Accelerated DNA Repair Research Articles

FGF2 mediates DNA repair in epidermoid carcinoma cells exposed to ionizing radiation

Purpose: Fibroblast growth factor 2 (FGF2) is a well-known survival factor. However, its role in DNA repair is poorly documented. The present study was designed to investigate in epidermoid carcinoma cells the potential role of FGF2 in DNA repair.Materials and methods: The side population (SP) with cancer stem cell-like properties and the main population (MP) were isolated from human A431 squamous carcinoma cells. Radiation-induced DNA damage and repair were assessed using the alkaline comet assay. FGF2 expression was quantified by enzyme linked immunosorbent assay (ELISA).Results: SP cells exhibited rapid repair of radiation induced DNA damage and a high constitutive level of nuclear FGF2. Blocking FGF2 signaling abrogated the rapid DNA repair. In contrast, in MP cells, a slower repair of damage was associated with low basal expression of FGF2. Moreover, the addition of exogenous FGF2 accelerated DNA repair in MP cells. When irradiated, SP cells secreted FGF2, whereas MP cells did not.Conclusions: FGF2 was found to mediate DNA repair in epidermoid carcinoma cells. We postulate that carcinoma stem cells would be intrinsically primed to rapidly repair DNA damage by a high constitutive level of nuclear FGF2. In contrast, the main population with a low FGF2 content exhibits a lower repair rate which can be increased by exogenous FGF2.

Low-Dose UVA Radiation-Induced Adaptive Response in Cultured Human Dermal Fibroblasts

Objective.To investigate the mechanism of the adaptive response induced by low-dose ultraviolet A (UVA) radiation.Methods.Cultured dermal fibroblasts were irradiated by a lethal dose of UVA (86.4 J/cm2) with preirradiation of single or repetitive low dose of UVA (7.2 J/cm2). Alterations of cellular morphology were observed by light microscope and electron microscope. Cell cycle and cellular apoptosis were assayed by flow cytometer. The extent of DNA damage was determined by single-cell gel electrophoresis (SCGE).Results.The cultured dermal fibroblasts, with pretreatment of single or repetitive irradiation of 7.2 J/cm2UVA relieved toxic reaction of cellular morphology and arrest of cell cycle, decreased apoptosis ratio, reduced DNA chain breakage, and accelerated DNA repair caused by subsequent 86.4 J/cm2UVA irradiation. Compared with nonpretreatment groups, all those differences were significant (P<0.01orP<0.05).Conclusions.The adaptation reaction might depend on the accumulated dose of low-dose UVA irradiation. Low-dose UVA radiation might induce adaptive response that may protect cultured dermal fibroblasts from the subsequent challenged dose of UVA damage. The duration and protective capability of the adaptive reaction might be related to the accumulated dose of low-dose UVA Irradiation.

Nucleotides Accelerate Mice Thymocyte DNA Repair in vitro

Wang, L., Song, X. and Gong, X. 2010. Nucleotides accelerate mice thymocyte DNA repair in vitro. J. Appl. Anim. Res., 38: 105–108. Effect of nucleotides on the repair of DNA damaged by N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) in mice thymocytes was evaluated using comet assay. Thymocytes from KunMing male mice were damaged with 0, 10 or 50 μmol/l MNNG. DNA damaged cells in each treatment were divided into four groups and cultured in medium supplemented with 0, 0.1, 1 and 10 mmol/l nucleotides. Nucleotide supplementation decreased the comet cells at 2 and 5 h and increased DNA repair on a dose dependent manner. These being more effective at 10 μmol/l MNNG level than 50 μmol level. Effect of nucleotides was more pronounced on comet tail size. The results indicated that the nucleotides supplementation accelerated DNA repair and the effect of nucleotides depended on the supplemental levels and the extent of DNA damage.

