Induced DNA DNA Double-strand Breaks Research Articles

Abstract 578: Plk1 decreases DNA double-strand break (DSB) formation during replication-stalling genotoxic stress.

Abstract Polo-like kinase 1 (Plk1) is a key regulator of mitosis and its over-expression/ deregulation is linked to cellular transformation. Recent studies highlight its role in resumption of cell cycle progression after DNA damage. Plk1 has also been shown to enhance DNA replication under stress of stalled replication forks. We reported Plk1 activation-induced G2/M checkpoint bypass in normal human lung cells (HLFs), in the presence of acute genotoxic stress. The present aim was to determine the effect of Plk1 on DNA double strand breaks (DSBs) during genotoxic stress. In the present study, we measured DNA DSB induction in HLFs by Cr(VI), etoposide, and H2O2 after Plk1 activation using the comet assay and γH2AX immunofluorescence staining. We used equitoxic concentrations of the different genotoxins and found that DNA DSBs increased > 2 fold after exposure to 3 μM Cr(VI), 12.5 μM etoposide, and 150 μM H2O2, respectively, and persisted for at least 4h. Transfection of a Plk1 constitutively active (c/a) mutant (T210D) abrogated both Cr(VI)- and etoposide-induced DNA DSBs as early as 30 min for the duration of treatment, in comparison to the vector-transfected cells. In sharp contrast, Plk1 had no effect on H2O2-induced DNA DSBs. Consistent with DNA DSB formation, Cr(VI) exposure was associated with G1 arrest, at least at 4h, as determined by BrdU incorporation. Moreover, Plk1 enhanced S phase progression during Cr(VI) treatment, which was accompanied by increased replication fork velocity and longer inter-origin distances as determined by using a single-molecule approach based on molecular combing. Expression of a Plk1 “kinase-dead” mutant (K82R) also abrogated DNA DSBs during Cr(VI) treatment, suggesting that this effect may be independent of Plk1 kinase activity. In summary, our data underline the ability of Plk1 to override DNA DSB-induced checkpoints, and to decrease DSBs after replication-stalling genotoxin exposure. We postulate these effects to be due to the replication-enhancing ability of Plk1, which may occur at the expense of genomic stability. This work was supported in part by NIH grants CA107972 and ES017334 (S.C.); ES05304 (S.R.P.); and a PhRMA Foundation predoctoral fellowship (G.C.K.). Citation Format: Daniel Gebreselassie, Gina Chun-Kost, Elisabetta Leo, Yves Pommier, Erika Saito, Steven Patierno, Susan M. Ceryak. Plk1 decreases DNA double-strand break (DSB) formation during replication-stalling genotoxic stress. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 578. doi:10.1158/1538-7445.AM2013-578

The effects of DNA double-strand breaks on mouse oocyte meiotic maturation

Both endogenous and exogenous factors can induce DNA double-strand breaks (DSBs) in oocytes, which is a potential risk for human-assisted reproductive technology as well as animal nuclear transfer. Here we used bleomycin (BLM) and laser micro-beam dissection (LMD) to induce DNA DSBs in germinal vesicle (GV) stage oocytes and compared the germinal vesicle breakdown (GVBD) rates and first polar body extrusion (PBE) rates between DNA DSB oocytes and untreated oocytes. Employing live cell imaging and immunofluorescence labeling, we observed the dynamics of DNA fragments during oocyte maturation. We also determined the cyclin B1 expression pattern in oocytes to analyze spindle assembly checkpoint (SAC) activity in DNA DSB oocytes. We used parthenogenetic activation to determine if the DNA DSB oocytes could be activated. As a result, we found that the BLM- or LMD-induced DSB oocytes showed lower GVBD rates and took a longer time to undergo GVBD compared with untreated oocytes. PBE was also delayed in DSB oocytes, but once GVBD had occurred, PBE was not affected, even in oocytes with severe DSBs. Compared with control oocytes, the DSB oocytes showed higher SAC activity, as indicated by less Ccnb1-GFP degradation during metaphase I to anaphase I transition. Parthenogenetic activation could activate the metaphase to interphase transition in the DNA DSB mature oocytes, but many oocytes contained multiple pronuclei or numerous micronuclei. These data suggest that DNA damage inhibits or delays the G2/M transition, but once GVBD occurs, DNA-damaged oocytes can complete chromosome separation and polar body extrusion even under a higher SAC activity, causing the formation of numerous micronuclei in early embryos.

