Formation Of Single-strand Breaks In DNA Research Articles

UV-induced photolesions elicit ATR-kinase-dependent signaling in non-cycling cells through nucleotide excision repair-dependent and -independent pathways

Activation of signaling pathways by UV radiation is a key event in the DNA damage response and initiated by different cellular processes. Here we show that non-cycling cells proficient in nucleotide excision repair (NER) initiate a rapid but transient activation of the damage response proteins p53 and H2AX; by contrast, NER-deficient cells display delayed but persistent signaling and inhibition of cell cycle progression upon release from G0 phase. In the absence of repair, UV-induced checkpoint activation coincides with the formation of single-strand DNA breaks by the action of the endonuclease Ape1. Although temporally distinct, activation of checkpoint proteins in NER-proficient and NER-deficient cells depends on a common pathway involving the ATR kinase. These data reveal that damage signaling in non-dividing cells proceeds via NER-dependent and NER-independent processing of UV photolesions through generation of DNA strand breaks, ultimately preventing the transition from G1 to S phase.

CARDIOMYOCYTIC APOPTOSIS FOLLOWING GLOBAL CARDIAC ISCHEMIA AND REPERFUSION CAN BE ATTENUATED BY PEROXISOME PROLIFERATOR-ACTIVATED RECEPTOR α BUT NOT γ ACTIVATORS

In this study, experiments were designed to determine if peroxisome proliferator-activated receptor (PPAR) alpha agonists could decrease myocardial ischemia/reperfusion injury after cardioplegia-induced cardiac arrest under cardiopulmonary bypass, attenuate the appearance of cardiomyocytic apoptosis, and decrease the damage of reactive oxygen species. Cardiomyocytic apoptosis occurs after cardiopulmonary bypass surgery. Reactive oxygen species and peroxynitrite generated during ischemia/reperfusion initiate the formation of single-strand DNA breaks. Peroxisome proliferator-activated receptors (PPARs) activators had an important role in alleviating myocardial apoptosis. Four groups of New Zealand white rabbits (10 in each group, each 2.5-3.5 kg) underwent cardiopulmonary bypass. Thirty minutes before surgery, one group received WY14643 (a PPAR-alpha agonist, 1 mg kg(-1)) and another received 15D-PGJ2 (a PPAR-gamma agonist; 0.3 mg kg(-1)). The ascending aorta was cross-clamped for 60 min, whereas intermittent cold crystalloid cardioplegic solution was infused into the aortic root every 20 min. The myocardium of the reperfused hearts and control hearts were harvested and studied in vitro for evidence of apoptosis using terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling method and Western blot analyses of cytochrome c and apoptosis-inducing factor. The reactive oxidative insults were checked using enzyme-linked immunosorbent assay to detect plasma cytokine levels. The occurrence of cardiomyocytic apoptosis and elevation of plasma cytokines were significantly lower in the group receiving PPAR-alpha agonists than in the other groups. Western blot analysis of apoptosis-inducing factor and cytochrome c revealed similar patterns. PPAR-alpha activation could diminish postischemic cardiomyocytic apoptosis and reactive oxygen species injuries after global cardiac arrest under cardiopulmonary bypass, possibly via prevention of both caspase-dependent and caspase-independent apoptotic pathways.

Bcl2 Suppresses DNA Repair by Enhancing c-Myc Transcriptional Activity

Bcl2 and c-Myc are two major oncogenic proteins that can functionally promote DNA damage, genetic instability, and tumorigenesis. However, the mechanism(s) remains unclear. Nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is the most potent carcinogen contained in cigarette smoke that induces cellular DNA damage. Here we found that Bcl2 potently suppresses the repair of NNK-induced abasic sites of DNA lesions in association with increased c-Myc transcriptional activity. The Bcl2 BH4 domain (amino acids 6-31) was found to bind directly to c-Myc MBII domain (amino acids 106-143), and this interaction is required for Bcl2 to enhance c-Myc transcriptional activity and inhibit DNA repair. In addition to mitochondria, Bcl2 is also expressed in the nucleus, where it co-localizes with c-Myc. Expression of nuclear-targeted Bcl2 enhances c-Myc transcriptional activity with suppression of DNA repair but fails to prolong cell survival. Depletion of c-Myc expression from cells overexpressing Bcl2 significantly accelerates the repair of NNK-induced DNA damage, indicating that c-Myc may be essential for the Bcl2 effect on DNA repair. It is known that apurinic/apyrimidinic endonuclease (APE1) plays a crucial role in the repair of abasic sites of DNA lesions. That overexpression of Bcl2 results in up-regulation of c-Myc and down-regulation of APE1 suggests APE1 may function as the downstream target of Bcl2/c-Myc in the DNA repair machinery. Thus, Bcl2, in addition to its survival function, may also suppress DNA repair in a novel mechanism involving c-Myc and APE1, which may lead to an accumulation of DNA damage in living cells, genetic instability, and tumorigenesis.