Bcl-2 and accelerated DNA repair mediates resistance of hair follicle bulge stem cells to DNA-damage-induced cell death

Adult stem cells (SCs) are at high risk of accumulating deleterious mutations because they reside and self-renew in adult tissues for extended periods. Little is known about how adult SCs sense and respond to DNA damage within their natural niche. Here, using mouse epidermis as a model, we define the functional consequences and the molecular mechanisms by which adult SCs respond to DNA damage. We show that multipotent hair-follicle-bulge SCs have two important mechanisms for increasing their resistance to DNA-damage-induced cell death: higher expression of the anti-apoptotic gene Bcl-2 and transient stabilization of p53 after DNA damage in bulge SCs. The attenuated p53 activation is the consequence of a faster DNA repair activity, mediated by a higher non-homologous end joining (NHEJ) activity, induced by the key protein DNA-PK. Because NHEJ is an error-prone mechanism, this novel characteristic of adult SCs may have important implications in cancer development and ageing.

Gene deletion of inositol hexakisphosphate kinase 2 predisposes to aerodigestive tract carcinoma

Inositol hexakisphosphate kinase 2 (IP6K2), a member of the inositol hexakisphosphate kinase family, functions as a growth suppressive and apoptosis-enhancing kinase during cell stress. We created mice with a targeted deletion of IP6K2; these mice display normal embryogenesis, development, growth and fertility. Chronic exposure to the carcinogen 4-nitroquinoline 1-oxide (4-NQO, a UV-mimetic compound) in drinking water resulted in fourfold increased incidence of invasive squamous cell carcinoma (SCC) formation in the oral cavity and esophagus of the knockout (KO) mice compared to the wild-type (WT) littermates. Paradoxically, KO mice displayed relative resistance to ionizing radiation and exhibit enhanced survival following 8-10 Gy total body irradiation. Primary KO fibroblasts displayed resistance to antiproliferative effects of interferon-beta and increased colony forming units following ionizing radiation. Radioresistance of KO fibroblasts was associated with accelerated DNA repair measured by comet assay. Direct microinjection of 5-PP-Ins(1,2,3,4,6)P(5) (the enzymatic product of IP6K2), but not InsP(6) (the substrate of IP6K2) induced cell death in SCC22A squamous carcinoma cells.

Statins Use a Novel Nijmegen Breakage Syndrome-1–Dependent Pathway to Accelerate DNA Repair in Vascular Smooth Muscle Cells

Although the hydroxymethylglutaryl-coenzyme A reductase inhibitors (statins) are widely used in atherosclerosis to reduce serum cholesterol, statins have multiple other effects, including direct effects on cells of the vessel wall. Recently, DNA damage, including telomere shortening, has been identified in vascular smooth muscle cells (VSMCs) in human atherosclerosis. Although statins reduce DNA damage in vitro, the mechanisms by which they might protect DNA integrity in VSMCs are unknown. We show that human atherosclerotic plaque VSMCs exhibit increased levels of double-stranded DNA breaks and basal activation of DNA repair pathways involving ataxia telangiectasia-mutated (ATM) and the histone H2AX in vivo and in vitro. Oxidant stress induced DNA damage and activated DNA repair pathways in VSMCs. Statin treatment did not reduce oxidant stress or DNA damage but markedly accelerated DNA repair. Accelerated DNA repair required both the Nijmegen breakage syndrome (NBS)-1 protein and the human double minute protein Hdm2, accompanied by phosphorylation of Hdm2, dissociation of NBS-1 and Hdm2, inhibition of NBS-1 degradation, and accelerated phosphorylation of ATM. Statin treatment reduced VSMC senescence and telomere attrition in culture, accelerated DNA repair and reduced apoptosis in vivo after irradiation, and reduced ATM/ATR (ATM and Rad3-related) activity in atherosclerosis. We conclude that statins activate a novel mechanism of accelerating DNA repair, dependent on NBS-1 stabilization and Hdm2. Statin treatment may delay cell senescence and promote DNA repair in atherosclerosis.

