DNA Damage Repair Kinetics Research Articles

Analysis of DNA damage and repair of plant extracts and phytochemicals, using yeasts as model organism

In this work the model organism Saccharomyces cerevisiae was used to characterise the biological activity and the mechanism of action of plant extracts and phytochemicals. One of the goals was to use mutant strains affected in oxidative stress response and DNA repair in order to uncover the molecular targets of plant extracts and phytochemicals. DNA damage was assessed with the comet assay [1,2], using “comet tail length” as a measure of DNA damage, which displayed a dose-response relationship with different DNA-damaging agents. Typical experiments involved incubation of yeast cells with plant extracts or phytochemicals before, during or after (for DNA repair kinetics) the oxidative shock with hydrogen peroxide. The results obtained with the comet assay showed that DNA damage was significantly decreased upon treatment with plant extracts and several phytochemicals in a dose-dependent manner (p< 0.001). In addition, DNA repair kinetics was significantly improved in cells incubated with an extract of leafs from Ginkgo biloba (GBE) [3]. However, in the mutant strain affected in CDC9, encoding a DNA ligase involved in the mechanisms of nucleotide excision repair and base excision repair, oxidative DNA damage repair kinetics was unchanged with GBE, suggesting that the activity of this extract involves one of these mechanisms, or both. As expected, GBE treatment improved survival of yeast cells when challenged with oxidative shock by H2O2. In addition, intracellular oxidation, measured by flow cytometry with the redox-sensitive fluorochrome dichlorofluorescein diacetate, was considerably decreased upon pre-treatment with GBE [3]. The use of simple eukaryotic organisms and the use of genetically manipulated strains can provide tools for the analysis of the mechanism of action of the biological activity of phytochemicals and plant extracts.

Reduced Activity of Double-Strand Break Repair Genes in Prostate Cancer Patients With Late Normal Tissue Radiation Toxicity

To investigate clinical parameters and DNA damage response as possible risk factors for radiation toxicity in the setting of prostate cancer. Clinical parameters of 61 prostate cancer patients, 34 with (overresponding, OR) and 27 without (non-responding, NR) severe late radiation toxicity were assembled. In addition, for a matched subset the DNA damage repair kinetics (γ-H2AX assay) and expression profiles of DNA repair genes were determined in ex vivo irradiated lymphocytes. Examination of clinical data indicated none of the considered clinical parameters to be correlated with the susceptibility of patients to develop late radiation toxicity. Although frequencies of γ-H2AX foci induced immediately after irradiation were similar (P=.32), significantly higher numbers of γ-H2AX foci were found 24 hours after irradiation in OR compared with NR patients (P=.03). Patient-specific γ-H2AX foci decay ratios were significantly higher in NR patients than in OR patients (P<.0001). Consequently, NR patients seem to repair DNA double-strand breaks (DSBs) more efficiently than OR patients. Moreover, gene expression analysis indicated several genes of the homologous recombination pathway to be stronger induced in NR compared with OR patients (P<.05). A similar trend was observed in genes of the nonhomologous end-joining repair pathway (P=.09). This is congruent with more proficient repair of DNA DSBs in patients without late radiation toxicity. Both gene expression profiling and DNA DSB repair kinetics data imply that less-efficient repair of radiation-induced DSBs may contribute to the development of late normal tissue damage. Induction levels of DSB repair genes (eg, RAD51) may potentially be used to assess the risk for late radiation toxicity.

Deficiency in Homologous Recombination Renders Mammalian Cells More Sensitive to Proton Versus Photon Irradiation

To investigate the impact of the 2 major DNA repair machineries on cellular survival in response to irradiation with the 2 types of ionizing radiation. The DNA repair and cell survival endpoints in wild-type, homologous recombination (HR)-deficient, and nonhomologous end-joining-deficient cells were analyzed after irradiation with clinically relevant, low-linear energy transfer (LET) protons and 200-keV photons. All cell lines were more sensitive to proton irradiation compared with photon irradiation, despite no differences in the induction of DNA breaks. Interestingly, HR-deficient cells and wild-type cells with small interfering RNA-down-regulated Rad51 were markedly hypersensitive to proton irradiation, resulting in an increased relative biological effectiveness in comparison with the relative biological effectiveness determined in wild-type cells. In contrast, lack of nonhomologous end-joining did not result in hypersensitivity toward proton irradiation. Repair kinetics of DNA damage in wild-type cells were equal after both types of irradiation, although proton irradiation resulted in more lethal chromosomal aberrations. Finally, repair kinetics in HR-deficient cells were significantly delayed after proton irradiation, with elevated amounts of residual γH2AX foci after irradiation. Our data indicate a differential quality of DNA damage by proton versus photon irradiation, with a specific requirement for homologous recombination for DNA repair and enhanced cell survival. This has potential relevance for clinical stratification of patients carrying mutations in the DNA damage response pathways.

