γH2AX Assay Research Articles

Stimulation of lactate receptor (HCAR1) affects cellular DNA repair capacity

Numerous G-protein coupled receptors have been reported to enhance cancer cell survival and resistance to clinically used chemotherapeutics. Recently, hydroxycarboxylic acid receptor 1 (HCAR1) was shown to drive lactate-dependent enhancement of cell survival and metastasis in pancreatic and breast cancers. Furthermore, our previous study confirmed the involvement of HCAR1 in lactate-related enhancement of DNA repair in cervical cancer cells. In the present study, we examined the possible mechanisms of HCAR1-mediated enhancement of DNA repair capacity. We observed that the HCAR1 agonist dihydroxybenzoic acid (DHBA) up-regulated BRCA1 (breast cancer type 1 susceptibility protein) and NBS1 (Nijmegen breakage syndrome 1) expression in HeLa cells. Moreover, HCAR1 silencing decreased mRNA and protein levels of BRCA1 by 30% and 20%, respectively. Immunocytochemical analyses of BRCA1, nibrin and DNA-PKcs indicated an increased accumulation of these proteins in cell nuclei after DHBA stimulation. Subsequently, these changes in the DNA repair protein levels translated into an enhanced DNA repair rate after doxorubicin treatment, as shown by γ-H2AX and comet assay experiments. In contrast, the down-regulation of HCAR1 decreased the efficiency of DNA repair. Finally, we observed the abrogation of DHBA-driven BRCA1 protein up-regulation and enhanced DNA repair following the preincubation of cells with the PKC inhibitor Gö6983. Taken together, our data indicate that lactate receptor/HCAR1 expression in cervical carcinoma cells may contribute to the modulation of cellular DNA repair mechanisms.

Effects of antioxidants on DNA double-strand breaks in human gingival fibroblasts exposed to dental resin co-monomer epoxy metabolites

ObjectiveEluted dental resin co-monomers can be metabolized to intermediate methacrylic acid (MA) and, further, to epoxy metabolites. Antioxidants have been studied previously, with the intention of decreasing the DNA double-strand breaks (DNA-DSBs) in human gingival fibroblasts (HGFs). In this study, the effects of the antioxidants, ascorbic acid (Asc) and N-acetylcysteine (NAC), were investigated on co-monomer metabolite-induced DNA-DSBs. MethodsHGFs were incubated with MA, 2,3-epoxy-2-methyl-propionicacid-methylester (EMPME) and 2,3-epoxy-2-methylpropionic acid (EMPA), respectively, in the presence or absence of antioxidants (Asc or NAC). EC50 Values were obtained from an XTT-based viability assay. DNA-DSBs were determined using a γ-H2AX assay. ResultsThe cytotoxicity of the compounds could be ranked in the following order (mean±SEM; n=4): EMPA>EMPME>MA. The average number of DSBs-foci/cell induced by each substance at EC50-concentration could be ranked in the following order (mean±SD; n=4): EMPA>EMPME>MA. EMPA (1.72mM) and EMPME (2.58mM) induced the highest number of DSBs-foci, that is 21-fold and 13-fold, respectively, compared to control (0.48±0.08 foci/cell). The addition of Asc (50; 100; 200μM) or NAC (50; 100; 200; 500μM) to MA (15.64; 5.21mM), EMPME (2.58mM), and EMPA (1.72; 0.57mM) significantly reduced the number of foci/cell in HGFs. The highest reduction could be found in HGFs with 1.72mM EMPA, the addition of NAC (50; 100; 200; 500μM) induced a 15-fold, 17-fold, 14-fold and 14-fold lower number of DSBs-foci/cell, respectively. SignificanceDental co-monomer epoxy metabolites, EMPME and EMPA, can induce DNA-DSBs. The addition of antioxidants (Asc or NAC) leads to reduction of DNA-DSBs, and NAC leads to more prominent reduction of DNA-DSBs compared to Asc.

Cellular and cell-free studies of catalytic DNA cleavage by ruthenium polypyridyl complexes containing redox-active intercalating ligands.