Homologous Recombination DNA Repair Genes Play a Critical Role in Reprogramming to a Pluripotent State

Induced pluripotent stem cells (iPSCs) hold great promise for personalized regenerative medicine. However, recent studies show that iPSC lines carry genetic abnormalities, suggesting that reprogramming may be mutagenic. Here, we show that the ectopic expression of reprogramming factors increases the level of phosphorylated histone H2AX, one of the earliest cellular responses to DNA double-strand breaks (DSBs). Additional mechanistic studies uncover a direct role of the homologous recombination (HR) pathway, a pathway essential for error-free repair of DNA DSBs, in reprogramming. This role is independent of the use of integrative or nonintegrative methods in introducing reprogramming factors, despite the latter being considered a safer approach that circumvents genetic modifications. Finally, deletion of the tumor suppressor p53 rescues the reprogramming phenotype in HR-deficient cells primarily through the restoration of reprogramming-dependent defects in cell proliferation and apoptosis. These mechanistic insights have important implications for the design of safer approaches to creating iPSCs.

Predictive markers for the response to 5-fluorouracil therapy in cancer cells: Constant-field gel electrophoresis as a tool for prediction of response to 5-fluorouracil-based chemotherapy

The prediction of response or severe toxicity and therapy individualisation are extremely important in cancer chemotherapy. There are few tools to predict chemoresponse or toxicity in cancer patients. We investigated the correlation between the induction and repair of DNA double-strand breaks (DSBs) using constant-field gel electrophoresis (CFGE) and evaluating cell cycle progression and the sensitivity of four cancer cell lines to 5-fluorouracil (5FU). Using a sulphorhodamine-B assay, colon carcinoma cells (HCT116) were found to be the most sensitive to 5FU, followed by liver carcinoma cells (HepG2) and breast carcinoma cells (MCF-7). Cervical carcinoma cells (HeLa) were the most resistant. As measured by CFGE, DSB induction, but not residual DSBs, exhibited a significant correlation with the sensitivity of the cell lines to 5FU. Flow cytometric cell cycle analysis revealed that 14% of HCT116 or HepG2 cells and 2% of MCF-7 cells shifted to sub-G1 phase after a 96-h incubation with 5FU. Another 5FU-induced cell cycle change in HCT116, HepG2 and MCF-7 cells was the mild arrest of cells in G1 and/or G2/M phases of the cell cycle. In addition, 5FU treatment resulted in the accumulation of HeLa cells in the S and G2/M phases. Determination of Fas ligand (Fas-L) and caspase 9 as representative markers for the extrinsic and intrinsic pathways of apoptosis, respectively, revealed that 5FU-induced apoptosis in HCT116 and HepG2 results from the expression of Fas-L (extrinsic pathway). Therefore, the induction of DNA DSBs by 5FU, detected using CFGE, and the induction of apoptosis are candidate predictive markers that may distinguish cancer cells which are likely to benefit from 5FU treatment and the measurement of DSBs using CFGE may aid the prediction of clinical outcome.

Adaptation of the neutral bacterial comet assay to assess antimicrobial-mediated DNA double-strand breaks in Escherichia coli

This study aimed to determine the mechanism of action of a natural antibacterial clay mineral mixture, designated as CB, by investigating the induction of DNA double-strand breaks (DSBs) in Escherichia coli. To quantify DNA damage upon exposure to soluble antimicrobial compounds, we modified a bacterial neutral comet assay, which associates the general length of an electrophoresed chromosome, or comet, with the degree of DSB-associated DNA damage. To appropriately account for antimicrobial-mediated strand fragmentation, suitable control reactions consisting of exposures to water, ethanol, kanamycin, and bleomycin were developed and optimized for the assay. Bacterial exposure to the CB clay resulted in significantly longer comet lengths, compared to water and kanamycin exposures, suggesting that the induction of DNA DSBs contributes to the killing activity of this antibacterial clay mineral mixture. The comet assay protocol described herein provides a general technique for evaluating soluble antimicrobial-derived DNA damage and for comparing DNA fragmentation between experimental and control assays.