Polydeoxyribonucleotide promotes cyclobutane pyrimidine dimer repair in UVB‐exposed dermal fibroblasts

DNA is the main cellular chromophore for ultraviolet B (UVB). Its absorption leads to the generation of typical photoproducts. The most frequent types (about 80%) are cyclobutane pyrimidine dimers (CPDs). Several studies have suggested that treatment with deoxyribonucleosides can protect some cell types from DNA damage. The aim of this work was to evaluate the ability of the polydeoxyribonucleotide (PDRN) to protect human dermal fibroblasts from UVB-induced DNA damage. Human dermal fibroblasts were irradiated with 600 mJ/cm(2) of UVB radiation. Cells were analyzed at increasing time points from irradiation to study the recovery from UVB-induced DNA photodamage. Damage repair was subsequently assessed by immunocytochemical analysis of CPDs levels and by measurement of p53 protein expression. The extracellular addition of 100 microg/ml PDRN immediately after irradiation caused a strong activation of p53 protein in the first 24 h. This signal was accompanied by an increase in CPDs repair rates at early time points of recovery. The addition of PDRN to the culture medium supports CPDs repair probably providing a faster supply of precursors for the deoxyribonucleotide triphosphates pool necessary to UVB-damaged cells. This condition could promote the action of the salvage pathway, thereby accelerating DNA repair, but other inducible responses linked to increased p53 expression could be involved.

Abstract 780: Atorvastatin Activates A Novel Nbs1-dependent Mechanism Of Accelerating Dna Repair In Atherosclerosis

There is increasing evidence that reactive oxidant species (ROS) and DNA damage promote the development and complications of atherosclerosis. Although statin therapy reduces both ROS and DNA damage in atherosclerosis, the mechanism of this effect is unknown. We first examined expression of DNA damage and repair markers in vascular smooth muscle cells (VSMCs) of human atherosclerotic plaques. With increasing disease severity, there was increased VSMC expression of the DNA repair markers P-ATM/ATR substrate and P-H2AX from 2.7%±2.2 and 0.5±0.71 [mean±SEM] (AHA Grade I/II), to 21%±3.5 and 36.5±2.1 (Grade III) lesions, and 86.5%±0.7 and 69.3±7.6 (Grade IV/V). Cultured plaque VSMCs also showed a 1.5 fold increased oxidant stress; a 4.4 fold increased double-stranded DNA breaks, and expression of P-H2AX by Western blots. ROS analogues induced a robust DNA damage response in VSMCs, characterised by lengthening of tails on COMET assay, and activation of ATM and P-H2AX, with completion of repair by 6 hours. Atorvastatin pre-treatment accelerated DNA repair by approximately 2 hours without inhibiting ROS induction or DNA damage, and markedly accelerated the kinetics of nibrin (NBS-1) and P-H2AX activation, both proteins recruited to sites of DNA damage, by preventing degradation of NBS-1. Atorvastatin induced phosphorylation of HDM2, an E3 ligase and putative regulator of NBS-1 stability, and siRNA knockdown of HDM2 replicated the effect of atorvastatin on NBS-1. The ability of atorvastatin to accelerate repair was completely dependent upon NBS-1, as atorvastatin was ineffective in cells either null or expressing constitutively active NBS-I. In summary, we have demonstrated a novel NBS-1-dependent mechanism by which statins accelerate DNA repair in atherosclerosis, through HDM2 phosphorylation and stabilisation of NBS-1. We believe that both NBS-1 and HDM2 are critical to DNA repair in atherosclerosis.

Naringin, a grapefruit flavanone, protects V79 cells against the bleomycin‐induced genotoxicity and decline in survival

