-BF) is a damaged DNA binding protein that is deficient in a subset of patients from complementation group E of xeroderma pigmentosum. The protein recognizes various forms of DNA damage including some cyclobutane pyrimidine dimers, 6-4 photoproducts, cis-diamminedichloroplatinum(II) adducts, and single-stranded DNA. We now show that it also recognizes damage induced by nitrogen mustard; N-methyl-N'-nitro-N-nitrosoguanidine, and depurination, but has no detectable affinity for DNA adducts generated by trans-diamminedichloroplatinum(II), 4-nitroquinoline-N-oxide, 8-methoxypsoralen, or enzymatically methylated cytosine and adenine. The failure to recognize 4-nitroquinoline-N-oxide and 8-methoxypsoralen adducts is consistent with previous reports that XPE cells carry out wild-type levels of repair synthesis after DNA damage by those drugs. These results demonstrate that XPE-BF is a versatile damage recognition protein, but suggest that other proteins must contribute to the recognition of DNA lesions for the human excision repair pathway.

4-Nitroquinoline-1-oxide↗

PubMed1994

The pathway regulating GADD153 induction in response to DNA damage is independent of protein kinase C and tyrosine kinases.

Treatment of cells with agents that damage DNA leads to the induction of numerous genes. Recent studies aimed at understanding the events preceding the transcriptional activation of some of these DNA damage-inducible genes in mammalian cells have demonstrated that various extranuclear protein kinases are involved in the signaling cascades. The mammalian GADD153 gene, a member of the CCAAT enhancer-binding protein family of transcription factors, is highly induced by a variety of DNA-damaging agents as well as by certain growth arrest conditions and oxidative stresses. We have examined the effects of numerous protein kinase and phosphatase inhibitors on the DNA damage-induced expression of GADD153, to identify the signal transduction components involved in its transcriptional regulation. In contrast to the transcriptional activation of c-jun and collagenase in response to DNA damage, GADD153 induction involves neither protein kinase C nor tyrosine kinases but does appear to require an unidentified serine-threonine-kinase. Elevation of intracellular glutathione levels by treatment with N-acetylcysteine did not affect the methyl methanesulfonate-induced expression of the GADD153 gene, although it did diminish cadmium chloride-induced expression. These findings suggest that oxidative stress and DNA damage regulate GADD153 transcription through different pathways. Based on our findings and those of others with respect to other DNA damage-inducible genes, we propose a model depicting the complex pathways which appear to be involved in the regulation of mammalian genes in response to genotoxic stress and in which the DNA damage-induced expression of GADD153 represents a unique pathway independent of either protein kinase C or tyrosine kinase.

Acetylcysteine↗

PubMed2001

DNA damage and cell cycle checkpoints in hyperoxic lung injury: braking to facilitate repair.

The beneficial use of supplemental oxygen therapies to increase arterial blood oxygen levels and reduce tissue hypoxia is offset by the knowledge that it injures and kills cells, resulting in increased morbidity and mortality. Although many studies have focused on understanding how hyperoxia kills cells, recent findings reveal that it also inhibits proliferation through activation of cell cycle checkpoints rather than through overt cytotoxicity. Cell cycle checkpoints are thought to be protective because they allow additional time for injured cells to repair damaged DNA and other essential molecules. During recovery in room air, the lung undergoes a burst of proliferation to replace injured and dead cells. Failure to terminate this proliferation has been associated with fibrosis. These observations suggest that growth-suppressive signals, which inhibit proliferation of injured cells and terminate proliferation when tissue repair has been completed, may play an important role in the pulmonary response to hyperoxia. Because DNA replication is coupled with DNA repair, activation of cell cycle checkpoints during hyperoxia may be a mechanism by which cells protect themselves from oxidant genotoxic stress. This review examines the effect of hyperoxia on DNA integrity, pulmonary cell proliferation, and cell cycle checkpoints activated by DNA damage.

Animals↗

PubMed1998

Evaluation of radiation-induced DNA damage and DNA repair in human lung cancer cell lines with different radiosensitivity using alkaline and neutral single cell gel electrophoresis.