Induction and repair of DNA double-strand breaks using constant-field gel electrophoresis and apoptosis as predictive markers for sensitivity of cancer cells to cisplatin

This study was designed to evaluate some parameters that may play a role in the prediction of cancer cells sensitivity to cisplatin (CIS). Sensitivity, induction and repair of DNA double-strand breaks (DSB), cell cycle regulation and induction of apoptosis were measured in four cancer cell lines with different sensitivities to CIS. Using a sulphorhodamine-B assay, the cervical carcinoma cells (HeLa) were found to be the most sensitive to CIS followed by breast carcinoma cells (MCF-7) and liver carcinoma cells (HepG2). Colon carcinoma HCT116 cells were the most resistant. As measured by constant-field gel electrophoresis (CFGE), DSB induction, but not residual DSB exhibited a significant correlation with the sensitivity of cells to CIS. Flow cytometric DNA ploidy analysis revealed that 67% of HeLa cells and 10% of MCF-7 cells shift to sub-G1 phase after incubation with CIS. Additionally, CIS induced the arrest of MCF-7 cells in S-phase and the arrest of HepG2 and HCT116 cells in both S phase and G2/M phase. Determination of the Fas-L level and Caspase-9 activity indicated that CIS-induced apoptosis results from the mitochondrial (intrinsic) pathway. These results, if confirmed using clinical samples, indicate that the induction of DNA DSB as measured by CFGE and the induction of apoptosis should be considered, along with other predictive markers, in future clinical trials to develop predictive assays for platinum -based therapy.

Abstract 2131: Plk1 decreases DNA double strand break (DSB) formation during replication- stalling genotoxic stress

Abstract Polo-like kinase 1 (Plk1) is a regulator of the mitotic checkpoint, and is involved in adaptation, which is the progression of cells into mitosis with damaged DNA. Deregulation of Plk1 activity is linked to cellular transformation. Recent studies suggest Plk1 activity to be critical for resumption of cell cycle progression during recovery from DNA damage. However, the ability of Plk1 to affect the DNA damage response remains relatively unclear. The aim of the present study was to determine the ability of Plk1 to affect the DNA damage response in the face of different forms of DSB-inducing genotoxic stress. We employed the respiratory carcinogen, hexavalent chromium [Cr(VI)], the anticancer drug and topoisomerase II inhibitor, etoposide, as well as the oxidizer, H2O2. We have previously shown that Plk1 activation was sufficient to bypass the G2/M checkpoint in normal human lung cells (HLFs), in the presence of Cr(VI)-induced acute genotoxic stress. In the present study, we measured DNA DSB induction in HLFs by the respective genotoxins under the condition of Plk1 activation by using the comet assay and γH2AX immunofluorescence staining. Transfection of HLFs with the constitutively active (c/a) Plk1 T210D mutant resulted in increased Plk1 protein expression 24h-48h post transfection. We used equitoxic concentrations of the different genotoxins and found that DNA DSBs increased > 2 fold after exposure to 3 μM Cr(VI), 12.5 μM etoposide, and 150 μM H2O2, respectively, and persisted for at least 4h. Consistent with DNA DSB formation, Cr(VI) exposure was associated with G1 arrest, at least at 4h, as determined by BrdU incorporation. Expression of the c/a T210D mutant increased Plk1 activity in vitro, and abrogated both Cr(VI) and etoposide-induced DNA DSBs as early as 30 min, and up to 4h post-treatment, in comparison to the vector control-transfected cells. In sharp contrast, treatment with 150 μM H2O2 induced a similar level of DNA DSBs in both the c/a T210D mutant- and vector-transfected cells. These data highlight the ability of Plk1 to decrease DNA DSBs uniquely under conditions of replication-stalling genotoxic stress. Given the documented roles of aberrant DNA damage response and Plk1 activation in cellular transformation, there is a critical need to define the molecular mechanism(s) by which Plk1 mediates decreased DNA DSBs after genotoxin exposure and delineate the pathways responsible for Plk1 activation. We postulate that the ability of Plk1 activation to abrogate DNA DSB formation after either Cr(VI) or etoposide exposure may occur at the expense of genomic stability. Supported by NIH grants CA107972 and ES017334 to SC and ES09961 and ES05304 to SRP and PhRMA foundation to GC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2131. doi:1538-7445.AM2012-2131