Lymphocytes of Patients with Alzheimer’s Disease Display Different DNA Damage Repair Kinetics and Expression Profiles of DNA Repair and Stress Response Genes

Alzheimer’s disease (AD) is a progressive neurodegenerative disorder, characterized by loss of memory and cognitive capacity. Given the limitations to analyze brain cells, it is important to study whether peripheral lymphocytes can provide biological markers for AD, an interesting approach, once they represent the overall condition of the organism. To that extent, we sought to find whether lymphocytes of AD patients present DNA damage and repair kinetics different from those found in elderly matched controls (EC group) under in vitro treatment with hydrogen peroxide. We found that AD patient cells indeed showed an altered DNA repair kinetics (comet assay). Real-time quantitative analysis of genes associated with DNA stress response also showed that FANCG and CDKN1A are upregulated in AD, while MTH1 is downregulated, compared with the control group. In contrast, the expression of ATM, ATR and FEN1 genes does not seem to differ between these groups. Interestingly, TP53 protein expression was increased in AD patients. Therefore, we found that kinetics of the stress response in the DNA were significantly different in AD patients, supporting the hypothesis that repair pathways may be compromised in AD and that peripheral lymphocytes can reveal this condition.

Hsp90 Inhibitors NVP-AUY922 and NVP-BEP800 May Exert a Significant Radiosensitization on Tumor Cells along with a Cell Type-Specific Cytotoxicity

Targeting heat shock protein 90 (Hsp90) provides a promising therapeutic approach to enhance the sensitivity of tumor cells to ionizing radiation (IR). To explore the impact of scheduling drug-IR administration, in the present study, we analyzed the response of lung carcinoma A549 and glioblastoma SNB19 cells to simultaneous drug-IR treatment followed by a long-term drug administration. Cellular response was evaluated at different time intervals after IR-alone, drug-alone, or combined drug-IR treatments by colony counts and expression profiles of Hsp90 and its clients, along with several apoptotic markers and cell cycle-related proteins, as well as by IR-drug-induced cell cycle arrest, DNA damage, and repair. A short 30-minute exposure to either Hsp90 inhibitor did not affect the radiosensitivity of both tumor cell lines. Increasing the duration of post-IR-drug treatment progressively enhanced the sensitivity of SNB19 cells to IR. In contrast, the response of A549 cells to drug-IR combination was largely determined by the cytotoxic effects of both drugs without radiosensitization. Combined drug-IR treatment induced more severe DNA damage in both tumor cell lines than each treatment alone and also protracted the kinetics of DNA damage repair in SNB19 cells. In addition to large cell cycle disturbances, drug-IR treatment also caused depletion of the antiapoptotic proteins Akt and Raf-1 in both cell lines, along with a decrease of survivin in A549 cells in case of NVP-AUY922. The data show that simultaneous Hsp90 inhibition and irradiation may induce cell type-specific radiosensitization as well as cytotoxicity against tumor cells.

The FA pathway counteracts oxidative stress through selective protection of antioxidant defense gene promoters

AbstractOxidative stress has been implicated in the pathogenesis of many human diseases including Fanconi anemia (FA), a genetic disorder associated with BM failure and cancer. Here we show that major antioxidant defense genes are down-regulated in FA patients, and that gene down-regulation is selectively associated with increased oxidative DNA damage in the promoters of the antioxidant defense genes. Assessment of promoter activity and DNA damage repair kinetics shows that increased initial damage, rather than a reduced repair rate, contributes to the augmented oxidative DNA damage. Mechanistically, FA proteins act in concert with the chromatin-remodeling factor BRG1 to protect the promoters of antioxidant defense genes from oxidative damage. Specifically, BRG1 binds to the promoters of the antioxidant defense genes at steady state. On challenge with oxidative stress, FA proteins are recruited to promoter DNA, which correlates with significant increase in the binding of BRG1 within promoter regions. In addition, oxidative stress-induced FANCD2 ubiquitination is required for the formation of a FA-BRG1–promoter complex. Taken together, these data identify a role for the FA pathway in cellular antioxidant defense.