The ruthenium(ii) polypyridyl complexes (RPCs), [(phen)2Ru(tatpp)]2+ (32+ ) and [(phen)2Ru(tatpp)Ru(phen)2]4+ (44+ ) are shown to cleave DNA in cell-free studies in the presence of a mild reducing agent, i.e. glutathione (GSH), in a manner that is enhanced upon lowering the [O2]. Reactive oxygen species (ROS) are involved in the cleavage process as hydroxy radical scavengers attenuate the cleavage activity. Cleavage experiments in the presence of superoxide dismutase (SOD) and catalase reveal a central role for H2O2 as the immediate precursor for hydroxy radicals. A mechanism is proposed which explains the inverse [O2] dependence and ROS data and involves redox cycling between three DNA-bound redox isomers of 32+ or 44+ . Cultured non-small cell lung cancer cells (H358) are sensitive to 32+ and 44+ with IC50 values of 13 and 15 μM, respectively, and xenograft H358 tumors in nude mice show substantial (∼80%) regression relative to untreated tumors when the mice are treated with enantiopure versions of 32+ and 44+ (Yadav et al. Mol Cancer Res, 2013, 12, 643). Fluorescence microscopy of H358 cells treated with 15 μM 44+ reveals enhanced intracellular ROS production in as little as 2 h post treatment. Detection of phosphorylated ATM via immunofluorescence within 2 h of treatment with 44+ reveals initiation of the DNA damage repair machinery due to the ROS insult and DNA double strand breaks (DSBs) in the nuclei of H358 cells and is confirmed using the γH2AX assay. The cell data for 32+ is less clear but DNA damage occurs. Notably, cells treated with [Ru(diphenylphen)3]2+ (IC50 1.7 μM) show no extra ROS production and no DNA damage by either the pATM or γH2AX even after 22 h. The enhanced DNA cleavage under low [O2] (4 μM) seen in cell-free cleavage assays of 32+ and 44+ is only partially reflected in the cytotoxicity of 32+ and 44+ in H358, HCC2998, HOP-62 and Hs766t under hypoxia (1.1% O2) relative to normoxia (18% O2). Cells treated with RPC 32+ show up to a two-fold enhancement in the IC50 under hypoxia whereas cells treated with RPC 44+ gave the same IC50 whether under hypoxia or normoxia.

Effects of prolonged exposure to low dose metformin in thyroid cancer cell lines.

Background: Thyroid cancer is generally associated with an excellent prognosis, but there is significant long-term morbidity with standard treatment. Some sub-types however have a poor prognosis. Metformin, an oral anti-diabetic drug is shown to have anti-cancer effects in several types of cancer (breast, lung and ovarian cancer). The proposed mechanisms include activation of the Adenosine Mono-phosphate-activated Protein Kinase (AMPK) pathway and inhibition of the mTOR pathway (which promotes growth and proliferation). By inhibiting hepatic gluconeogenesis and increasing glucose uptake by muscles, metformin decreases blood glucose and circulating Insulin levels.Aims: Explore the effect of metformin on the growth and proliferation of thyroid cancer cell lines.Methods: The effects of metformin on thyroid cancer cell lines (FTC-133, K1E7, RO82-W-1, 8305C and TT) and normal thyroid follicular cells (Nthy-ori 3-1) were investigated using the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) assay for cell proliferation; clonogenic assays; FACS analysis for apoptosis and cell cycle, H2A.X phosphorylation (γH2AX) assay for DNA repair and scratch assay for cell migration.Results: Metformin inhibited cell proliferation and colony formation at 0.03 mM and above and inhibited cell migration at 0.3 mM. At concentrations of 0.1 mM and above metformin increased the percentage of apoptotic cells and induced cell cycle arrest in G0/G1 phase at minimum concentration of 0.3 mM. Unlike previous reports, no effect on DNA repair response was demonstrated.Conclusion: Metformin suppressed growth of all thyroid cancer cell lines, at concentrations considered to be within in the therapeutic range for diabetic patients on metformin (<0.3 mM).