No Evidence for DNA Double-strand Breaks Caused by Endodontic Sealers

IntroductionOn extrusion, endodontic sealers might come into close contact with the periapical tissues for long periods. The objective of this study was to test possible mutagenicity of resin-based endodontic sealers by evaluating their potential to induce DNA double-strand breaks (DSBs). MethodsHuman gingival fibroblasts were exposed to subtoxic concentrations of eluates from 1 epoxy resin–based endodontic sealer (AH Plus Jet) and 2 methacrylate-based endodontic sealers (EndoRez and Real Seal). As control, Calcicur, a Ca(OH)2-based sealer, was used. The γ-H2AX immunofluorescence assay was used to microscopically detect DNA DSBs, and a custom algorithm was developed to quantify them. ResultsThe cytotoxicity of the 24-hour eluates could be ranked in the following order: AH Plus Jet > Real Seal > EndoRez >> Calcicur. The γ-H2AX assay revealed that 1.3%–4.3% of the cell nucleus was occupied by foci when the cells were exposed to the eluates of the endodontic sealers. This was not significantly different from the negative control group in which the cells had been exposed to medium (2.1%). ConclusionsNo indications for increased risk of genotoxicity of resin-based root canal sealers caused by the induction of DNA DSBs were found in this study.

Inhibition of poly (ADP-ribose) polymerase-1 (PARP-1) to increase radiosensitivity of human pancreatic cancer (PAC) cell lines proficient in homology-directed repair (HDR).

204 Background: Radiation-induced single strand DNA breaks (SSBs) are primarily repaired by base excision repair, of which PARP1 is a key component. Unrepaired SSBs may cause collapse of stalled replication forks during DNA replication generating DNA double-strand breaks (DSBs). PARP inhibition has been shown to potentiate the effect of radiotherapy (RT) in a variety of human cancers in vitro and in vivo. We investigated the ability of the PARP inhibitor olaparib to radiosensitize BRCA1/2 wild-type human PAC cell lines and quantified repair capability of an induced DSB by HDR using the I-SceI-induced DSB DR-GFP assay. Methods: MiaPaCa -2 and ASPC-1 human PAC cells lines were exposed to RT administered in a single fraction by 157-gammacell irradiator at doses of 2, 4, 6 and 8 Gy in the presence or absence of olaparib at a dose of 500nM (4 hours pre and 20 hours post RT). Plates were incubated at 37C for 10-14 days and stained with Giemsa. Clonogenic survival assays following olaparib continuous exposure at concentrations up to 10 µM for 10-14 days were also performed. HDR of an induced DNA DSB was assessed in ASPC1 and MiaPaCa cell lines by co-transfection with an I-SceI expression vector and the DR-GFP reporter plasmid(Pierce et al., 1999), and in BRCA2 deficient CAPAN-1 cells harboring a stably integrated chromosomal DR-GFP gene (Moynahan et al. 2001). Results: Linear quadratic models were fitted to the data sets to generate survival curves. The half maximal inhibitory concentration (IC 50) of RT was 2Gy for both cell lines. The sensitivity enhancement ratio (SER) at 25% survival was 1.44 and 1.53 for MiaPaCa-2 and ASPC-1 cell lines respectively. Both cell lines were resistant to treatment with single agent olaparib to dose of 10µM and were proficient in HDR of DNA DSBs when compared to the BRCA2-deficient Capan-1 cell line. Conclusions: Inhibition of PARP increases radiosensitivity in human PAC cell lines proficient in HDR of DNA DSBs and resistant to treatment with single agent PARP inhibitor. Therapeutic strategies incorporating PARP inhibition and RT offer potential to improve clinical outcomes for patients presenting with localized sporadic and BRCA mutated PAC.

DNA Double Strand Breaks in Gastric Epithelium withHelicobacter pyloriInfection

DNA double strand breaks (DSB) is one of the critical types of DNA damage. If unrepaired, DSB is accumulated in the nucleus of cells, the cells become apoptotic or transform to tumor by way of genomic instability. Some of malignant cancers and its premalignant lesions were proven to have DSB in their nuclei. There was no report that Helicobacter pylori (H. pylori), the gastric carcinogen, induce DNA DSB in gastric epithelium in vivo. The aim of this study was to investigate whether H. pylori induce DSB in the gastric epithelial cells of chronic gastritis. Immunohistochemical stains were performed for the DSB markers, phospho-53BP1 and gH2AX, in the gastric epithelium derived from 44 peptic ulcer disease patients before and after H. pylori eradication. DNA fragmentation assay was performed in the cell line to investigate the DNA damage by H. pylori infection. The mean expression score of gH2AX was significantly higher in the H. pylori infected gastric epithelium as compared to the H. pylori eradicated gastric epithelium (8.8±5.5 vs. 6.2±5.3 respectively; p=0.008). The expression score of phospho-53BP1 between before and after eradication of H. pylori was not statistically different, but tended to be higher in H. pylori infection. DNA fragmentation was developed significantly more in the cell lines after infection with H. pylori. DSB of DNA damage was typical feature of H. pylori infection in the gastric epithelium.

