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DNA replication is an essential process for cell division and as such it is a process that is directly targeted by several anticancer drugs. CDC7 plays an essential role in the activation of replication origins and has recently been proposed as a novel target for drug discovery. The MCM DNA helicase complex (MCM2-7) is a key target of the CDC7 kinase, and MCM phosphorylation status at specific sites is a reliable biomarker of CDC7 cellular activity. In this work we describe a cell-based assay that utilizes the "In Cell Western Technique'' (ICW) to identify compounds that affect cellular CDC7 activity. By screening a library of approved drugs and kinase inhibitors we found several compounds that can affect CDC7-dependent phosphorylation of MCM2 in HeLa cells. Among these, Mitoxantrone, a topoisomerase inhibitor, and Ryuvidine, previously described as a CDK4 inhibitor, cause a reduction in phosphorylated MCM2 levels and a sudden blockade of DNA synthesis that is accompanied by an ATM-dependent checkpoint response. This study sheds light on the previously observed cytotoxity of Ryuvidine, strongly suggesting that it is related to its effect of causing DNA damage.

Plants play important roles in human life not only as suppliers of oxygen but also as a fundamental resource to sustain the human race on this earthly plane. Plants also play a major role in our nutrition by converting energy from the sun during photosynthesis. In addition, plants have been used extensively in traditional medicine since time immemorial. Information in the biomedical literature has indicated that many natural herbs have been investigated for their efficacy against lethal irradiation. Pharmacological studies by various groups of investigators have shown that natural herbs possess significant radioprotective activity. In view of the immense medicinal importance of natural product based radioprotective agents, this review aims at compiling all currently available information on radioprotective agents from medicinal plants and herbs, especially the evaluation methods and mechanisms of action. In this review we particularly emphasize on ethnomedicinal uses, botany, phytochemistry, mechanisms of action and toxicology. We also describe modern techniques for evaluating herbal samples as radioprotective agents. The usage of herbal remedies for combating lethal irradiation is a green antiirradiation approach for the betterment of human beings without high cost, side effects and toxicity.

Hadrontherapy is a form of external radiation therapy, which uses beams of charged particles such as carbon ions. Compared to conventional radiotherapy with photons, the main advantage of carbon ion therapy is the precise dose localization along with an increased biological effectiveness. The first results obtained from prostate cancer patients treated with carbon ion therapy showed good local tumor control and survival rates. In view of this advanced treatment modality we investigated the effects of irradiation with different beam qualities on gene expression changes in the PC3 prostate adenocarcinoma cell line. For this purpose, PC3 cells were irradiated with various doses (0.0, 0.5 and 2.0 Gy) of carbon ions (LET=33.7 keV/μm) at the beam of the Grand Accélérateur National d'Ions Lourds (Caen, France). Comparative experiments with X-rays were performed at the Belgian Nuclear Research Centre. Genome-wide gene expression was analyzed using microarrays. Our results show a downregulation in many genes involved in cell cycle and cell organization processes after 2.0 Gy irradiation. This effect was more pronounced after carbon ion irradiation compared with X-rays. Furthermore, we found a significant downregulation of many genes related to cell motility. Several of these changes were confirmed using qPCR. In addition, recurrence-free survival analysis of prostate cancer patients based on one of these motility genes (FN1) revealed that patients with low expression levels had a prolonged recurrence-free survival time, indicating that this gene may be a potential prognostic biomarker for prostate cancer. Understanding how different radiation qualities affect the cellular behavior of prostate cancer cells is important to improve the clinical outcome of cancer radiation therapy.

