Constant-field Gel Electrophoresis Research Articles

Evaluation of the Yield of DNA Double-Strand Breaks for Carbon Ions Using Monte Carlo Simulation and DNA Fragment Distribution

The aim of this work was to provide a method to evaluate the yield of DNA double-strand breaks (DSBs) for carbon ions, overcoming the bias in existing methods due to the nonrandom distribution of DSBs. A previously established biophysical program based on the radiation track structure and a multilevel chromosome model was used to simulate DNA damage induced by x-rays and carbon ions. The fraction of activity retained (FAR) as a function of absorbed dose or particle fluence was obtained by counting the fraction of DNA fragments larger than 6 Mbp. Simulated FAR curves for the 250 kV x-rays and carbon ions at various energies were compared with measurements using constant-field gel electrophoresis. The doses or fluences at the FAR of 0.7 based on linear interpolation were used to estimate the simulation error for the production of DSBs. The relative difference of doses at the FAR of 0.7 between simulation and experiment was -8.5% for the 250 kV x-rays. The relative differences of fluences at the FAR of 0.7 between simulations and experiments were -17.5%, -42.2%, -18.2%, -3.1%, 10.8%, and -14.5% for the 34, 65, 130, 217, 2232, and 3132 MeV carbon ions, respectively. In comparison, the measurement uncertainty was about 20%. Carbon ions produced remarkably more DSBs and DSB clusters per unit dose than x-rays. The yield of DSBs for carbon ions, ranging from 10 to 16 Gbp-1Gy-1, increased with linear energy transfer (LET) but plateaued in the high-LET end. The yield of DSB clusters first increased and then decreased with LET. This pattern was similar to the relative biological effectiveness for cell survival for heavy ions. The estimated yields of DSBs for carbon ions increased from 10 Gbp-1Gy-1 in the low-LET end to 16 Gbp-1Gy-1 in the high-LET end with 20% uncertainty.

Cellular susceptibility and oxidative stress response to menadione of logarithmic, quiescent, and nonquiescent Saccharomyces cerevisiae cell populations

The aim of the present study was to compare cellular susceptibility and oxidative stress response of S. cerevisiae logarithmic (log), quiescent (Q), and non-quiescent (NQ) cell populations to menadione – a well-known inducer of oxidative stress. Three main approaches were used: microbiological – cell survival, molecular – constant field gel electrophoresis for detection of DNA double-strand breaks (DSB), and biochemical – measurement of reactive oxygen species (ROS) levels, oxidized proteins, lipid peroxidation, glutathione, superoxide dismutase (SOD) and catalase on S. cerevisiae haploid strain BY4741. The doses causing 20% (LD20) and 50% (LD50) lethality were calculated. The effect of menadione as a well-known oxidative stress inducer is compared in the log, Q, and NQ cells. Survival data reveal that Q cells are the most susceptible to menadione with LD50 corresponding to 9 µM menadione. On the other hand, dose-dependent DSB induction is found only in Q cells confirming the results shown above. No effect on DSBs levels is observed in log and NQ cells. Further, the oxidative stress response of the cell populations is clarified. Results show significantly higher levels of SOD and ROS in Q cells than in log cells after the treatment with 100 µM menadione. On the other side, higher induction of oxidized proteins, malondialdehyde, and glutathione is observed after menadione treatment of log cells. Our study provides evidence that Saccharomyces cerevisiae quiescent cells are the most susceptible to the menadione action. It might be suggested that the DNA damaging and genotoxic action of menadione in Saccharomyces cerevisiae quiescent cells could be related to ROS production.