No Formation of DNA Double-Strand Breaks and No Activation of Recombination Repair with UVA

Longwave UVA is an independent class I carcinogen. A complete understanding of UVA-induced DNA damage and how this damage is processed in skin cells is therefore of utmost importance. A particular question that has remained contentious is whether UVA induces DNA double-strand breaks (DSBs), either directly or through processing of other types of DNA damage, such as recombination repair of replication forks stalled at DNA photoproducts. We therefore studied activation of the recombination repair pathway by solar available doses of UVA and assessed formation of DNA DSBs in primary skin fibroblasts. We found that, unlike ionizing radiation or UVB, UVA does not activate the Fanconi anemia/BRCA DNA damage response pathway or the "recombinase" RAD51 in primary skin fibroblasts. The fact that this pathway mediates recombination repair of DNA DSBs suggests that DNA DSBs are not formed by UVA. This is further supported by findings that UVA did not induce DNA DSBs, as assayed by neutral single-cell electrophoresis or by formation of γ-H2AX nuclear foci, considered the most sensitive assay for DNA DSBs. The lack of sufficient evidence for formation of DNA DSBs underlines the pivotal role of UVA-induced DNA photoproducts in UVA mutagenesis and carcinogenesis.

Abstract 3914: Plk1 enhances DNA double strand break (DSB) repair during acute genotoxin exposure

Abstract Polo-like kinase 1 (Plk1) is a regulator of the mitotic checkpoint, and is involved in adaptation, which is the progression of cells into mitosis with damaged DNA. Deregulation of Plk1 activity is linked to cellular transformation. Recent studies suggest Plk1 activity to be critical for resumption of cell cycle progression in cancer cells during recovery from DNA damage. However, the ability of Plk1 to affect the DNA damage response remains relatively unclear. In this study we employed the respiratory carcinogen, hexavalent chromium [Cr(VI)], which is a well-documented genotoxin of occupational and environmental concern. We have previously shown that Plk1 activation was sufficient to bypass the G2/M checkpoint in normal human lung cells (HLFs), in the presence of Cr(VI)-induced acute genotoxic stress. In the present study we measured DNA DSB induction by Cr(VI) in HLFs under the condition of Plk1 activation by using the comet assay and gamma H2AX immunofluorescence staining. Transfection of HLFs with the constitutively active (c/a) Plk1 T210D mutant resulted in increased Plk1 protein expression 24h-48h post transfection. We found that DNA DSBs increased > 2 fold 15 min after 3 μM Cr(VI) exposure, and persisted for at least 4h. Consistent with DNA DSB formation, Cr(VI) exposure was associated with G1 arrest, at least at 4h, as determined by BrdU incorporation. Expression of the c/a T210D mutant increased Plk1 activity in vitro, and abrogated Cr(VI)-induced DNA DSBs as early as 30 min, and up to 4h post-treatment, in comparison to the vector control transfected cells. Since Cr(VI)-induced DNA DSBs were equally formed by 15 min in both vector- and Plk1 T210D-transfected cells, these data highlight the heretofore unreported ability of Plk1 to enhance DNA repair. Our previous report found that Plk1 expression increased survival and mutagenesis of wt S. cerevisiae treated with Cr(VI). Therefore, we also determined the ability of Plk1 to “rescue” repair deficient rad52 yeast from Cr(VI)-induced lethality. S. cerevisae transformed with Plk1-Gal-HA-EGFP plasmid grown under inducible conditions overexpress Plk1. We found that induction of Plk1 in rad52 yeast significantly protected them from Cr(VI) clonogenic lethality. Given the documented roles of aberrant DNA damage response and Plk1 activation in cellular transformation, there is a critical need to define the molecular mechanism(s) by which Plk1 mediates DNA damage repair after genotoxin exposure and delineate the pathways responsible for Plk1 activation. We postulate that the ability of Plk1 activation to provoke DNA DSB repair after Cr(VI) exposure occurs at the expense of genomic stability. Supported by NIH grants CA107972 to SC and ES09961 and ES05304 to SRP and PhRMA foundation to GC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3914. doi:10.1158/1538-7445.AM2011-3914

Abstract 5081: Inhibiting DNA double strand break repair for alpha radioimmunotherapy