We investigate the gas-phase metallicity and Lyman Continuum (LyC) escape fraction of a strongly gravitationally lensed, extreme emission-line galaxy at z=3.417, J1000+0221S, recently discovered by the CANDELS team. We derive ionization and metallicity sensitive emission-line ratios from H+K band LBT/LUCI medium resolution spectroscopy. J1000+0221S shows high ionization conditions, as evidenced by its enhanced [OIII]/[OII] and [OIII]/Hbeta ratios. Consistently, strong-line methods based on the available line ratios suggest that J1000+0221S is an extremely metal-poor galaxy, with a metallicity of 12+log(O/H) < 7.44 (< 5% solar), placing it among the most metal-poor star-forming galaxies at z > 3 discovered so far. In combination with its low stellar mass (2x10^8 Msun) and high star formation rate (5 Msun/yr), the metallicity of J1000+0221S is consistent with the extrapolation to low masses of the mass-metallicity relation traced by Lyman-break galaxies at z > 3, but it is 0.55 dex lower than predicted by the fundamental metallicity relation at z < 2.5. These observations suggest the picture of a rapidly growing galaxy, possibly fed by the massive accretion of pristine gas. Additionally, deep LBT/LBC in the UGR bands are used to derive a limit to the LyC escape fraction, thus allowing us to explore for the first time the regime of sub-L* galaxies at z > 3. We find a 1sigma upper limit to the escape fraction of 23%, which adds a new observational constraint to recent theoretical models predicting that sub-L* galaxies at high-z have high escape fractions and thus are the responsible for the reioization of the Universe., 5 pages, 3 figures and 1 table. Accepted for publication in ApJ Letters

Aims. We investigate the effects of ionising photons on accretion and stellar mass growth in a young star forming region, using a Monte Carlo radiation transfer code coupled to a smoothed particle hydrodynamics (SPH) simulation. Methods. We introduce the framework with which we correct stellar cluster masses for the effects of photoionising (PI) feedback and compare to the results of a full ionisation hydrodynamics code. Results. We present results of our simulations of star formation in the spiral arm of a disk galaxy, including the effects of photoionising radiation from high mass stars. We find that PI feedback reduces the total mass accreted onto stellar clusters by approximately 23 per cent over the course of the simulation and reduces the number of high mass clusters, as well as the maximum mass attained by a stellar cluster. Mean star formation rates (SFRs) drop from 0.042 solar masses per year in our control run to 0.032 solar masses per year after the inclusion of PI feedback with a final instantaneous SFR reduction of 62 per cent. The overall cluster mass distribution appears to be affected little by PI feedback. Conclusions. We compare our results to the observed extra-galactic Schmidt-Kennicutt relation and the observed properties of local star forming regions in the Milky Way and find that internal photoionising (PI) feedback is unlikely to reduce star formation rates by more than a factor of approximately 2 and thus may play only a minor role in regulating star formation., Comment: 10 pages, 11 figures, A&A, in press

Purpose: To provide an update on the recent knowledge about the molecular mechanisms of thermal radiosensitization and its possible relevance to thermoradiotherapy. Summary: Hyperthermia is probably the most potent cellular radiosensitizer known to date. Heat interacts with radiation and potentiates the cellular action of radiation by interfering with the cells' capability to deal with radiation-induced DNA damage. For ionizing irradiation, heat inhibits the repair of all types of DNA damage. Genetic and biochemical data suggest that the main pathways for DNA double-strand break (DSB) rejoining, non-homologous end-joining and homologous recombination, are not the likely primary targets for heat-induced radiosensitization. Rather, heat is suggested to affect primarily the religation step of base excision repair. Subsequently additional DSB arise during the DNA, repair process in irradiated and heated cells and these additional DSB are all repaired with slow kinetics, the repair of which is highly error prone. Both mis- and non-rejoined DSB lead to an elevated number of lethal chromosome aberrations, finally causing additional cell killing. Heal-induced inhibition of DNA repair is considered riot to result from altered signalling or enzyme inactivation but rather from alterations in higher-order chromatin structure. Although, the detailed mechanisms are not yet known, a substantial body of indirect and correlative data suggests that heat-induced protein aggregation at the level of attachment of looped DNA to the nuclear matrix impairs the accessibility of the damaged DNA for the repair machinery or impairs the processivity of the repair machinery itself. Conclusion: Since recent phase III clinical trials have shown significant benefit of adding hyperthermia to radiotherapy regimens for a number of malignancies, it will become more important again to determine the molecular effects underlying this success. Such information could eventually also improve treatment quality in terms of patient selection, improved sequencing of the heat and radiation treatments, the number of heat treatments, and multimodality treatments (i.e. thermochemoradiotherapy).