On the mode of action of Origanum vulgare spp. hirtum methanolic extract and essential oil on Chlamydomonas reinhardtii

Aim: To reveal whether methanolic extract and essential oil from Origanum vulgare subsp. hirtum in doses causing even low levels of mortality in aphids, would have harmful effects on plants-genotoxic, mutagenic and/or DNA damaging. Materials and methods: Aerial parts of Origanum vulgare ssp. hirtum from the ex-situ collection of IBER, BAS during flowering were collected. Extraction and isolation procedures, as well as GC/MS analysis of essential oil and methanolic extract were performed by standard protocols. The components were identified by comparing their relative retention times to the retention times of authentic standards, and with mass spectra with the NIST. Test system: Chlamydomonas reinhardtii strain 137 C+ (WT). Endpoints: “clonal” assay, the test of “visible mutations”, constant field gel electrophoresis. Statistics: GraphPad Prism version 6.04 (San Diego, USA) and One-way Analysis of Variance ANOVA with multiple comparisons using the Tukey method. Results: A good correlation was observed between chemical composition of essential oil and methanolic extract, and their mode of action. Our genotoxic and double strand breaks results demonstrated mild genotoxic and statistically non-significant DNA damaging potential of methanolic extract and concentration-dependent well - expressed genotoxic and DNA damaging potential of essential oil. A good relationship between increased double strand breaks levels and decreased survival might be related to one of the main constituents of essential oil, suspected to be carvacrol. No mutagenic effect for ME and EO was found. Conclusion: Well-expressed toxic/genotoxic capacity of essential oil, as well as its capacity to damage DNA inducing double strand breaks, but the absence of mutagenic potential, could be considered as a good reason to recommend Origanum vulgare subsp. hirtum essential oil as a promising candidate for purposes of “green” technologies.

Human shelterin protein POT1 prevents severe telomere instability induced by homology-directed DNA repair.

The evolutionarily conserved POT1 protein binds single-stranded G-rich telomeric DNA and has been implicated in contributing to telomeric DNA maintenance and the suppression of DNA damage checkpoint signaling. Here, we explore human POT1 function through genetics and proteomics, discovering that a complete absence of POT1 leads to severe telomere maintenance defects that had not been anticipated from previous depletion studies in human cells. Conditional deletion of POT1 in HEK293E cells gives rise to rapid telomere elongation and length heterogeneity, branched telomeric DNA structures, telomeric R-loops, and telomere fragility. We determine the telomeric proteome upon POT1-loss, implementing an improved telomeric chromatin isolation protocol. We identify a large set of proteins involved in nucleic acid metabolism that engage with telomeres upon POT1-loss. Inactivation of the homology-directed repair machinery suppresses POT1-loss-mediated telomeric DNA defects. Our results unravel as major function of human POT1 the suppression of telomere instability induced by homology-directed repair.

DNA susceptibility of Saccharomyces cerevisiae to Zeocin depends on the growth phase.

The aim of this study was to evaluate the level of Zeocin-induced double-strand breaks (DSBs) in Saccharomyces cerevisiae cells in a different growth phase, using constant-field gel electrophoresis (CFGE). Saccharomyces cerevisiae diploid strain D7ts1 with enhanced cellular permeability was used. The effects of growth phase and treatment time were evaluated based on Zeocin-induced DSBs, measured by CFGE. Survival assay was also applied. No protoplast isolation was necessary for the detection of DSBs in strain D7ts1. Differences in the response of cells depending on the growth phase were obtained. Cells in exponential growth phase had increased DSB levels only after Zeocin treatment with concentrations equal or higher than 200μgml-1. Increasing treatment time did not result in higher DSB levels. Oppositely, treatment of cells at the beginning of stationary phase with Zeocin concentrations resulted in more than 1.5-fold increase in DSB levels in comparison with those in untreated cells. Increased DSB levels were measured for all the treatment times. A dose-dependent decrease in cell survival was observed after Zeocin treatment with concentrations in the range of lethality LD20-LD50. A strong negative correlation was calculated between the levels of DSBs and cell survival. New information is provided concerning DNA susceptibility depending on the growth phase. DNA susceptibility is higher in cells at the beginning of stationary phase than those in exponential phase. Data presented here illustrate that the optimized by us CFGE protocol is sensitive and could be used successfully for DSB measurement in Saccharomyces cerevisiae strains with enhanced cellular permeability.