Abstract Metastasis to distant organs is the major cause of mortality in breast cancer patients. Median overall survival remains poor (5 year survival 26.1%) despite recent advances in new targeted biological therapy. Radiolabeled antibody offers an effective approach to deliver cytotoxic radiation doses to eliminate micrometastatic tumors. Alpha particle emitters are promising against small micrometastases because of their high potency and specificity (high linear energy transfer, LET>80 keV/µm, particle range ∼ 80 μm). Alpha particles typically cause DNA double strand breaks (DSBs) when they traverse the cell nucleus. Our experience with alpha particle emitter labeled antibody showed that it significantly prolonged survival in a mouse model of metastatic breast cancer with eventual recurrence at estimated micromet dose of 4.4 Gy. Understanding cellular repair of alpha radiation induced DNA DSBs could help us rationally design more effective strategy by inhibiting components of the DNA DSB repair pathways to sensitize cancer cells to alpha radiation. Under this hypothesis, we evaluated the role of DNA dependent protein kinase catalytic subunit (DNA-PKcs) inhibition on targeted alpha-emitter therapy. Inhibition, with small molecule inhibitor NU7441, of this key enzyme in the non-homologous end joining (NHEJ) pathway sensitizes triple negative breast cancer cells to alpha particle emitter 213Bi labeled anti-EGFR antibody. Immuno-fluorescent staining of γH2AX foci confirms a reduction in the DNA DSB repair rate in these cells. Using a cell-line deficient in the homologous recombination (HR) pathway (BRCA-1 deficient), we also have preliminary evidence suggesting that HR deficiency reduces the incremental effect of NHEJ inhibition but the combination increases overall cell kill. This data suggest inhibiting components of DNA double strand repair pathways is an effective approach to enhance the efficacy of alpha particle emitter labeled monoclonal antibody. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5081. doi:10.1158/1538-7445.AM2011-5081

Emodin Triggers DNA Double-Strand Breaks by Stabilizing Topoisomerase II-DNA Cleavage Complexes and by Inhibiting ATP Hydrolysis of Topoisomerase II

Emodin, an anthraquinone derived from a plant and fungi, has been reported to possess potential genotoxicity, but the mechanism is not entirely clear. Here, we report that emodin causes DNA double-strand breaks (DSBs) through stabilization of topoisomerase (Topo) II-DNA cleavage complexes and inhibition of ATP hydrolysis. In our study, emodin did not induce mutagenecity in the salmonella mutation assay but caused genotoxicity in the thymidine kinase gene mutation assay and in the micronucleus test. Moreover, emodin induced DNA DSBs demonstrated by induction of comet tails, the expression of phosphorylated histone H2AX, and phosphorylation of ataxia telangiectasia mutated. Our studies also revealed that emodin exerted strong inhibitory activity against Topo II in the supercoiled pBR322 relaxation assay and in Topo II-mediated kinetoplast DNA decatenation, similar to the previous report. We also showed that the inhibitory effect of emodin on Topo II was because of its ability to stabilize Topo II-DNA complexes and to inhibit the ATP hydrolysis of Topo II. Furthermore, emodin was found to trigger DNA DSBs in a Topo II-dependent manner using the Topo II catalytic inhibitor aclarubicin and in Topo II-deficient mitoxantrone-resistant variant HL-60/MX2 cells. Together, these results suggest that in emodin-induced DNA DSBs and genotoxicity, stabilization of Topo II-DNA cleavage complexes and inhibition of ATP hydrolysis play an important role.

H2AX phosphorylation screen of cells from radiosensitive cancer patients reveals a novel DNA double-strand break repair cellular phenotype

Background:About 1–5% of cancer patients suffer from significant normal tissue reactions as a result of radiotherapy (RT). It is not possible at this time to predict how most patients’ normal tissues will respond to RT. DNA repair dysfunction is implicated in sensitivity to RT particularly in genes that mediate the repair of DNA double-strand breaks (DSBs). Phosphorylation of histone H2AX (phosphorylated molecules are known as γH2AX) occurs rapidly in response to DNA DSBs, and, among its other roles, contributes to repair protein recruitment to these damaged sites. Mammalian cell lines have also been crucial in facilitating the successful cloning of many DNA DSB repair genes; yet, very few mutant cell lines exist for non-syndromic clinical radiosensitivity (RS).Methods:Here, we survey DNA DSB induction and repair in whole cells from RS patients, as revealed by γH2AX foci assays, as potential predictive markers of clinical radiation response.Results:With one exception, both DNA focus induction and repair in cell lines from RS patients were comparable with controls. Using γH2AX foci assays, we identified a RS cancer patient cell line with a novel ionising radiation-induced DNA DSB repair defect; these data were confirmed by an independent DNA DSB repair assay.Conclusion:γH2AX focus measurement has limited scope as a pre-RT predictive assay in lymphoblast cell lines from RT patients; however, the assay can successfully identify novel DNA DSB repair-defective patient cell lines, thus potentially facilitating the discovery of novel constitutional contributions to clinical RS.