Detection of DNA damage by space radiation in human fibroblasts flown on the International Space Station

Although charged particles in space have been detected with radiation detectors on board spacecraft since the discovery of the Van Allen Belts, reports on the effects of direct exposure to space radiation in biological systems have been limited. Measurement of biological effects of space radiation is challenging due to the low dose and low dose rate nature of the radiation environment, and due to the difficulty in distinguishing the radiation effects from microgravity and other space environmental factors. In astronauts, only a few changes, such as increased chromosome aberrations in their lymphocytes and early onset of cataracts, are attributed primarily to their exposure to space radiation. In this study, cultured human fibroblasts were flown on the International Space Station (ISS). Cells were kept at 37°C in space for 14 days before being fixed for analysis of DNA damage with the γ-H2AX assay. The 3-dimensional γ-H2AX foci were captured with a laser confocal microscope. Quantitative analysis revealed several foci that were larger and displayed a track pattern only in the Day 14 flight samples. To confirm that the foci data from the flight study was actually induced from space radiation exposure, cultured human fibroblasts were exposed to low dose rate γ rays at 37°C. Cells exposed to chronic γ rays showed similar foci size distribution in comparison to the non-exposed controls. The cells were also exposed to low- and high-LET protons, and high-LET Fe ions on the ground. Our results suggest that in G1 human fibroblasts under the normal culture condition, only a small fraction of large size foci can be attributed to high-LET radiation in space.

The comparative in vitro assessment of e-cigarette and cigarette smoke aerosols using the γH2AX assay and applied dose measurements

DNA damage can be caused by a variety of external and internal factors and together with cellular responses, can establish genomic instability through multiple pathways. DNA damage therefore, is considered to play an important role in the aetiology and early stages of carcinogenesis. The DNA-damage inducing potential of tobacco smoke aerosols in vitro has been extensively investigated; however, the ability of e-cigarette aerosols to induce DNA damage has not been extensively investigated.E-cigarette use has grown globally in recent years and the health implications of long term e-cigarette use are still unclear. Therefore, this study has assessed the induction of double-strand DNA damage in vitro using human lung epithelial cells to e-cigarette aerosols from two different product variants (a “cigalike” and a closed “modular” system) and cigarette smoke. A Vitrocell® VC 10 aerosol exposure system was used to generate and dilute cigarette smoke and e-cigarette aerosols, which were delivered to human bronchial epithelial cells (BEAS-2Bs) housed at the air-liquid-interface (ALI) for up to 120min exposure (diluting airflow, 0.25–1L/min). Following exposure, cells were immediately fixed, incubated with primary (0.1% γH2AX antibody in PBS) and secondary antibodies (DyLight™ 549 conjugated goat anti-mouse IgG) containing Hoechst dye DNA staining solution (0.2% secondary antibody and 0.01% Hoechst in PBS), and finally screened using the Cellomics Arrayscan VTI platform.The results from this study demonstrate a clear DNA damage-induced dose response with increasing smoke concentrations up to cytotoxic levels. In contrast, e-cigarette aerosols from two product variants did not induce DNA damage at equivalent to or greater than doses of cigarette smoke aerosol. In this study dosimetry approaches were used to contextualize exposure, define exposure conditions and facilitate comparisons between cigarette smoke and e-cigarette aerosols. Quartz crystal microbalance (QCM) technology and quantified nicotine delivery were both assessed at the exposure interface. Nicotine was eluted from the QCM surface to give a quantifiable measure of exposure to support deposited mass. Dose measured as deposited mass (μg/cm2) and nicotine (ng/mL) demonstrated that in vitro e-cigarette exposures were conducted at doses up to 12–28 fold to that of cigarette smoke and demonstrated a consistent negative finding.

Homologous recombination preferentially repairs heat-induced DNA double-strand breaks in mammalian cells

Purpose: Heat shock induces DNA double-strand breaks (DSBs), but the precise mechanism of repairing heat-induced damage is unclear. Here, we investigated the DNA repair pathways involved in cell death induced by heat shock.Materials and methods: B02, a specific inhibitor of human RAD51 (homologous recombination; HR), and NU7026, a specific inhibitor of DNA-PK (non-homologous end-joining; NHEJ), were used for survival assays of human cancer cell lines with different p53-gene status. Mouse embryonic fibroblasts (MEFs) lacking Lig4 (NHEJ) and/or Rad54 (HR) were used for survival assays and a phosphorylated histone H2AX at Ser139 (γH2AX) assay. MEFs lacking Rad51d (HR) were used for survival assays. SPD8 cells were used to measure HR frequency after heat shock.Results: Human cancer cells were more sensitive to heat shock in the presence of B02 despite their p53-gene status, and the effect of B02 on heat sensitivity was specific to the G2 phase. Rad54-deficient MEFs were sensitive to heat shock and showed prolonged γH2AX signals following heat shock. Rad51d-deficient MEFs were also sensitive to heat shock. Moreover, heat shock-stimulated cells had increased HR.Conclusions: The HR pathway plays an important role in the survival of mammalian cells against death induced by heat shock via the repair of heat-induced DNA DSBs.