Glioblastoma (GBM) is a devastating cancer with a median survival of around 15 months. Significant advances in treatment have not been achieved yet, even with a host of new therapeutics under investigation. Therefore, the quest for a cure for GBM remains as intense as ever. Of particular interest for GBM therapy is the selective induction of apoptosis using the pro-apoptotic tumour necrosis factor-related apoptosis-inducing ligand (TRAIL). TRAIL signals apoptosis via its two agonistic receptors TRAIL-R1 and TRAIL-R2. TRAIL is normally present as homotrimeric transmembrane protein, but can also be processed into a soluble trimeric form (sTRAIL). Recombinant sTRAIL has strong tumouricidal activity towards GBM cells, with no or minimal toxicity towards normal human cells. Unfortunately, GBM is a very heterogeneous tumour, with multiple genetically aberrant clones within one tumour. Consequently, any single agent therapy is likely to be not effective enough. However, the anti-GBM activity of TRAIL can be synergistically enhanced by a variety of conventional and novel targeted therapies, making TRAIL an ideal candidate for combinatorial strategies. Here we will, after briefly detailing the biology of TRAIL/TRAIL receptor signalling, focus on the promises and pitfalls of recombinant TRAIL as a therapeutic agent alone and in combinatorial therapeutic approaches for GBM.

Glioblastoma (GBM) is a devastating cancer with a median survival of around 15 months. Significant advances in treatment have not been achieved yet, even with a host of new therapeutics under investigation. Therefore, the quest for a cure for GBM remains as intense as ever. Of particular interest for GBM therapy is the selective induction of apoptosis using the pro-apoptotic tumour necrosis factor-related apoptosis-inducing ligand (TRAIL). TRAIL signals apoptosis via its two agonistic receptors TRAIL-R1 and TRAIL-R2. TRAIL is normally present as homotrimeric transmembrane protein, but can also be processed into a soluble trimeric form (sTRAIL). Recombinant sTRAIL has strong tumouricidal activity towards GBM cells, with no or minimal toxicity towards normal human cells. Unfortunately, GBM is a very heterogeneous tumour, with multiple genetically aberrant clones within one tumour. Consequently, any single agent therapy is likely to be not effective enough. However, the anti-GBM activity of TRAIL can be synergistically enhanced by a variety of conventional and novel targeted therapies, making TRAIL an ideal candidate for combinatorial strategies. Here we will, after briefly detailing the biology of TRAIL/TRAIL receptor signalling, focus on the promises and pitfalls of recombinant TRAIL as a therapeutic agent alone and in combinatorial therapeutic approaches for GBM.

The dependence on age of both the basal and the X-radiation-induced levels of apoptosis was examined in human peripheral blood mononuclear cells (PBMC). In the same samples, the base value and the extent of induced DNA single-strand breaks were determined, using a sensitive and fast microplate assay. PBMC were isolated from blood of donors of various age groups (20-30, 40-60 and > 70 years of age) and X-irradiated ex vivo using a 6 MV linear accelerator to give a total exposure of 4 Gy. The mean basal levels of apoptosis in PBMC from donors in the 40-60 year age group and the > 70 year age group were found to be only slightly higher (by 20-10%) compared to that of the 20-30 year age group, whereas the extent of DNA damage strongly and significantly (P < 0.01) increased with age by up to 2-fold. In contrast to the extent of induced DNA damage, which steadily increased in the course of ageing by up to 1.8-fold, there was only a transient increase in the level of induced apoptosis to 1.5-fold in PBMC from X-irradiated blood (4 Gy photons) from donors aged 40-60 followed by a decrease to 0.9-fold in PBMC from old donors (> 70), compared to age group 20-30. The results show that X-ray-induced apoptosis and DNA damage in PBMC are not correlated during ageing.