High resolution imaging of changes in the structure and spatial organization of chromatin, γ-H2A.X and the MRN complex within etoposide-induced DNA repair foci

The focal accumulation of DNA repair factors, including the MRE11/Rad50/NBS1 (MRN) complex and the phospho-histone variant γ-H2A.X, is a key cytological feature of the DNA damage response (DDR). Although these foci have been extensively studied by light microscopy, there is comparatively little known regarding their ultrastructure. Using correlative light microscopy and electron spectroscopic imaging (LM/ESI) we have characterized the ultrastructure of chromatin and DNA repair foci within the nuclei of normal human fibroblasts in response to DNA double-strand breaks (DSBs). The induction of DNA DSBs by etoposide leads to a global decrease in chromatin density, which is accompanied by the formation of invaginations of the nuclear envelope as revealed by live-cell microscopy. Using LM/ESI and the immunogold localization of γ-H2A.X and MRE11 within repair foci, we also observed decondensed 10nm chromatin fibers within repair foci and the accumulation of large non-chromosomal protein complexes over three hours recovery from etoposide. At 18 h after etoposide treatment, we observed a close juxtapositioning of PML nuclear bodies and late repair foci of γ-H2A.X, which exhibited a highly organized chromatin arrangement distinct from earlier repair foci. Finally, the dual immunogold labeling of MRE11 with either γ-H2A.X or NBS1 revealed that γ-H2A.X and the MRN complex are sub-compartmentalized within repair foci at the sub-micron scale. Together these data provide the first ultrastructural comparison of γ-H2A.X and MRN DNA repair foci, which are structurally dynamic over time and strikingly similar in organization.

DNA Double-Strand Breaks by Asbestos, Silica, and Titanium Dioxide

DNA double-strand breaks (DSBs) can result in cell death or genetic alterations when cells are subjected to radiation, exposure to toxins, or other environmental stresses. A complex DNA-damage-response pathway is activated to repair the damage, and the inability to repair these breaks can lead to carcinogenesis. One of the earliest responses to DNA DSBs is the phosphorylation of a histone, H2AX, at serine 139 (gamma-H2AX), which can be detected by a fluorescent antibody. A study was undertaken to compare the induction of DNA DSBs in normal (small airway epithelial) cells and cancer cells (A549) after exposure to asbestos (crocidolite), a proven carcinogen, silica, a suspected carcinogen, and titanium dioxide (TiO(2)), an inert particle recently reported to be carcinogenic in animals. The results indicate that crocidolite induced greater DNA DSBs than silica and TiO(2), regardless of cell type. DNA DSBs caused by crocidolite were higher in normal cells than in cancer cells. Silica and TiO(2) induced higher DNA DSBs in cancer cells than in normal cells. The production of reactive oxygen species was found to be highest in cells exposed to crocidolite, followed, in potency, by silica and TiO(2). The generation of reactive oxygen species was higher in normal cells than in cancer cells. Cell viability assay indicated that crocidolite caused the greatest cytotoxicity in both cell types. Apoptosis, measured by caspase 3/7 and poly (ADP-Ribose) polymerase activation, was highest in crocidolite-exposed cells, followed by TiO(2) and silica. The results of this study indicate that crocidolite has a greater carcinogenic potential than silica and TiO(2), judged by its ability to cause sustained genomic instability in normal lung cells.