Cells Deficient in the Fanconi Anemia Protein FANCD2 are Hypersensitive to the Cytotoxicity and DNA Damage Induced by Coffee and Caffeic Acid.

Epidemiological studies have found a positive association between coffee consumption and a lower risk of cardiovascular disorders, some cancers, diabetes, Parkinson and Alzheimer disease. Coffee consumption, however, has also been linked to an increased risk of developing some types of cancer, including bladder cancer in adults and leukemia in children of mothers who drink coffee during pregnancy. Since cancer is driven by the accumulation of DNA alterations, the ability of the coffee constituent caffeic acid to induce DNA damage in cells may play a role in the carcinogenic potential of this beverage. This carcinogenic potential may be exacerbated in cells with DNA repair defects. People with the genetic disease Fanconi Anemia have DNA repair deficiencies and are predisposed to several cancers, particularly acute myeloid leukemia. Defects in the DNA repair protein Fanconi Anemia D2 (FANCD2) also play an important role in the development of a variety of cancers (e.g., bladder cancer) in people without this genetic disease. This communication shows that cells deficient in FANCD2 are hypersensitive to the cytotoxicity (clonogenic assay) and DNA damage (γ-H2AX and 53BP1 focus assay) induced by caffeic acid and by a commercial lyophilized coffee extract. These data suggest that people with Fanconi Anemia, or healthy people who develop sporadic mutations in FANCD2, may be hypersensitive to the carcinogenic activity of coffee.

The Microbiota of Breast Tissue and Its Association with Breast Cancer

ABSTRACTIn the United States, 1 in 8 women will be diagnosed with breast cancer in her lifetime. Along with genetics, the environment contributes to disease development, but what these exact environmental factors are remains unknown. We have previously shown that breast tissue is not sterile but contains a diverse population of bacteria. We thus believe that the host's local microbiome could be modulating the risk of breast cancer development. Using 16S rRNA amplicon sequencing, we show that bacterial profiles differ between normal adjacent tissue from women with breast cancer and tissue from healthy controls. Women with breast cancer had higher relative abundances of Bacillus, Enterobacteriaceae and Staphylococcus. Escherichia coli (a member of the Enterobacteriaceae family) and Staphylococcus epidermidis, isolated from breast cancer patients, were shown to induce DNA double-stranded breaks in HeLa cells using the histone-2AX (H2AX) phosphorylation (γ-H2AX) assay. We also found that microbial profiles are similar between normal adjacent tissue and tissue sampled directly from the tumor. This study raises important questions as to what role the breast microbiome plays in disease development or progression and how we can manipulate this for possible therapeutics or prevention. IMPORTANCE This study shows that different bacterial profiles in breast tissue exist between healthy women and those with breast cancer. Higher relative abundances of bacteria that had the ability to cause DNA damage in vitro were detected in breast cancer patients, as was a decrease in some lactic acid bacteria, known for their beneficial health effects, including anticarcinogenic properties. This study raises important questions as to the role of the mammary microbiome in modulating the risk of breast cancer development.

Frequency of gamma H2AX foci in healthy volunteers and health workers occupationally exposed to X-irradiation and its relevance in biological dosimetry.

Gamma-H2AX (γ-H2AX) assay is a marker to measure double-strand breaks in the deoxyribonucleic acid. Variables such as age, oxidative stress, temperature, genetic factors and inter-individual variation have been reported to influence the baseline γ-H2AX focus levels. Therefore, knowledge on baseline frequency of γ-H2AX foci in a targeted population would facilitate reliable radiation triage and dose estimation. The objective of the present study was to establish the baseline data using blood samples from healthy volunteers (n=130) differing in age, occupation and lifestyle as well as from occupationally exposed health workers (n=20). The γ-H2AX focus assay was performed using epifluorescence microscopy. In vitro dose-response curve for γ-H2AX foci was constructed in blood samples (n=3) exposed to X-rays (30min post-exposure). The mean γ-H2AX focus frequency obtained in healthy volunteers was 0.042±0.001 and showed an age-related increase (p<0.001). Significantly higher (p<0.005) focus frequencies were observed in health workers (0.066±0.005) than in healthy volunteers. A sub-group analysis did not show a significant (p>0.1) difference in γ-H2AX focus frequency among sexes. Blood exposed in vitro to X-rays showed dose-dependent increase in γ-H2AX foci frequency (Y=0.1902±0.1363+2.9020±0.3240*D). Baseline frequency of γ-H2AX foci obtained from different age groups showed a significant (p<0.01) influence on the dose-response coefficients. The overall results demonstrated that the γ-H2AX assay can be used as a reliable biomarker for radiation triage and estimating the radiation absorbed dose by considering variables such as age, occupation and lifestyle factors.