Modulation of DNA damage response and induction of apoptosis mediates synergism between doxorubicin and a new imidazopyridine derivative in breast and lung cancer cells

DNA damage response machinery (DDR) is an attractive target of cancer therapy. Modulation of DDR network may alter the response of cancer cells to DNA damaging anticancer drugs such as doxorubicin. The aim of the present study is to investigate the effects of a newly developed imidazopyridine (IAZP) derivative on the DDR after induction of DNA damage in cancer cells by doxorubicin. Cytotoxicity sulphrhodamine-B assay showed a weak anti-proliferative effect of IAZP alone on six cancer cell lines (MCF7, A549, A549DOX11, HepG2, HeLa and M8) and a normal fibroblast strain. Combination of IAZP with doxorubicin resulted in synergism in lung (A549) and breast (MCF7) cancer cells but neither in the other cancer cell lines nor in normal fibroblasts. Molecular studies revealed that synergism is mediated by modulation of DNA damage response and induction of apoptosis. Using constant-field gel electrophoresis and immunofluorescence detection of γ-H2AX foci, IAZP was shown to inhibit the repair of doxorubicin-induced DNA damage in A549 and MCF7 cells. Immunoblot analysis showed that IAZP suppresses the phosphorylation of the ataxia lelangiectasia and Rad3 related (ATR) protein, which is an important player in the response of cancer cells to chemotherapy-induced DNA damage. Moreover, IAZP augmented the doxorubicin-induced degradation of p21, activation of p53, CDK2, caspase 3/7 and phosphorylation of Rb protein. These effects enhanced doxorubicin-induced apoptosis in both cell lines. Our results indicate that IAZP is a promising agent that may enhance the cytotoxic effects of doxorubicin on some cancer cells through targeting the DDR. It is a preliminary step toward the clinical application of IAZP in combination with anticancer drugs and opens the avenue for the development of compounds targeting the DDR pathway that might improve the therapeutic index of anticancer drugs and enhance their cure rate.

Hsp90 inhibitor is a good candidate for effective combination therapy with carbon ions

Purpose/Background: To further improve the effectiveness of heavy ion radio-therapy, we studied the biology of a combined treatment using Hsp 90 inhibitor and carbon ion irradiation. We have previously reported that an Hsp90 inhibitor 17AAG can be an effective radio-sensitizer with X-rays for certain human tumor cells, while normal cells were not sensitized by this drug [ 1]. The underlying mechanism for this was demonstrated to be inhibition of DNA double-strand break (DSB) repair by 17AAG; particularly, homologous recombination repair (HRR) pathway was shown to be affected by this agent. In vivo mouse xenograft study also indicated a better tumor control with the combined treatment when compared with X-ray treatment alone.Materials and Methods: Cell lines used were SQ5 human lung cancer cells and HFL III normal human fibroblasts as control. For irradiation, X-rays and 290 MeV/n carbon ions were used at 50–70 keV/µm LET setting. As Hsp90 inhibitors, 17AAG and PU-H71 were used for pre-irradiation treatment for 24 h. Colony formation assay was used for radiation sensitivity studies. Repair of DNA DSBs was measured by constant field gel electrophoresis, and the appearance/disappearance of Rad51 foci was analyzed for HRR efficiency. For IRB-approved mouse xenograft study, BALB/c nu/nu mice were used to implant SQ5 cells for local irradiation.Results/Discussion: SQ5 tumor cells were better controlled with the combination of 17 AAG and carbon ion irradiation in vitro and in vivo xenograft model when compared with carbon irradiation alone. The cause of this radio-sensitizing effect seems to come from inhibition of repair of DNA DSBs by 17AAG as in X-rays. Likewise, HRR pathway was affected by addition of 17AAG in carbon irradiated tumor cells. The effect of 17AAG pre-treatment is shown below where the appearance of an HRR key protein Rad51 was significantly delayed after carbon ion irradiation in the drug-treated samples when compared with samples with carbon alone (Fig. 1).We also started to investigate PU-H71 with carbon ion irradiation in vitro and in vivo. PU-H71 alone is currently under phase I clinical trial. Our data indicate that PU-H71 pre-treatment also showed a significant radio-sensitization in SQ5 human lung tumor cells exposed to carbon ions as well as to X-rays. As shown with 17AAG, normal human cells were not significantly affected with this drug. PU-H71 also seems to affect repair of radiation-induced DSBs. Further mechanism studies and in vivo experiments are currently underway.Conclusion: Hsp90 inhibitor would be a good candidate for the effective combined treatment with carbon ion radio-therapy. Fig. 1.Post-irradiation Rad51 foci appearance kinetics in SQ5 cells irradiated with 2 Gy carbon ions compared with the combined treatment of 17AAG and carbon radiation (2 Gy).