The effect of naringin, a grapefruit flavonone was studied on bleomycin-induced genomic damage and alteration in the survival of cultured V79 cells. Exposure of V79 cells to bleomycin induced a concentration dependent elevation in the frequency of binucleate cells bearing micronuclei (MNBNC) and a maximum number of MNBNCs were observed in the cells treated with 50 microg ml(-1) bleomycin, the highest concentration evaluated. This genotoxic effect of bleomycin was reflected in the cell survival, where a concentration dependent decline was observed in the cells treated with different concentrations of bleomycin. Treatment of cells with 1 mm naringin before exposure to different concentrations of bleomycin arrested the bleomycin-induced decline in the cell survival accompanied by a significant reduction in the frequency of micronuclei when compared with bleomycin treatment alone. The cell survival and micronuclei induction were found to be inversely correlated. The repair kinetics of DNA damage induced by bleomycin was evaluated by exposing the cells to 10 microg ml(-1) bleomycin using single cell gel electrophoresis. Treatment of V79 cells with bleomycin resulted in a continuous increase in DNA damage up to 6 h post-bleomycin treatment as evident by migration of more DNA into the tails (% tail DNA) of the comets and a subsequent increase in olive tail moment (OTM), an index of DNA damage. Treatment of V79 cells with 1 mm naringin reduced bleomycin-induced DNA damage and accelerated DNA repair as indicated by a reduction in % tail DNA and OTM with increasing assessment time. A maximum reduction in the DNA damage was observed at 6 h post-bleomycin treatment, where it was 5 times lower than bleomycin alone. Our study, which was conducted on the basis of antioxidant, free radical scavenging and metal chelating properties of naringin demonstrates that naringin reduced the genotoxic effects of bleomycin and consequently increased the cell survival and therefore may act as a chemoprotective agent in clinical situations.

Prophylaxis of oxidative DNA damage by formamidopyrimidine‐DNA glycosylase

Lying at the gas-exchange interface, lung epithelia may be at risk of oxidation-induced mutagenesis. Further, inflammation processes possibly consequent on smoking liberate reactive oxygen species that multiply the carcinogenic effects of tobacco. DNA repair mechanisms play a major role in counteracting the deleterious effects of oxidative DNA damage. Some studies find positive associations between lung cancer and variations in the human 8-oxoguanine DNA glycosylase (hOGG1) gene that encodes a major DNA glycosylase for oxidized lesions with sluggish kinetics properties. The bacterial homologue formamidopyrimidine-DNA glycosylase (FPG) is 80-fold faster than hOGG1 in repairing mutagenic oxidative lesions. Cell-culture studies have shown that FPG can be expressed in mammalian cells, where it accelerates DNA repair and abates mutagenicity of a wide range of DNA-damaging agents. Prophylaxis of oxidative DNA damage and mutation could be achieved in lung epithelia and other tissues of at-risk individuals by expression of the FPG protein. Currently available vehicles for this peculiar type of gene therapy are briefly surveyed.

Introduction of human telomerase reverse transcriptase to normal human fibroblasts enhances DNA repair capacity.

From numerous reports on proteins involved in DNA repair and telomere maintenance that physically associate with human telomerase reverse transcriptase (hTERT), we inferred that hTERT/telomerase might play a role in DNA repair. We investigated this possibility in normal human oral fibroblasts (NHOF) with and without ectopic expression of hTERT/telomerase. To study the effect of hTERT/telomerase on DNA repair, we examined the mutation frequency rate, host cell reactivation rate, nucleotide excision repair capacity, and DNA end-joining activity of NHOF and NHOF capable of expressing hTERT/telomerase (NHOF-T). NHOF-T was obtained by transfecting NHOF with hTERT plasmid. Compared with parental NHOF and NHOF transfected with empty vector (NHOF-EV), we found that (a) the N-methyl-N'-nitro-N-nitrosoguanidine-induced mutation frequency of an exogenous shuttle vector was reduced in NHOF-T, (b) the host cell reactivation rate of N-methyl-N'-nitro-N-nitrosoguanidine-damaged plasmids was significantly faster in NHOF-T; (c) the nucleotide excision repair of UV-damaged DNA in NHOF-T was faster, and (d) the DNA end-joining capacity in NHOF-T was enhanced. We also found that the above enhanced DNA repair activities in NHOF-T disappeared when the cells lost the capacity to express hTERT/telomerase. These results indicated that hTERT/telomerase enhances DNA repair activities in NHOF. We hypothesize that hTERT/telomerase accelerates DNA repair by recruiting DNA repair proteins to the damaged DNA sites.