High throughput toxicity screening and intracellular detection of nanomaterials

With the growing numbers of nanomaterials (NMs), there is a great demand for rapid and reliable ways of testing NM safety—preferably using in vitro approaches, to avoid the ethical dilemmas associated with animal research. Data are needed for developing intelligent testing strategies for risk assessment of NMs, based on grouping and read‐across approaches. The adoption of high throughput screening (HTS) and high content analysis (HCA) for NM toxicity testing allows the testing of numerous materials at different concentrations and on different types of cells, reduces the effect of inter‐experimental variation, and makes substantial savings in time and cost. HTS/HCA approaches facilitate the classification of key biological indicators of NM‐cell interactions. Validation of in vitro HTS tests is required, taking account of relevance to in vivo results. HTS/HCA approaches are needed to assess dose‐ and time‐dependent toxicity, allowing prediction of in vivo adverse effects. Several HTS/HCA methods are being validated and applied for NM testing in the FP7 project NANoREG, including Label‐free cellular screening of NM uptake, HCA, High throughput flow cytometry, Impedance‐based monitoring, Multiplex analysis of secreted products, and genotoxicity methods—namely High throughput comet assay, High throughput in vitro micronucleus assay, and γH2AX assay. There are several technical challenges with HTS/HCA for NM testing, as toxicity screening needs to be coupled with characterization of NMs in exposure medium prior to the test; possible interference of NMs with HTS/HCA techniques is another concern. Advantages and challenges of HTS/HCA approaches in NM safety are discussed. WIREs Nanomed Nanobiotechnol 2017, 9:e1413. doi: 10.1002/wnan.1413For further resources related to this article, please visit the WIREs website.

TU‐H‐CAMPUS‐TeP3‐01: Gold Nanoparticle‐Enhanced Radiation Therapy in In Vitro A549 Lung Carcinoma: Studies in Both Traditional Monolayer and Three Dimensional Cell Culture Models

Purpose:To measure the increase in in vitro radiosensitivity for A549 lung carcinoma cells due to gold nanoparticle (GNP) radiation dose enhancement in both traditional monolayer and three dimensional (3D) cell culture models.Methods:A γH2AX immunofluorescence assay is performed on monolayer A549 cell culture and quantitatively analyzed to measure the increase in double strand breaks (DSBs) resulting from GNP dose enhancement. A clonogenic survival assay (CSA) is then performed on monolayer A549 cell culture to assess true viability after treatment. And lastly, another γH2AX assay is performed on 3D A549 multicellular nodules overlaid on a bed of growth factor reduced matrigel to measure dose response in a model that better recapitulates treatment response to actual tumors in vivo.Results:The first γH2AX assay performed on the monolayer cell culture shows a significant increase in DSBs due to GNP dose enhancement. The maximum average observed increase in normalized fluorescent intensity for monolayer cell culture is 171% for the 6Gy‐treatment groups incubated in 0.556 mg Au/ml solution. The CSA performed on monolayer cell culture also shows considerable GNP dose enhancement. The maximum decrease in the normalized surviving fraction is 12% for the 4Gy‐treatment group incubated in 0.556 mg Au/ml. And lastly, the GNP dose enhancement is confirmed to be mitigated in three dimensional cell culture models as compared to the traditional monolayer model. The maximum average observed dose enhancement for 3D cell culture is 19% for the 6Gy‐treatment groups and incubated in 0.556 mg Au/ml.Conclusion:A marked increase in radiosensitivity is observed for A549 lung carcinoma cells when treated with GNPs plus radiation as opposed to radiation alone. Traditional monolayer cell culture also shows a much more pronounced radiation dose enhancement than 3D cell culture.