Using the comet assay, radiation-induced DNA strand breaks were evaluated in human lung cancer cell lines with different radiosensitivity (U-1285, U-1906E, U-1752 and U-1810). Single strand breaks were more sensitive indicators of the radiation-induced damage than double strand breaks. However, there was no consistent pattern in the way the various cell lines responded to 1-5 Gy of gamma-irradiation and all cell lines showed a remarkably efficient DNA repair after 1 h. In a separate study of the repair kinetics of DNA double strand breaks, the radioresistant cell line U-1810 showed a more efficient initial strand rejoining than the radiosensitive cell line U-1285 after irradiation at 2 Gy. The latter finding suggests that the detection of early DNA repair may be useful when monitoring the intrinsic radiosensitivity of human lung cancer cells.

DNA↗

PubMed1997

Induced N- and C-terminal cleavage of p53: a core fragment of p53, generated by interaction with damaged DNA, promotes cleavage of the N-terminus of full-length p53, whereas ssDNA induces C-terminal cleavage of p53.

p53 is able to recognize and bind sites of DNA damage and, in some way, damage to cellular DNA activates a p53 response leading to G1 arrest or apoptosis. We have previously shown that 'damaged DNA' induces N-terminal cleavage of p53 to generate p40(DeltaN) and p35 (core) protein products. We now show that the p35 product has protease activity and is able to cleave between residues 23 and 24 of full-length p53 to generate a novel product, p50(DeltaN23). This activity was inhibited by bestatin, an aminopeptidase inhibitor. Residues 23 and 24 lie within the mdm-2 binding domain of p53 and the possibility that p50(DeltaN23) may be resistant to feedback regulation by mdm-2 is discussed. Unexpectedly, interaction with ssDNA induced two further cleavage products of p53, generated by C-terminal cleavage and designated p50(DeltaC) and p40(DeltaC). In vivo generation of a C-terminal cleavage product of endogenous p53 similar in size to p50(DeltaC) correlated with up-regulation of p21 expression in ML-1 cells exposed to either adriamycin or cisplatin. The possible significance of the various p53 cleavage products in relation to the cellular response to DNA damage is discussed.

Amino Acid Sequence↗

PubMed2006

Severe oxidatively damaged DNA after cisplatin treatment of cancer patients.

There is growing evidence suggesting that cytotoxic activity of cisplatin is closely associated with increased generation of reactive oxygen species (ROS). Therefore, this study was undertaken to examine oxidative DNA damage, which arises as a result of chemotherapy with cisplatin. Using HPLC prepurification/isotope dilution GC/MS methodology, we examined the amount of 8-oxoGua and 8-oxodG excreted into urine in cancer patients (n = 66) who received chemotherapy with cisplatin. One day after the infusion of the drug, significant increase in the amount of 8-oxoGua and 8-oxodG in urine of the patients was observed, when compared to the initial value (78%, p < 0.0001 and 22%, p = 0.0051). In the "nadir days" (when the most distinct cell death based on hematological cell counts can be observed), the level of modified base and nucleoside decreased in comparison with the aforementioned time point. These results, for the first time, indicate that oxidatively damaged DNA may be, at least in part, responsible for cisplatin induced cytotoxicity. Our results also demonstrate that cell death does not contribute to urinary 8-oxoGua and 8-oxodG in humans.

Antineoplastic Agents↗

PubMed2006

ECNIS-sponsored workshop on biomarkers of exposure and cancer risk: DNA damage and DNA adduct detection and 6th GUM-32P-postlabelling workshop, German Cancer Research Center, Heidelberg, Germany, 29-30 September 2006.

Of all the chemicals classified as carcinogenic to humans by the International Agency for Research on Cancer (IARC), 90% exert their biological effects through binding of their metabolically activated intermediates to DNA forming covalent DNA adducts. As a consequence DNA adducts are generally considered to be causative and directly related to tumour formation. DNA adduct analyses reflect tissue-specific rates of adduct formation and removal, which depend on carcinogen uptake, metabolic activation, DNA repair, adduct instability and tissue turnover and are thus useful markers of carcinogen exposure. The measurement of carcinogen-DNA adduct levels is central to the understanding of chemical carcinogenesis both in animals and humans to determine molecular mechanisms and exposure. Sensitive methods for DNA adduct analysis used to date are based on (32)P-post-labelling, immunoassay, mass spectrometry and laser-induced fluorescence. The aim of this workshop held over 2 days (29-30 September 2006) at the German Cancer Research Center (DKFZ) in Heidelberg, Germany, was to discuss methodological improvements of DNA adduct detection with emphasis on the (32)P-post-labelling procedure as well as new findings achieved by applying the methods to studies on understanding human cancer mechanisms and to elucidate the relationship between adduct formation and human cancer risk.