Chromium-Induced Genotoxicity and Interference in Human Lymphoblastoid Cell (TK6) Repair Processes

Two model chromium (Cr) compounds, one hexavalent (sodium chromate) and one trivalent (chromium chloride), were investigated in a human lymphoblastoid cell line (TK6) to increase our knowledge regarding Cr-induced genotoxicity mechanisms. Both selected compounds were genotoxic using the comet assay, although the percentage of DNA in tail obtained after treatment with CrVI was significantly higher than that obtained with CrIII, at the higher concentrations tested. To determine the nature of the induced damage, enzymes recognizing oxidized bases were used. Treatments with formamidopyrimidine (FPG) and endonuclease III (EndoIII) displayed a greater degree of DNA damage, indicating that the induction of oxidized bases accounts for an important proportion of the damage induced by Cr compounds. In addition, the kinetic repair studies showed that generated DNA damage is removed in approximately 8 h, with the damage induced by CrIII being removed/repaired more rapidly than damage produced by CrVI. To detect Cr interferences with the repair process, a post-treatment was applied after exposure to 2 Gy gamma radiation. Post-treatment significantly delayed the repair kinetics of DNA damage induced by radiation. This interference effect induced by CrVI was more pronounced. In conclusion, evidence indicates that a high proportion of the Cr-induced DNA damage is correlated with oxidative damage, and that both Cr compounds interfere with repair mechanisms involved in repair of DNA damage induced by gamma radiation.

The role of FOXO3 in DNA damage response in thyrocytes

Members of the forkhead box-O (FOXO) transcription factors family play an important role in stress defence. FOXO3 deregulation has recently been identified as a hallmark of thyroid carcinogenesis. In this study, we explore the role of FOXO3 in defence of oxidative stress in normal thyrocytes. Stable rat thyroid cell lines were generated expressing either the human wild-type FOXO3, a constitutively activating FOXO3 mutant, or the empty control vector. Cell clones were characterised for proliferation, function and morphology. Hydrogen peroxide and UV irradiation were used to induce oxidative stress. Changes in FOXO3 activity, induction of cell cycle arrest or apoptosis and kinetics of DNA damage repair were analysed. Upregulation of FOXO3 in thyrocytes resulted in decreased proliferation and changes in morphology, but did not affect differentiation. Hydrogen peroxide stimulated the expression of the FOXO3 target genes growth arrest and DNA damage-inducible protein 45 α (Gadd45α) and Bcl-2 interacting mediator of cell death (BIM) and induced programmed cell death in cells with overexpression of the human wild-type FOXO3. In contrast, UV irradiation resulted in a distinct cellular response with activation of FOXO3-c-Jun-N-terminal kinase-Gadd45α signalling and induction of cell cycle arrest at the G(2)-M-checkpoint. This was accompanied by FOXO3-induced DNA damage repair as evidenced by lower DNA breaks over time in a comet assay in FOXO3 cell clones compared with control cells. In conclusion, FOXO3 is a pivotal relay in the coordination of the cellular response to genotoxic stress in the thyroid. Depending on the stimulus, FOXO3 induces either cell cycle arrest or apoptosis. Conversely, FOXO3 inactivation in thyroid cancers is consistent with genomic instability and loss of cell cycle control.

Multiscale iterative voting for differential analysis of stress response for 2D and 3D cell culture models

Three-dimensional (2D) cell culture models have emerged as the basis for improved cell systems biology. However, there is a gap in robust computational techniques for segmentation of these model systems that are imaged through confocal or deconvolution microscopy. The main issues are the volume of data, overlapping subcellular compartments and variation in scale or size of subcompartments of interest, which lead to ambiguities for quantitative analysis on a cell-by-cell basis. We address these ambiguities through a series of geometric operations that constrain the problem through iterative voting and decomposition strategies. The main contributions of this paper are to (i) extend the previously developed 2D radial voting to an efficient 3D implementation, (ii) demonstrate application of iterative radial voting at multiple subcellular and molecular scales, and (iii) investigate application of the proposed technology to two endpoints between 2D and 3D cell culture models. These endpoints correspond to kinetics of DNA damage repair as measured by phosphorylation of γH2AX, and the loss of the membrane-bound E-cadherin protein as a result of ionizing radiation. Preliminary results indicate little difference in the kinetics of the DNA damage protein between 2D and 3D cell culture models; however, differences between membrane-bound E-cadherin are more pronounced.