Differences in quantification of DNA double-strand breaks assessed by 53BP1/γH2AX focus formation assays and the comet assay in mammalian cells treated with irradiation and N-acetyl-L-cysteine.

The biological effect of ionizing radiation (IR) on genomic DNA is thought to be either direct or indirect; the latter is mediated by IR induction of free radicals and reactive oxygen species (ROS). This study was designed to evaluate the effect of N-acetyl-L-cysteine (NAC), a well-known ROS-scavenging antioxidant, on IR induction of genotoxicity, cytotoxicity and ROS production in mammalian cells, and aimed to clarify the conflicting data in previous publications. Although we clearly demonstrate the beneficial effect of NAC on IR-induced genotoxicity and cytotoxicity (determined using the micronucleus assay and cell viability/clonogenic assays), the data on NAC's effect on DNA double-strand break (DSB) formation were inconsistent in different assays. Specifically, mitigation of IR-induced DSBs by NAC was readily detected by the neutral comet assay, but not by the γH2AX or 53BP1 focus assays. NAC is a glutathione precursor and exerts its effect after conversion to glutathione, and presumably it has its own biological activity. Assuming that the focus assay reflects the biological responses to DSBs (detection and repair), while the comet assay reflects the physical status of genomic DNA, our results indicate that the comet assay could readily detect the antioxidant effect of NAC on DSB formation. However, NAC's biological effect might affect the detection of DSB repair by the focus assays. Our data illustrate that multiple parameters should be carefully used to analyze DNA damage when studying potential candidates for radioprotective compounds.

Phosphorylation-Dependent Regulation of the DNA Damage Response of Adaptor Protein KIBRA in Cancer Cells.

Multifunctional adaptor proteins encompassing various protein-protein interaction domains play a central role in the DNA damage response pathway. In this report, we show that KIBRA is a physiologically interacting reversible substrate of ataxia telangiectasia mutated (ATM) kinase. We identified the site of phosphorylation in KIBRA as threonine 1006, which is embedded within the serine/threonine (S/T) Q consensus motif, by site-directed mutagenesis, and we further confirmed the same with a phospho-(S/T) Q motif-specific antibody. Results from DNA repair functional assays such as the γ-H2AX assay, pulsed-field gel electrophoresis (PFGE), Comet assay, terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) assay, and clonogenic cell survival assay using stable overexpression clones of wild-type (wt.) KIBRA and active (T1006E) and inactive (T1006A) KIBRA phosphorylation mutants showed that T1006 phosphorylation on KIBRA is essential for optimal DNA double-strand break repair in cancer cells. Further, results from stable retroviral short hairpin RNA-mediated knockdown (KD) clones of KIBRA and KIBRA knockout (KO) model cells generated by a clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 system showed that depleting KIBRA levels compromised the DNA repair functions in cancer cells upon inducing DNA damage. All these phenotypic events were reversed upon reconstitution of KIBRA into cells lacking KIBRA knock-in (KI) model cells. All these results point to the fact that phosphorylated KIBRA might be functioning as a scaffolding protein/adaptor protein facilitating the platform for further recruitment of other DNA damage response factors. In summary, these data demonstrate the imperative functional role of KIBRAper se(KIBRA phosphorylation at T1006 site as a molecular switch that regulates the DNA damage response, possibly via the nonhomologous end joining [NHEJ] pathway), suggesting that KIBRA could be a potential therapeutic target for modulating chemoresistance in cancer cells.

Polymorphisms in double strand break repair related genes influence radiosensitivity phenotype in lymphocytes from healthy individuals.