Purpose: To test whether induction of DNA damage is correlated with tumour-cell radiosensitivity. Materials and methods: Initial DNA damage caused by X-irradiation was measured in ten human tumour cell lines, which largely differed in radiosensitivity, using either the pulsed-field gel electrophoresis assay or the halo technique. Results: None of the parameters of DNA damage correlated with any parameter of cellular radiosensitivity. This was not only true when the analysis was performed on all data but also when the analysis was performed after separating the cell lines into radioresistant and sensitive groups. Even when differences in chromosome number, ploidy and cell cycle distribution were taken into account, no significant correlations were obtained. Conclusions: Contrary to previous suggestions, differences in the number of double-strand breaks induced or chromatin-related 'presentation' of DNA lesions, measured by pulsed-field gel electrophoresis or halo respectively, are generally not the dominant factors determining tumour-cell radiosensitivity.

The focus of the study is an intercomparison of laboratories' dose-assessment performances using the cytokinesis-block micronucleus (CBMN) assay as a diagnostic triage tool for individual radiation dose assessment. Homogenously X-irradiated (240 kVp, 1 Gy/min) blood samples for establishing calibration data (0.25-5 Gy) as well as blind samples (0.1-6.4 Gy) were sent to the participants. The CBMN assay was performed according to protocols individually established and varying among participating laboratories. The time taken to report dose estimates was documented for each laboratory. Additional information concerning laboratory organization/characteristics as well as assay performance was collected. The mean absolute difference (MAD) was calculated and radiation doses were merged into four triage categories reflecting clinical aspects to calculate accuracy, sensitivity and specificity. The earliest report time was 4 days after sample arrival. The CBMN dose estimates were reported with high accuracy (MAD values of 0.20-0.50 Gy at doses below 6.4 Gy for both manual and automated scoring procedures), but showed a limitation of the assay at the dose point of 6.4 Gy, which resulted in a clear dose underestimation in all cases. The MAD values (without 6.4 Gy) differed significantly (P = 0.03) between manual (0.25 Gy, SEM = 0.06, n = 4) or automated scoring procedures (0.37 Gy, SEM = 0.08, n = 5), but lowest MAD were equal (0.2 Gy) for both scoring procedures. Likewise, both scoring procedures led to the same allocation of dose estimates to triage categories of clinical significance (about 83% accuracy and up to 100% specificity).

Classical radiation biology research has centred on nuclear DNA as the main target of radiation-induced damage. Over the past two decades, this has been challenged by a significant amount of scientific evidence clearly showing radiation-induced cell signalling effects to have important roles in mediating overall radiobiological response. These effects, generally termed radiation-induced bystander effects (RIBEs) have challenged the traditional DNA targeted theory in radiation biology and highlighted an important role for cells not directly traversed by radiation. The multiplicity of experimental systems and exposure conditions in which RIBEs have been observed has hindered precise definitions of these effects. However, RIBEs have recently been classified for different relevant human radiation exposure scenarios in an attempt to clarify their role in vivo. Despite significant research efforts in this area, there is little direct evidence for their role in clinically relevant exposure scenarios. In this review, we explore the clinical relevance of RIBEs from classical experimental approaches through to novel models that have been used to further determine their potential implications in the clinic.

Centrosomes, the principal microtubule-organizing centers of animal somatic cells, consist of two centrioles embedded in the pericentriolar material (PCM). Pericentrin is a large PCM protein that is required for normal PCM assembly. Mutations in PCNT cause primordial dwarfism. Pericentrin has also been implicated in the control of DNA damage responses. To test how pericentrin is involved in cell cycle control after genotoxic stress, we disrupted the Pcnt locus in chicken DT40 cells. Pericentrin-deficient cells proceeded through mitosis more slowly, with a high level of monopolar spindles, and were more sensitive to spindle poisons than controls. Centriole structures appeared normal by light and electron microscopy, but the PCM did not recruit gamma-tubulin efficiently. Cell cycle delays after ionizing radiation (IR) treatment were normal in pericentrin-deficient cells. However, pericentrin disruption in Mcph1(-/-) cells abrogated centrosome hyperamplification after IR. We conclude that pericentrin controls genomic stability by both ensuring appropriate mitotic spindle activity and centrosome regulation.