Prevention and repair of DNA damage by selected phytochemicals as measured by single cell gel electrophoresis.

We assessed the ability of some natural products--namely, curcumin, resveratrol, indole-3-carbinol, and ellagic acid--to modify the DNA damaging ability of the alkylating carcinogen N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) in cultured Chinese hamster lung fibroblast cells (CH V-79). MNNG produced DNA single strand breaks in a dose- and time-dependent manner, as observed by increase in the tail moments of the comet, when the cells were subjected to alkaline single cell gel electrophoresis. When the cells were treated in the presence of each of the natural compounds, the DNA damage caused by MNNG was considerably reduced. This effect was found to be dose related. Preincubation of cells with each of these compounds individually afforded significant protection to DNA against damage caused by subsequent treatment with MNNG, indicating a true chemopreventive role of these substances. The most remarkable aspect of the present study was that all four compounds helped in the recovery of DNA damage by accelerating DNA repair efficiency in the damaged cells. This was further substantiated by the observation on unscheduled DNA synthesis. Our results suggest that these agents are chemopreventive by virtue of their ability to protect DNA as well as to induce DNA repair.

Poly(ADP-ribosyl)ation accelerates DNA repair in a pathway dependent on Cockayne syndrome B protein.

Activation of poly(ADP-ribose)polymerases 1 and 2 (PARP-1 and PARP-2) is one of the earliest responses of mammalian cells to DNA damage by numerous genotoxic agents. We have analysed the influence of PARP inhibition, either achieved by over-expression of the DNA binding domain of PARP-1 or by treatment with 3,4-dihydro-5-[4-(1-piperidinyl)butoxyl]-1(2H)-isoquinolinone, on the repair of single-strand breaks (SSB), pyrimidine dimers and oxidative base modifications sensitive to Fpg protein (mostly 8-hydroxyguanine) in mammalian cells at very low, non-cytotoxic levels of DNA damage. The data show that the repair rates of all three types of DNA damage are significantly lower in PARP-inhibited cells. Importantly, the retardation of the repair of base modifications is not associated with accumulation of intermediates such as SSB or abasic sites. Moreover, the influence of the PARP inhibition is not observed in cells deficient in Cockayne syndrome B protein (Csb). The results indicate that PARP activation and Csb are both involved in a novel mechanism that accelerates the global repair of various types of DNA modifications.

Alkylator-induced DNA excision repair in human leukemia CCRF-CEM cells in vitro, measured using the single-cell gel electrophoresis (comet) assay.

The capacity to repair DNA damage is an important factor that affects the therapeutic outcome in cancer treatment. To clarify the cellular repair response, we investigated the kinetics of DNA excision repair initiated by 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) in human leukemia CCRF-CEM cells at an exponential growth phase in vitro. Using the alkaline single-cell gel electrophoresis (comet) assay, we quantitated the repair kinetics as the amount of DNA single-strand breaks that were generated from the incision and were diminished by the rejoining in the repair process. CEM cells could initiate DNA excision repair in response to BCNU by starting an incision reaction. However, the incision capacity came to a plateau at a concentration of 80 to 100 microM or after an incubation time of 90 to 120 minutes. When the cells were pulsed with 40 microM BCNU, the maximal incision occurred at the end of the incubation period, and the repair process was completed within 4 hours When cells were treated with 100 microM BCNU, the incised DNA was not rejoined at 4 hours, suggesting that the repair was not completed. Higher concentrations might surpass the cellular capacity for repair and would be associated with increased cell death. Evaluation of the repair process may provide a clue for therapeutic strategies to improve clinical efficacy if accelerated DNA repair is responsible for the drug resistance.

In vitro evidence for homologous recombinational repair in resistance to melphalan.