Reduction of acute photodamage in skin by topical application of a novel PARP inhibitor

The ultraviolet (UV) components of sunlight induce damage to the DNA in skin cells, which is considered to be the initiating step in the harmful biological effects of UV radiation. Repair of DNA damage results in the formation of single-strand DNA breaks, which activate the nuclear poly(ADP-ribose) polymerase (PARP). Overactivation of PARP worsens the oxidative cell damage and impairs the energy metabolism, raising the possibility that moderation of PARP activation following DNA damage may protect skin cells from UV radiation. The topical effects of the novel PARP inhibitor O-(3-pyperidino-2-hydroxy-1-propyl) pyridine-3-carboxylic acid amidoxime monohydrochloride (BGP-15M) were investigated on UV-induced skin damage in a hairless mouse model. For evaluation of the UV-induced acute photodamage to the skin and the potential protective effect of BGP-15M, DNA injury was detected by measuring the formation of single-strand DNA breaks and counting the resulting sunburn (apoptotic) cells. The ADP-ribosylation of PARP was assessed by Western blot analysis and then quantified. In addition, the UV-induced immunosuppression was investigated by the immunostaining of tumor necrosis factor alpha and interleukin-10 expressions in epidermal cells. The signs of inflammation were examined clinically and histochemically. Besides its primary effect in decreasing the activity of nuclear PARP, topically applied BGP-15M proved to be protective against solar and artificial UV radiation-induced acute skin damage. The DNA injury was decreased ( P<0.01). An inhibition of immunosuppression was observed by down-regulation of the epidermal production of cytokines IL-10 and TNFα. In the mouse skin, clinical or histological signs of UV-induced inflammation could not be observed. These data suggest that BGP-15M directly interferes with UV-induced cellular processes and modifies the activity of PARP. The effects provided by topical application of the new PARP-regulator BGP-15M indicate that it may be a novel type of agent in photoprotection of the skin.

Aqueous extracts of cigarette tar containing the tar free radical cause DNA nicks in mammalian cells.

The ability of aqueous extracts of cigarette tar to nick DNA was investigated using viable mammalian cells. Tar extracts contain a radical with a stable electron spin resonance (ESR) signal at g = 2.0036 characteristic of a semiquinone. The association of the tar component that carries the ESR signal with DNA was demonstrated using viable rat alveolar macrophages. The formation of single-strand DNA breaks caused by cigarette tar extracts in viable rat thymocytes follows saturation kinetics, indicating a tar component associates with DNA and then nicks it. These studies support our hypothesis that tar components that contain the cigarette tar radical can enter cells, associate with, and then nick DNA.

Cytotoxicity of, and DNA damage by, active oxygen species produced by xanthine oxidase

Toxicity to Raji cells of the xanthine oxidase/hypoxanthine system is related to the formation of single-strand DNA breaks. DNA damage was proportional to the concentration of xanthine oxidase and to the time of exposure. It was prevented by the absence of hypoxanthine, or by the presence of allopurinol, or both superoxide dismutase and catalase. The release of 51Cr from damaged cells was detectable 12h after the inhibition of cloning efficiency and the production of DNA breakage. These data suggest that DNA damage induced by the oxygen products precedes the severe lesion to the cellular membrane.

DNA strand breaks induced by hydrogen peroxide in isolated rat hepatocytes.

It has been proposed that increased rates of hepatic hydrogen peroxide (H2O2) production may initiate or promote the liver tumors that appear following chronic exposure of rodents to chemicals that cause peroxisome proliferation. However, the effect of H2O2 on the structural integrity of DNA in parenchymal hepatocytes, the target cells of peroxisome proliferator-induced carcinogenesis, is largely uncharacterized. Furthermore, oxidant-induced cellular damage has been invoked as causal of a number of hepatotoxic effects associated with exposure to chemicals other than peroxisome proliferators. For these reasons, alkaline elution analysis was used to study the action of H2O2, added exogenously, on DNA of intact, isolated rat hepatocytes. Addition of a bolus of H2O2 (0.01-1.0 mM) to monolayer cultures of hepatocytes caused concentration-dependent increases in single-strand DNA breaks (SSDB), which were maximal within 30 min of exposure. Cytotoxicity, as measured by lactate dehydrogenase (LDH) release, was minimal during a 1-h exposure to H2O2 concentrations less than 1 mM, but the efflux of oxidized glutathione was increased. Formation of SSDB was nearly linear with respect to H2O2 concentration in the range 0.1-1.0 mM. No double-strand DNA breaks or DNA-protein crosslinks were identified at H2O2 concentrations of 1 mM or less. Repair of SSDB in H2O2-free medium occurred in a rapid, linear manner only for the first 15 min, resulting in disappearance of 65% of the SSDB. A second, slower phase of SSDB rejoining occurred between 20 and 60 min of incubation in H2O2-free media; at 60 min rejoining was maximal (80% repair). These results define a specific type of DNA damage associated with H2O2 exposure of hepatocytes and suggest that primary cultures of rat hepatocytes are a suitable model for characterizing the potential genotoxic effects of oxidants, particularly excess H2O2 that may occur in the livers of animals exposed chronically to peroxisome proliferators.

Effects of sodium arsenite on single-strand DNA break formation and post-replication repair in E. coli following UV irradiation

A non-lethal dose of sodium arsenite is found to inhibit the formation of single-strand DNA breaks in Escherichia coli WP2 wild-type and WP6 polA strains after UV irradiation. Inhibition of single-strand breakage follows a dose-dependent relationship with respect to increasing sodium arsenite concentration. ATP level in WP2 cells is decreased in the presence of sodium arsenite and therefore the inhibition of DNA break formation may be mediated through lowered ATP levels in the irradiated cells. In the presence of a non-lethal dose of sodium aresenite, post-replication repair in WP2 uvrA strains after UV irradiation is also inhibited.