Regulation of ATM in DNA double strand break repair accounts for the radiosensitivity in human cells exposed to high linear energy transfer ionizing radiation

High linear energy transfer (LET) radiation shows different biological effects from low-LET radiation. The complex nature of high LET radiation-induced damage, especially the clustered DNA damage, brings about slow repair of DNA double strand breaks (DSBs), which finally lead to higher lethality and chromosome aberration. Ionizing radiation (IR) induced DNA DSBs are repaired by both non-homologous end-joining (NHEJ) and homologous recombination repair (HRR) pathways in mammalian cells. The novel function of ataxia telangiectasia-mutated (ATM) protein is its involvement in the DSB repair of slow kinetics for “dirty” breaks rejoining by NHEJ, this suggests that ATM may play a more important role in high LET radiation-induced DNA damage. We show here that KU55933, an ATM inhibitor could distinctly lower the clonogenic survival in normal human skin fibroblast cells exposed to carbon ion radiation and dramatically impair the normal process for DSB repair. We also implicated the involvement of ATM in the two pathways of DNA DSB repair, with DNA-PKcs and Rad51 as the representative proteins. The phosphorylation of DNA-PKcs at Thr-2609 with both immunoblotting and immunofluorescent staining indicated an ATM-dependent change, while for Rad51, KU55933 pretreatment could postpone the formation of nuclear Rad51 foci. Interestingly, we also found that pretreatment with chloroquine, an ATM stimulator could protect cells from carbon ion radiation only at lower doses. For doses over 1 Gy, protection was no longer observed. There was a dose-dependent increase for ATM kinase activity, with saturation at about 1 Gy. Chloroquine pretreatment prior to 1 Gy of carbon ion radiation did not enhance the autophosphorylation of ATM at serine 1981. The function of ATM in G2/M checkpoint arrest facilitated DSB repair in high-LET irradiation. Our results provide a possible mechanism for the direct involvement of ATM in DSB repair by high-LET irradiation.

H2AX Phosphorylation and Kinetics of Radiation-Induced DNA Double Strand Break Repair in Human Primary Thyrocytes

Papillary thyroid carcinoma (PTC) represents the most frequent thyroid neoplasia and is associated with radiation exposure. A consistent proportion of PTC is characterized by chromosome rearrangements producing RET and TRK oncogenes, but the mechanisms underlying the thyrocyte propensity to such alterations are poorly understood. Diminished capacity of thyrocytes to repair radiation-induced DNA double strand breaks (DSBs) might play a role in chromosome rearrangements; however, this question has not been fully addressed due to the lack of physiologically relevant experimental models. Several normal human thyroid primary cultures were produced and characterized for their capacity to repair the DNA DSBs induced by ionizing radiation. For comparison, normal human bronchial epithelial cells were used. We employed the gammaH2AX foci assay; counts were determined at different time points after irradiation. All the thyrocyte samples analyzed showed similar DNA DSBs induction. The rate of gammaH2AX foci clearance was homogenous, showing only small differences among samples. This work reports the first characterization of DNA DSB repair in human primary thyrocytes, a relevant clinical model for thyroid carcinogenesis, and represents an important step toward dissection of the link between DNA DSB repair and thyroid-specific oncogenic rearrangements.

Characteristics and modifying factors of asbestos‐induced oxidative DNA damage

Respiratory exposure to asbestos has been linked with mesothelioma in humans. However, its carcinogenic mechanism is still unclear. Here we studied the ability of chrysotile, crocidolite and amosite fibers to induce oxidative DNA damage and the modifying factors using four distinct approaches. Electron spin resonance analyses revealed that crocidolite and amosite containing high amounts of iron, but not chrysotile, catalyzed hydroxyl radical generation in the presence of H(2)O(2), which was enhanced by an iron chelator, nitrilotriacetic acid, and suppressed by desferal. Natural iron chelators, such as citrate, adenosine 5'-triphosphate and guanosine 5'-triphosphate, did not inhibit this reaction. Second, we used time-lapse video microscopy to evaluate how cells cope with asbestos fibers. RAW264.7 cells, MeT-5 A and HeLa cells engulfed asbestos fibers, which reached not only cytoplasm but also the nucleus. Third, we utilized supercoiled plasmid DNA to evaluate the ability of each asbestos to induce DNA double strand breaks (DSB). Crocidolite and amosite, but not chrysotile, induced DNA DSB in the presence of iron chelators. We cloned the fragments to identify break sites. DSB occurred preferentially within repeat sequences and between two G:C sequences. Finally, i.p. administration of each asbestos to rats induced not only formation of nuclear 8-hydroxy-2'-deoxyguanosine in the mesothelia, spleen, liver and kidney but also significant iron deposits in the spleen. Together with the established carcinogenicity of i.p. chrysotile, our data suggest that asbestos-associated catalytic iron, whether constitutional or induced by other mechanisms, plays an important role in asbestos-induced carcinogenesis and that chemoprevention may be possible through targeting the catalytic iron.