The generation of DNA interstrand cross-links is thought to be important in the cytotoxicity of nitrogen mustard alkylating agents, such as melphalan, which have antitumor activity. Cell lines with mutations in recombinational repair pathways are hypersensitive to nitrogen mustards. Thus, resistance to melphalan may require accelerated DNA repair by either recombinational repair mechanisms involving Rad51-related proteins (including x-ray repair cross-complementing proteins Xrcc2, Xrcc3, and Rad52) or by nonhomologous endjoining involving DNA-dependent protein kinase (DNA-PK) and Ku proteins. We investigated the role of DNA repair in melphalan resistance in epithelial tumor cell lines. Melphalan cytotoxicity was determined in 14 epithelial tumor cell lines by use of the sulforhodamine assay. Homologous recombinational repair involving Rad51-related proteins was investigated by determining the levels of Rad51, Rad52, and Xrcc3 proteins and the density of nuclear melphalan-induced Rad51 foci, which represent sites of homologous recombinational repair. Nonhomologous endjoining was investigated by determining the levels of Ku70 and Ku86 proteins and DNA-PK activity. Linear regression analysis was used to analyze correlations between the various protein levels, DNA-PK activity, or Rad51 foci formation and melphalan cytotoxicity. All statistical tests were two-sided. Melphalan resistance was correlated with Xrcc3 levels (r =.587; P =.027) and the density of melphalan-induced Rad51 foci (r =.848; P =.008). We found no correlation between melphalan resistance and Rad51, Rad52, or Ku protein levels or DNA-PK activity. Correlations of melphalan resistance in epithelial tumor cell lines with Xrcc3 protein levels and melphalan-induced Rad51 foci density suggest that homologous recombinational repair is involved in resistance to this nitrogen mustard.

The radioprotective effect of BBI is associated with the activation of DNA repair-relevant genes

Purpose: To investigate the molecular mechanisms of the radioprotective effect of the Bowman-Birk proteinase inhibitor (BBI) in normal human skin fibroblasts (HSF). Material and methods: The effect of BBI pre-treatment on p53 protein level and on mRNA levels of downstream genes (ERCC3, Gadd45 and p53) was investigated. Results: As indicated by time-course experiments based on clonogenic assays, a 6 h pre-incubation with BBI before irradiation of HSF with a single dose of 6 Gy resulted in maximum radioprotection. In non-irradiated cells, pre-incubation with BBI resulted in an increased level of p53 protein. Concomitantly, enhanced mRNA levels of the ERCC3 and the Gadd45 genes were observed. As a consequence, BBI-treated cells showed accelerated DNA repair compared with untreated cells when irradiated. Conclusions: The radioprotective effect of the Bowman-Birk proteinase inhibitor was accompanied by elevated mRNA expression of repair-relevant genes prior to irradiation. Activation of the DNA-repair machinery induced by pre-treatment with BBI is one possible mechanism of the radioprotective effect of BBI.

Cyclin D1-mediated inhibition of repair and replicative DNA synthesis in human fibroblasts.

Cyclin D1 is a key regulator of the G1 phase of the cell cycle. Inhibition of cyclin D1 function results in cell cycle arrest, whereas unregulated expression of the protein accelerates G1. Cyclin D1 is localized to the nucleus during G1. We found that during repair DNA synthesis, subsequent to UV-induced DNA damage, G1 cells readily lost their cyclin D1 while the proliferating cell nuclear antigen (PCNA) tightly associated with nuclear structures. Microinjection of cyclin D1 antisense accelerated DNA repair, whereas overexpression of cyclin D1 prevented DNA repair and the relocation of PCNA after DNA damage. Coexpression of cyclin D1 with its primary catalytic subunit, Cdk4, or with Cdk2, also prevented repair. In contrast, coexpression of PCNA, which is also a cyclin D1-associated protein, restored the ability of cells to repair their DNA. Acute overexpression of cyclin D1 in fibroblasts prevented them from entering S phase. Again, these effects were abolished by coexpression of cyclin D1 together with PCNA, but not with Cdk4 or Cdk2. Altogether, these results indicate that down-regulation of cyclin D1 is necessary for PCNA relocation and repair DNA synthesis as well as for the start of DNA replication. Cyclin D1 appears to be an essential component of a G1-checkpoint.