Animals↗

PubMed1990

Expression of the Saccharomyces cerevisiae DNA repair gene RAD6 that encodes a ubiquitin conjugating enzyme, increases in response to DNA damage and in meiosis but remains constant during the mitotic cell cycle.

The RAD6 gene of Saccharomyces cerevisiae encodes a ubiquitin-conjugating (E2) enzyme and is required for the repair of damaged DNA, mutagenesis, and sporulation. Here, we report our studies on the regulation of RAD6 gene expression after UV damage, during the mitotic cell cycle, in meiosis, and following heat shock and starvation. RAD6 mRNA levels became elevated in cells exposed to UV light, and at all UV doses the increase in mRNA levels was rapid and occurred within 30 min after exposure to UV. RAD6 mRNA levels also increased in sporulating MATa/MAT alpha cells, and the period of maximal accumulation of RAD6 mRNA during meiosis is coincident with the time during which recombination occurs. However, RAD6 mRNA levels showed no periodic fluctuation in the mitotic cell cycle, were not elevated upon heat shock, and fell in cells in the stationary phase of growth. These observations suggest that RAD6 activity is required throughout the cell cycle rather than being restricted to a specific stage, and that during meiosis, high levels of RAD6 activity may be needed at a stage coincident with genetic recombination. The observation that RAD6 transcription is not induced by heat and starvation, treatments that activate stress responses, suggests that the primary role of RAD6 is in the repair of damaged DNA rather than in adapting cells to stress situations.

DNA Damage↗

PubMed2003

Potential role of MLH1 in the induction of p53 and apoptosis by blocking transcription on damaged DNA templates.

Defects in DNA mismatch repair (MMR) are common in human cancers, confer tolerance to certain types of chemotherapeutic agents, and lead to genomic instability. In addition to their mismatch-correcting roles during DNA replication, MMR proteins can bind to certain DNA lesions and signal p53 and apoptosis by an unknown mechanism. To further study the mechanism by which the MMR protein MLH1 is involved in the induction of p53 and apoptosis, we exposed the colon carcinoma cell line HCT116 (MLH1-deficient) and mlh1-corrected HCT116 sublines to alkylating agents or hydrogen peroxide (H2O2). It was found that while alkylating agents induced both apoptosis and phosphorylation of the Ser-15 site of p53 in a MLH1-dependent manner, induction of apoptosis, but not p53 phosphorylation, was MLH1 dependent following treatment with H2O2. The MLH1-dependent induction of p53 phosphorylation by alkylating agents did not appear to be cell cycle dependent, arguing against a futile repair mechanism operating during S phase as the sole mechanism for the MLH1-dependent DNA damage signaling. Importantly, we found that both alkylating agents and H2O2 caused significant inhibition of mRNA synthesis in MLH1-expressing but not in MLH1-deficient cells. These findings suggest a novel mechanism of MLH1 in the induction p53 and apoptosis by inhibiting RNA polymerase II-dependent transcription on damaged DNA templates.

Adaptor Proteins, Signal Transducing↗

PubMed1991

Mutagenicity, DNA damage and DNA adduct formation by N-nitroso-2-hydroxyalkylamine and corresponding aldehydes.

The potent carcinogen N-nitrosodiethanolamine (NDELA) becomes mutagenic to Salmonella typhimurium TA98 and TA100 when activated by alcohol dehydrogenase from yeast or horse liver. Metabolic pathways different from alpha-oxidation might therefore be important for the activation of N-nitroso-2-hydroxyalkylamines such as NDELA. In an in-vitro test system (Namalva cells), neither NDELA nor N-nitrosoethyl-2-hydroxyethylamine was genotoxic, whereas the corresponding metabolites from alcohol dehydrogenase-mediated oxidation, N-nitroso-2-hydroxymorpholine and N-nitrosoethylethanalamine, induced single-strand breaks even at low doses. An immuno-slot-blot assay was used to study the formation of O6-2-hydroxyethyldeoxyguanosine in rat liver after oral administration of different N-nitroso-2-hydroxyalkylamines. When given at equimolar doses (0.375 mmol/kg), DNA hydroxyethylation was considerably lower (6.7 mumol/mol deoxyguanosine) with NDELA than with N-nitrosoethyl-2-hydroxyethylamine (48.7 mumol/mol deoxyguanosine) or N-nitrosomethyl-2-hydroxyethylamine (72.1 mumol/mol deoxyguanosine). N-Nitroso-2-hydroxymorpholine did not form detectable levels of O6-2-hydroxyethyldeoxyguanosine.