Biophysical studies of the effect of high power ultrasound on the DNA solution

Stability and molecular size of the DNA double helical structure were studied on an aqueous solution of DNA after exposure to high power doses of continuous wave ultrasound at frequency of 20 kHz. Thermal transition spectrophotometry (UV-melting), constant-field gel electrophoresis (CFGE), differential scanning calorimetry (DSC) and dielectric properties measurements were used to evaluate the ultrasound-induced changes in the DNA double helical structure. The thermal transition spectrophotometry (UV-melting) and differential scanning calorimetry (DSC) results showed that ultrasound power caused loss of DNA double helical structure and the DNA double strands melting temperature decreased as the ultrasound power increased, indicating a decrease in the stability of the double helical structure of DNA. The constant-field gel electrophoresis (CFGE) results showed that the molecular size of the DNA fragments decreased as the ultrasound power increased. The dielectric data in the frequency range from 20 Hz to 100 kHz for the native DNA showed that dispersion at frequency of about 500 Hz resulted from polarization induced by counterions. The decrease in the dielectric increment indicated a decrease in length of DNA molecule after exposure to ultrasound power.

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.

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 3920: Oxidatively-induced complex DNA damage and repair in human colon adenocarcinoma

Abstract Colorectal cancer is the third leading cause of cancer mortality in the United States. Oxidative stress and DNA damage have been associated with a variety of human pathophysiological conditions, including colon cancer. Complex DNA damage may manifest in double strand breaks (DSBs) and non-DSB, bi-stranded, oxidatively-induced clustered DNA lesions (OCDLs). To date, there is very limited knowledge regarding the possible accrual of OCDLs in human colon tumors or the OCDL repair efficiency of primary colonic malignant cells. Therefore, the impact of oxidatively-induced clustered DNA damage in the predisposition to or progression of colon cancer remains undefined. We hypothesized that colon adenocarcinoma tissue and cells would have increased levels of DNA damage compared with normal counterparts. Furthermore, we expected that malignant colon cells would exhibit a defective Base Excision Repair (BER) pathway. To test this hypothesis, normal and malignant colonic tissue from 10 consented female patients was collected and number of DNA lesions measured using neutral constant field gel electrophoresis. Quantitative measurement of OCDL difference in each group was achieved using QuantiScan analysis. DNA repair efficiency of colon tissue-derived primary normal and malignant cells was assessed by single-cell gel electrophoresis. Expression and localization of BER-associated proteins was determined through Western blot analysis and immunohistochemistry. We found that degree of complex DNA damage varied among individual subjects. Forty percent of patients had tumors with increased levels of APE1-induced OCDLs compared to normal counterparts. The vast majority (80%) of malignant tissue expressed higher levels of APE1, XRCC1 and PARP1, ranging from 3- to >10-fold, compared to normal peri-tumoral tissue. However, 20% of malignant tissue had reduced levels of the aforementioned proteins as compared to peri-tumoral matched normal tissue. Oxidatively-induced DNA damage was more efficiently repaired in primary cells derived from normal tissue compared to cells from paired malignant tissue (1 hour versus >6 hours) following treatment with 6 mM H2O2. Since the expression of most BER-associated proteins (e.g., XRCC1, OGG1, etc.) was similar in both malignant and normal cells, the DNA repair defect may be the result of a 2.5-fold lower expression of APE1 protein measured in these cancerous cells compared to normal primary cells. Furthermore, a differential cytoplasmic to nuclear translocation of APE1 was also observed in cancerous cells compared to normal cells during the DNA damage and repair process. In conclusion, these findings strongly indicate that there is variation in oxidatively-induced complex DNA damage – perhaps mediated through a defective BER pathway – among patients with colon cancer, thus underscoring the need for patient-specific approaches to diagnosis and treatment of this disease. 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 3920. doi:10.1158/1538-7445.AM2011-3920