A range of individual radiosensitivity observed in humans can influence individual's susceptibility toward cancer risk and radiotherapy outcome. Therefore, it is important to measure the variation in radiosensitivity and to identify the genetic factors influencing it. By adopting a pathway specific genotype-phenotype design, we established the variability in cellular radiosensitivity by performing γ-H2AX foci assay in healthy individuals. Further, we genotyped ten selected SNPs in candidate genes XRCC3 (rs861539), XRCC4 (rs1805377), XRCC5 (rs3835), XRCC6 (rs2267437), ATM (rs3218698, rs1800057), LIG4 (rs1805388), NBN (rs1805794), RAD51 (rs1801320) and PRKDC (rs7003908), and analysed their influence on observed variation in radiosensitivity. The rs2267437 polymorphisms in XRCC6 was associated (P=0.0326) with increased DSB induction while rs1805388 in LIG4 (P=0.0240) was associated with increased radioresistance. Further, multiple risk alleles decreased the DSB repair capacity in an additive manner. Polymorphisms in candidate DSB repair genes can act individually or in combination to the efficacy of DSB repair process, resulting in variation of cellular radiosensitivity. Current study suggests that γ-H2AX assay may fulfil the role of a rapid and sensitive biomarker that can be used for epidemiological studies to measure variations in radiosensitivity. DSB repair gene polymorphisms can impact the formation and repair of DSBs. γ-H2AX foci analysis as well as DSBs repair gene polymorphisms can be used to assess cellular radiosensitivity, which will be useful in population risk assessment, disease prediction, individualization of radiotherapy and also in setting the radiation protection standards.

Sinuleptolide inhibits proliferation of oral cancer Ca9-22 cells involving apoptosis, oxidative stress, and DNA damage

ObjectiveSinuleptolide, a soft corals-derived bioactive norditerpenoid, is a marine natural product with a potent anti-inflammatory effect. We evaluate the potential anti-oral cancer effects of sinuleptolide and investigate the possible mechanisms involved. DesignsCell viability, cell cycle, apoptosis, reactive oxygen species (ROS), mitochondrial membrane potential (MMP), and DNA damage analyses were performed. ResultsIn a cell viability assay, we found that sinuleptolide is dose-responsively antiproliferative against oral gingival cancer Ca9-22 cells but less harmful to normal human gingival fibroblast (HGF-1) cells (P<0.001). In cell cycle analysis, sinuleptolide induced subG1 accumulation at a higher dose and led to G2/M arrest of Ca9-22 cells (P<0.005). Apoptosis was significantly increased in sinuleptolide-treated Ca9-22 cells based on annexin V and poly(ADP-ribose) polymerase (PARP) expressions (P<0.05–0.0001). Based on flow cytometer analysis, sinuleptolide also induced the generation of ROS and decreased MMP in a dose-responsive manner (P<0.05–0.0001). DNA damage increased dose-responsively after sinuleptolide treatments (P < 0.001) based on comet and γH2AX assays. ConclusionSinuleptolide can induce an antiproliferation of oral cancer Ca9-22 cells involving apoptosis, oxidative stress and DNA damage, suggesting that sinuleptolide represents a potential chemotherapeutic drug for oral cancer treatment.

Exponential Increase in Relative Biological Effectiveness Along Distal Edge of a Proton Bragg Peak as Measured by Deoxyribonucleic Acid Double-Strand Breaks

To quantify the relative biological effectiveness (RBE) of the distal edge of the proton Bragg peak, using an in vitro assay of DNA double-strand breaks (DSBs). U2OS cells were irradiated within the plateau of a spread-out Bragg peak and at each millimeter position along the distal edge using a custom slide holder, allowing for simultaneous measurement of physical dose. A reference radiation signal was generated using photons. The DNA DSBs at 3 hours (to assess for early damage) and at 24 hours (to assess for residual damage and repair) after irradiation were measured using the γH2AX assay and quantified via flow cytometry. Results were confirmed with clonogenic survival assays. A detailed map of the RBE as a function of depth along the Bragg peak was generated using γH2AX measurements as a biological endpoint. At 3 hours after irradiation, DNA DSBs were higher with protons at every point along the distal edge compared with samples irradiated with photons to similar doses. This effect was even more pronounced after 24 hours, indicating that the impact of DNA repair is less after proton irradiation relative to photons. The RBE demonstrated an exponential increase as a function of depth and was measured to be as high as 4.0 after 3 hours and as high as 6.0 after 24 hours. When the RBE-corrected dose was plotted as a function of depth, the peak effective dose was extended 2-3 mm beyond what would be expected with physical measurement. We generated a highly comprehensive map of the RBE of the distal edge of the Bragg peak, using a direct assay of DNA DSBs in vitro. Our data show that the RBE of the distal edge increases with depth and is significantly higher than previously reported estimates.