Cell cycle checkpoint defects contribute to genomic instability in PTEN deficient cells independent of DNA DSB repair

Chromosomes in PTEN deficient cells display both numerical as well as structural alterations including regional amplification. We found that PTEN deficient cells displayed a normal DNA damage response (DDR) as evidenced by the ionizing radiation (IR)-induced phosphorylation of Ataxia Telangiectasia Mutated (ATM) as well as its effectors. PTEN deficient cells also had no defect in Rad51 expression or DNA damage repair kinetics post irradiation. In contrast, caffeine treatment specifically increased IR-induced chromosome aberrations and mitotic index only in cells with PTEN, and not in cells deficient for PTEN, suggesting that their checkpoints were defective. Furthermore, PTEN-deficient cells were unable to maintain active spindle checkpoint after taxol treatment. Genomic instability in PTEN deficient cells could not be attributed to lack of PTEN at centromeres, since no interaction was detected between centromeric DNA and PTEN in wild type cells. These results indicate that PTEN deficiency alters multiple cell cycle checkpoints possibly leaving less time for DNA damage repair and/or chromosome segregation as evidenced by the increased structural as well as numerical alterations seen in PTEN deficient cells.

Attenuated DNA Damage Repair by Trichostatin A through BRCA1 Suppression

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

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.

Radioadaptive response: Efficient repair of radiation-induced DNA damage in adapted cells

To verify the hypothesis that the induction of a novel, efficient repair mechanism for chromosomal DNA breaks may be involved in the radioadaptive response, the repair kinetics of DNA damage has been studied in cultured Chinese hamster V79 cells with single-cell gel electrophoresis. The cells were adapted by priming exposure with 5 cGy of γ-rays and 4-h incubation at 37°C. There were no indication of any difference in the initial yields of DNA double-strand breaks induced by challenging doses from non-adapted cells and from adapted cells. The rejoining of DNA double-strand breaks was monitored over 120 min after the adapted cells were challenged with 5 or 1.5 Gy, doses at the same level to those used in the cytogenetical adaptive response. The rate of DNA damage repair in adapted cells was higher than that in non-adapted cells, and the residual damage was less in adapted cells than in non-adapted cells. These results indicate that the radioadaptive response may result from the induction of a novel, efficient DNA repair mechanism which leads to less residual damage, but not from the induction of protective functions that reduce the initial DNA damage.

Induction and repair of DNA damage in UV-irradiated human lymphocytes. Spectral differences and repair kinetics

The alkaline elution assay has been employed to study the induction and repair kinetics of DNA damage in human lymphocytes after irradiation with biologically relevant doses of UVB (297 and 302 nm) or UVA (365 nm) radiation. At 365 nm, when the predominant lesions are single-strand breaks, the rate of lesion induction was 1.5 × 10 −3 per 10 8 Da per kJ m −2. The number of breaks decayed with a half-life of about 50 min after a dose of 20 kJ m −2. In the UVB region, cyclobutyl pyrimidine dimers and 6-4 photoproducts are formed, both of which are repairable via the nucleotide excision repair pathway. By using repair inhibitors, the rate of induction of such lesions at 297 and 302 nm was found to be 0.07 per 10 8 Da per J m −2. Lesions were removed with a half-life of about 100 min. Mathematical modelling of the excision repair process revealed a time-dependent polymerization-ligation rate: after an initial lag phase the polymerization-ligation rate increased, reaching 50% of its maximum rate at 80–100 min after the start of repair incubation. This course of development might be due to a damage-associated regulation of DNA precursors synthesis.

Photodynamic effects of haematoporphyrin derivative on DNA repair in murine L929 fibroblasts.

Illumination with red light of murine L929 fibroblasts that had been sensitized with haematoporphyrin derivative caused DNA single-strand breaks after a lag time of about 20 min, as revealed by alkaline elution. The cells appeared not to be capable of recovering from this damage. The photodynamic effect of haematoporphyrin derivative on DNA repair was assessed by monitoring the repair kinetics of DNA damage induced by either X-rays, u.v. light (254 nm) or methyl methanesulphonate treatment subsequent to a non-DNA-damaging photodynamic treatment with haematoporphyrin derivative. On 'post-incubation', the normally rapid repair of X-ray-induced DNA strand breaks did not occur, whereas with u.v. light and methyl methanesulphonate treatment after photodynamic treatment prolonged post-incubation resulted in an increase in the number of strand breaks rather than the normally observed decrease. This clearly shows that, after a photodynamic treatment with haematoporphyrin derivative that itself did not cause strand breaks, excision repair in L929 cells is severely inhibited at a stage beyond the incision step.