Abstract 5544: ASPM is a novel participant in DNA double-strand break repair and a potential target for radiotherapy

Abstract Comprehensive serach for human cellular response to ionizing radiation (IR) is indispensable strategy to understand the molecular basis under tumor radiotherapy. In a previous study using HiCEP, we found that ASPM (Abnormal spindle-like microcephaly-associated) or the most common-type microcephaly (MCPH5) gene is selectively down-regulated by IR. Here we first demonstrate that down-regulation of ASPM by siRNA synergistically enhanced radiosensitivity in some immortalized human cell lines. Both constant-field gel electrophoresis and gamma-H2AX foci assay exhibited impaired DNA double-strand breaks (DSB) in the cells treated with the siRNA. Greater extent of chromosomal instability was also common in the treated cells than untreated control. Getting together, ASPM (MCPH5) is a novel participant in the DNA double-strand break repair pathways. Although roles of microcephalins (MCPH1, pericentrin, ATR) have recently been proposed on DNA damage repair and check point regulation, this is the first report on ASPM. In clinical aspects, ASPM would be a promising target for the future cancer treatment as well as a useful biomarker in the prognostic strategy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5544.

Enhanced radiation-induced cell killing by Herbimycin A pre-treatment

Herbimycin A (HA), as in Geldanamycin, binds to conserved pockets of heat shock protein 90 (Hsp90) and inhibits its chaperone functions. Hsp90 plays an integral role in cancer cell growth and survival, because it maintains the stability of several key proteins by its chaperone's activity. It is known that some of the proteins associated with radiation responses are functionally stabilized by Hsp90. In this study, we investigated the effect of HA on radiosensitivity in human cancer cells and the mechanism related to the sensitization. In order to gain a mechanistic insight of this sensitization, we examined repair of DNA double-strand breaks (DSBs) in irradiated human cancer cells pre-treated with HA, as unrepaired DSBs are thought to be the main cause of radiation-induced cell death. Cellular radiosensitivity was determined by clonogenic assay, and the DSB rejoining kinetics was examined by constant field gel electrophoresis. SQ-5, a lung squamous carcinoma cell line, showed synergistic increase in radiosensitivity when cells were pre-treated with HA. In addition, HA significantly inhibited repair of radiation-induced DSBs. These results suggest that the combination of HA and ionizing radiation may be a useful therapeutic strategy for treating certain cancer cells.

Chemopreventive agent sulforaphane enhances radiosensitivity in human tumor cells

Sulforaphane (SFN), an isothiocyanate derived from broccoli and other cruciferous vegetables, is a positive regulator of phase II detoxification enzymes and is highly effective in protection against chemically induced cancers by inducing apoptosis and cell cycle arrest. Here, we report that SFN also enhances radiosensitivity in human tumor cells. Cell survival in HeLa human cervix carcinoma cells pretreated with SFN was significantly lower than in cells treated with radiation only. Constant-field gel electrophoresis and a gamma-H2AX foci assay showed marked inhibition of DSB repair in irradiated cells treated with SFN, while little inhibition was observed in cells with DMSO (control). In addition, immunofluorescence experiments revealed a significant delay in Rad51 (a key protein for homologous recombination repair) foci formation and disappearance in irradiated cells treated with SFN when compared to the cells with X-irradiation alone. The dephosphorylation of DNA-PKcs (a critical nonhomologous end joining protein) was also markedly delayed by SFN pretreatment in irradiated cells. These DSB repair inhibition data partially support the high apoptotic frequency of irradiated cells pretreated with SFN. Furthermore, the combined treatment of X-rays and SFN (i.p. 300 micromol/kg) in the xenograft model with HeLa cells showed efficient inhibition of in vivo tumor growth. To the best of our knowledge, our study is the first report showing SFN-enhanced radiosensitivity of tumor cells in vitro and in vivo, which opens the door for a multitude of clinical applications for chemoradiotherapy using SFN.