In vitro and in vivo genotoxic effects of straight versus tangled multi-walled carbon nanotubes

Some multi-walled carbon nanotubes (MWCNTs) induce mesothelioma in rodents, straight MWCNTs showing a more pronounced effect than tangled MWCNTs. As primary and secondary genotoxicity may play a role in MWCNT carcinogenesis, we used a battery of assays for DNA damage and micronuclei to compare the genotoxicity of straight (MWCNT-S) and tangled MWCNTs (MWCNT-T) in vitro (primary genotoxicity) and in vivo (primary or secondary genotoxicity). C57Bl/6 mice showed a dose-dependent increase in DNA strand breaks, as measured by the comet assay, in lung cells 24 h after a single pharyngeal aspiration of MWCNT-S (1–200 μg/mouse). An increase was also observed for DNA strand breaks in lung and bronchoalveolar lavage (BAL) cells and for micronucleated alveolar type II cells in mice exposed to aerosolized MWCNT-S (8.2–10.8 mg/m3) for 4 d, 4 h/d. No systemic genotoxic effects, assessed by the γ-H2AX assay in blood mononuclear leukocytes or by micronucleated polychromatic erythrocytes (MNPCEs) in bone marrow or blood, were observed for MWCNT-S by either exposure technique. MWCNT-T showed a dose-related decrease in DNA damage in BAL and lung cells of mice after a single pharyngeal aspiration (1–200 μg/mouse) and in MNPCEs after inhalation exposure (17.5 mg/m3). In vitro in human bronchial epithelial BEAS-2B cells, MWCNT-S induced DNA strand breaks at low doses (5 and 10 μg/cm2), while MWCNT-T increased strand breakage only at 200 μg/cm2. Neither of the MWCNTs was able to induce micronuclei in vitro. Our findings suggest that both primary and secondary mechanisms may be involved in the genotoxicity of straight MWCNTs.

In Vitro Methods for Studying the Mechanisms of Resistance to DNA-Damaging Therapeutic Drugs.

Most commonly used anticancer drugs exert their effects mainly by causing DNA damage. The enhancement in DNA damage response (DDR) is considered a key mechanism that enables cancer cells to survive through eliminating the damaged DNA lesions and thereby developing resistance to DNA-damaging agents. This chapter describes the four experimental approaches for studying DDR and genotoxic drug resistance, including the use of γ-H2AX and comet assays to monitor DNA damage and repair capacity as well as the use of clonogenic and β-galactosidase staining assays to assess long-term cell fate after DNA-damaging treatment. Finally, we also present examples of these methods currently used in our laboratory for studying the role of FOXM1 in DNA damage-induced senescence and epirubicin resistance.

Mechanisms of Action and Reduced Cardiotoxicity of Pixantrone; a Topoisomerase II Targeting Agent with Cellular Selectivity for the Topoisomerase IIα Isoform.

Pixantrone is a new noncardiotoxic aza-anthracenedione anticancer drug structurally related to anthracyclines and anthracenediones, such as doxorubicin and mitoxantrone. Pixantrone is approved in the European Union for the treatment of relapsed or refractory aggressive B cell non-Hodgkin lymphoma. This study was undertaken to investigate both the mechanism(s) of its anticancer activity and its relative lack of cardiotoxicity. Pixantrone targeted DNA topoisomerase IIα as evidenced by its ability to inhibit kinetoplast DNA decatenation; to produce linear double-strand DNA in a pBR322 DNA cleavage assay; to produce DNA double-strand breaks in a cellular phospho-histone γH2AX assay; to form covalent topoisomerase II-DNA complexes in a cellular immunodetection of complex of enzyme-to-DNA assay; and to display cross-resistance in etoposide-resistant K562 cells. Pixantrone produced semiquinone free radicals in an enzymatic reducing system, although not in a cellular system, most likely due to low cellular uptake. Pixantrone was 10- to 12-fold less damaging to neonatal rat myocytes than doxorubicin or mitoxantrone, as measured by lactate dehydrogenase release. Three factors potentially contribute to the reduced cardiotoxicity of pixantrone. First, its lack of binding to iron(III) makes it unable to induce iron-based oxidative stress. Second, its low cellular uptake may limit its ability to produce semiquinone free radicals and redox cycle. Finally, because the β isoform of topoisomerase II predominates in postmitotic cardiomyocytes, and pixantrone is demonstrated in this study to be selective for topoisomerase IIα in stabilizing enzyme-DNA covalent complexes, the attenuated cardiotoxicity of this agent may also be due to its selectivity for targeting topoisomerase IIα over topoisomerase IIβ.