Aldehydes↗

PubMed2000

DNA damage checkpoints and DNA replication controls in Saccharomyces cerevisiae.

In response to genotoxic agents and cell cycle blocks all eukaryotic cells activate a set of surveillance mechanims called checkpoints. A subset of these mechanisms is represented by the DNA damage checkpoint, which is triggered by DNA lesions. The activation of this signal transduction pathway leads to a delay of cell cycle progression to prevent replication and segregation of damaged DNA molecules, and to induce transcription of several DNA repair genes. The yeast Saccharomyces cerevisiae has been invaluable in genetically dissecting the DNA damage checkpoint pathway and recent findings have provided new insights into the architecture of checkpoint protein complexes, in their order of function and in the mechanisms controlling DNA replication in response to DNA damage.

Cell Cycle↗

PubMed1994

Nuclear accumulation of p53 in response to treatment with DNA-damaging agents.

A number of agents which damage DNA also trigger the nuclear accumulation of the tumor suppressor protein p53. Here we show the correlation with different p53 detection methods. As an example we investigated the effects of the cancer therapy drug mitomycin C on different mammalian cell lines. Our findings demonstrate that either the immunofluorescence techniques (indirect immunofluorescence staining or flow cytometric analysis) or ELISA or immunoblot assays are useful methods in detecting p53 accumulation. Simultaneously we measured DNA damage with the terminal deoxynucleotidyl transferase assay. Compatible data were obtained. Thus p53 accumulation may be used as indicator of DNA injury.

3T3 Cells↗

PubMed1985

Molecular mechanisms for the recognition of damaged DNA regions by peptides and proteins.

DNA damages can lead to drastic perturbations of living cell cycle (e.g., in carcinogenesis) by inducing mutations in the genetic information. Therefore DNA repair processes play an important role during cell life by eliminating DNA damages before mutation fixation. Different repair processes are briefly presented in this review. Two probes were used to provide information on the mechanisms involved in the specific recognition of damaged DNA by proteins and enzymes of the DNA repair machinery. It will be shown that a simple tripeptide Lys-Trp-Lys is able to mimic two repair systems, namely, the photosensitized splitting of pyrimidine dimers and the cleavage of phosphodiester bonds at apurinic sites.

DNA↗

PubMed2000

Detection of oxidative base DNA damage by a new biochemical assay.

Reactive oxygen species (ROS) damage DNA which appears to represent the major target involved in mutagenesis, carcinogenesis, and aging cell responses. Various DNA modifications are generated by ROS, but 8-hydroxy-2'-deoxyguanosine (8-oxoG) has retained a lot of attention in the last few years. Therefore, numerous methods have been developed to detect and quantify the extent of 8-oxoG in DNA, most of them requiring a significant amount of DNA that might be limiting in the case of biological samples. 8-oxoG is repaired in Escherichia coli by a specific glycosylase, the Fpg (formamidopyrimidine DNA glycosylase) protein, in a reaction that requires a covalent intermediate favored under reducing conditions. We set up a new assay based on the capture of plasmid DNA into sensitized microplate wells. DNA damaged by photoactivation of methylene blue was adsorbed on a polylysine-treated plastic well. Then the Fpg protein was added, allowed to fix on the damage by taking advantage of minimized glycosylase activity at low temperature and the reductive trapping of the covalent intermediate, yielding to a stable DNA-protein interaction. The trapped protein was subsequently recognized by a specific antibody. A secondary antibody coupled with horseradish peroxidase was used to detect the complex and the measurement was carried out by chemiluminescence. This new assay offers various potentialities, specifically in the field of technology of ROS producers.

8-Hydroxy-2'-Deoxyguanosine↗