Effect of high power ultrasound on aqueous solution of DNA

The effect of (20 kHz continuous wave) ultrasound (power from 17 to 77 W) on the damage of nitrogen bases, the loss of double-helical structure, strand breaks and fragmentation of Calf spleen DNA were studied in vitro. The relative ultrasound sensitivity of the hydrogen bonded base pairs A-T and G-C were also studied. The techniques used in this investigation were ultraviolet absorption spectroscopy and constant-field gel electrophoresis (CFGE). The results showed that ultrasound decreased the hydrogen-bonded nucleotides. The ultrasound sensitivity was greater for the A-T base pairs than for the G-C base pairs. It was also shown that high power ultrasound damaged the nitrogen bases. The loss of the double helical structure by the ultrasound was mainly due to the separation of certain sections of the DNA by single strand breaks (SSB) and also to a small extent due to base damage. The size of the DNA fragments decreased as the ultrasound power increased and finally a plateau effect was seen at the highest powers. Key words: DNA damage, ultrasound, spectroscopy.

The extreme radiosensitivity of the squamous cell carcinoma SKX is due to a defect in double-strand break repair

Purpose Squamous cell carcinomas (SCCs) are characterized by moderate radiosensitivity. We have established the human head & neck SCC cell line SKX, which shows an exceptionally high radiosensitivity. It was the aim of this study to understand the underlying mechanisms. Materials & methods Experiments were performed with SKX and FaDu, the latter taken as a control of moderate radiosensitivity. Cell lines were grown as xenografts as well as cell cultures. For xenografts, radiosensitivity was determined via local tumour control assay, and for cell cultures using colony assay. For cell cultures, apoptosis was determined by Annexin V staining and G1-arrest by BrdU labelling. Double-strand breaks (DSBs) were detected by both constant-field gel electrophoresis (CFGE) and γH2AX-foci technique; DSB rejoining was also assessed by in vitro rejoining assay; chromosomal damage was determined by G01-assay. Results Compared to FaDu, SKX cells are extremely radiosensitive as found for both xenografts (TCD 50 for 10 fractions 46.0 Gy [95% C.I.: 39; 54 Gy] vs. 18.9 Gy [95% C.I.: 13; 25 Gy]) and cell cultures (D 0.01; 7.1 vs. 3.5 Gy). Both cell lines showed neither radiation-induced apoptosis nor radiation-induced permanent G1-arrest. For DSBs, there was no difference in the induction but for repair with SKX cells showing a higher level of both, slowly repaired DSBs and residual DSBs. The in vitro DSB repair assay revealed that SKX cells are defective in nonhomologous endjoining (NHEJ), and that more than 40% of DSBs are rejoined by single-strand annealing (SSA). SKX cells also depicted a two-fold higher number of lethal chromosomal aberrations when compared to FaDu cells. Conclusions The extreme radiosensitivity of the SCC SKX seen both in vivo and in vitro can be ascribed to a reduced DNA double-strand break repair, resulting from a defect in NHEJ. This defect might be due to preferred usage of other pathways, such as SSA, which prevents efficient endjoining.

Sulforaphane induces DNA double strand breaks predominantly repaired by homologous recombination pathway in human cancer cells

Cytotoxicity and DNA double strand breaks (DSBs) were studied in HeLa cells treated with sulforaphane (SFN), a well-known chemo-preventive agent. Cell survival was impaired by SFN in a concentration and treatment time-dependent manner. Both constant field gel electrophoresis (CFGE) and γ-H2AX assay unambiguously indicated formation of DSBs by SFN, reflecting the cell survival data. These DSBs were predominantly processed by homologous recombination repair (HRR), judging from the SFN concentration-dependent manner of Rad51 foci formation. On the other hand, the phosphorylation of DNA-PKcs, a key non-homologous end joining (NHEJ) protein, was not observed by SFN treatment, suggesting that NHEJ may not be involved in DSBs induced by this chemical. G2/M arrest by SFN, a typical response for cells exposed to ionizing radiation was also observed. Our new data indicate the clear induction of DSBs by SFN and a useful anti-tumor aspect of SFN through the induction of DNA DSBs.

Down regulation of BRCA2 causes radio‐sensitization of human tumor cells in vitro and in vivo

In order to study the role of BRCA2 protein in homologous recombination repair and radio-sensitization, we utilized RNA interference strategy in vitro and in vivo with human tumor cells. HeLa cells transfected with small-interfering BRCA2 NA (BRCA2 siRNA) (Qiagen) as well as negative-control siRNA for 48 h were irradiated, and several critical end points were examined. The radiation cell survival level was significantly reduced in HeLa cells with BRCA2 siRNA when compared with mock- or negative-control siRNA transfected cells. DNA double strand break repair as measured by constant field gel-electrophoresis showed a clear inhibition in cells with BRCA2 siRNA, while little inhibition was observed in cells with negative control siRNA. Our immuno-staining experiments revealed a significant delay in Rad51 foci formation in cells with BRCA2 siRNA when compared with the control populations. However, none of the non-homologous end joining proteins nor the phosphorylation of DNA-dependent protein kinase catalytic subunit was affected in cells transfected with BRCA2 siRNA. In addition, the combined treatment with radiation and BRCA2 siRNA in xenograft model with HeLa cells showed an efficient inhibition of in vivo tumor growth. Our results demonstrate down-regulation of BRCA2 leads to radio-sensitization mainly through the inhibition of homologous recombination repair type double-strand break repair; a possibility of using BRCA2 siRNA as an effective radiosensitizer in tumor radiotherapy may arise.

DNA Double-Strand Break Induction and Repair in Irradiated Lymphoblastoid, Fibroblast Cell Lines and White Blood Cells from ATM, NBS and Radiosensitive Patients

DNA double-strand breaks (dsbs) in lymphoblastoid cell lines (LCLs), fibroblasts and white blood cells from healthy donors, cancer patients with and without late effects of grade 3-4 (RTOG) as well as donors with known radiosensitivity syndromes were examined with the aim to detect dsb repair ability as a marker for radiosensitivity. LCLs from six healthy donors, seven patients with a heterozygous or homozygous genotype for ataxia-telangiectasia (ATM) and Nijmegen breakage syndrome (NBS), two patients with a late toxicity of grade 3-4 (RTOG), and one cell line with a ligase IV-/- status and its parental cell line were examined. Furthermore, fibroblasts from patients with ATM, NBS, two healthy control individuals, and leukocytes from 16 healthy and 22 cancer patients including seven patients with clinical hypersensitivity grade 3 (RTOG) were examined. Cells were irradiated in vitro with 0-150 Gy. Initial damage as well as remaining damage after 8 and 24 h were measured using constant field gel electrophoresis. In contrast to cells derived from patients homozygous for NBS, impaired dsb repair ability could be detected both in fibroblast and lymphoblastoid cells from ATM and ligase IV-/- patients. The dsb repair ability of all 38 leukocyte cell lines (patients with grade 3-4 late effects and controls) was similar, whereas the initial damage among healthy donors was less. Despite showing a clinically elevated radiosensitivity after irradiation, the DNA repair of the patients with clinical hypersensitivity grade 3 (RTOG) appeared to be normal. Other mechanisms such as mutations, altered cell cycle or defective apoptosis could play a critical role toward determining radiosensitivity.

Induction of DNA double-strand breaks by zeocin in Chlamydomonas reinhardtii and the role of increased DNA double-strand breaks rejoining in the formation of an adaptive response

This study aimed to test the potential of the radiomimetic chemical zeocin to induce DNA double-strand breaks (DSB) and "adaptive response" (AR) in Chlamydomonas reinhardtii strain CW15 as a model system. The AR was measured as cell survival using a micro-colony assay, and by changes in rejoining of DSB DNA. The level of induced DSB was measured by constant field gel electrophoresis based on incorporation of cells into agarose blocks before cell lysis. This avoids the risk of accidental induction of DSB during the manipulation procedures. Our results showed that zeocin could induce DSB in C. reinhardtii strain CW15 in a linear dose-response fashion up to 100 microg ml(-1) which marked the beginning of a plateau. The level of DSB induced by 100 microg ml(-1) zeocin was similar to that induced by 250 Gy of gamma-ray irradiation. It was also found that, similar to gamma rays, zeocin could induce AR measured as DSB in C. reinhardtii CW15 and this AR involved acceleration of the rate of DSB rejoining, too. To our knowledge, this is the first demonstration that zeocin could induce AR in some low eukaryotes such as C. reinhardtii.