A personal dosimetry service that evaluates the occupational doses for external and internal radiation of the radiation workers is one of the main components of radiation protection programme. The education and training (E&T) activities in this field are basic aspect of the optimization of all exposures to radiation. The E&T activities in the field of occupational radiation protection at the national and international level are mainly interested and implemented by Ankara Nuclear Research and Training Center (ANRTC) in Turkey. This study describes the Turkish experience in education and training of the staff of dosimetry services, postgraduate students and medical physics experts. The first individual monitoring training course realized in 2012 in Turkey. The aim of this study is to provide a structured description of postgraduate courses that addressed to qualified experts and medical physics experts, and the modules mainly dedicated to individual monitoring.

Objective: Ionizing radiation interacts with biological systems to induce excessive fluxes of free radicals that attack various cellular components. Melatonin has been shown to be a direct free radical scavenger and indirect antioxidant via its stimulatory actions on the antioxidant system.The aim of this study was to evaluate the antioxidant role of melatonin against radiation-induced oxidative injury to the rat liver after whole body irradiation. Materials and Methods: In this experimental study,thirty-two rats were divided into four groups. Group 1 was the control group, group 2 only received melatonin (30 mg/kg on the first day and 30 mg/kg on the following days), group 3 only received whole body gamma irradiation of 10 Gy, and group 4 received 30 mg/kg melatonin 30 minutes prior to radiation plus whole body irradiation of 10 Gy plus 30 mg/kg melatonin daily through intraperitoneal (IP) injection for three days after irradiation. Three days after irradiation, all rats were sacrificed and their livers were excised to measure the biochemical parameters malondialdehyde (MDA) and glutathione (GSH). Each data point represents mean ± standard error on the mean (SEM) of at least eight animals per group. A one-way analysis of variance (ANOVA) was performed to compare different groups, followed by Tukey’s multiple comparison tests (p

Three human tumour cell lines (HX142, RT112 and MGH-U1) with different radiosensitivities were tested for differences in the rate and/or extent of DNA unwinding in alkali as well as for differences in the induction of DNA double strand breaks by means of the pulsed field gel electrophoresis, after X-irradiation. Unlike that which has been found using the non-denaturing filter elution technique (NDE, McMillan et al., 1990), no differences in initial DNA damage (the extent of alkaline unwinding and the induction of double strand breaks) were found for the three cell lines. These data suggest that rather than a different number of DNA lesions per Da per Gy between these cell lines, structural differences in chromatin structure (related to radiosensitivity) might impair the detectability of lesions in some assays like the NDE. The nature of such structure differences remains unclear. However, the differences did not affect alkaline unwinding profiles, as all three cell lines showed identical rates of DNA unwinding after exposure to X-rays. Furthermore, the three cell lines did not show significant differences in the kinetics of DNA strand break rejoining nor in the amounts of damage remaining after 24 h repair. The results obtained in this study, together with other findings, suggest that the three cell lines may differ in their 'presentation' of DNA damage.

The interaction between moderate-dose radiation and cisplatin or carboplatin was studied in a cisplatin-sensitive (GLC(4)) and -resistant (GLC(4)-CDDP) human small-cell lung cancer cell line. Cellular toxicity was analysed under oxic conditions with the microculture tetrazolium assay. For the platinum and radiation toxicity with the clinically relevant dose ranges applied, this assay was used to obtain information on cell survival after the treatments. Apart from effects on cell survival effects on DNA were also investigated. Configurational DNA changes could be induced by platinum drugs and thereby these drugs might change the frequency of DNA double-strand breaks (dsbs). DNA fragmentation assayed with the clamped homogeneous electric field (CHEF) technique was used as a measure for dsbs in DNA. The radiosensitising effect of the platinum drugs was expressed as enhancement ratio (ER) calculated directly from survival levels of the initial slope of the curve. The highest ER for cisplatin in GLC(4) was 1.39 and in GLC(4)-CDDP 1.38. These were all at 75% cell survival. Carboplatin showed increased enhancement with prolonged incubation up to 1.21 in GLC(4) and was equally effective as cisplatin in GLC(4)-CDDP. According to isobologram analysis, prolonged incubation with both platinum drugs showed at least additivity with radiation for both cell lines at clinically achievable doses. GLC(4)-CDDP showed cross-resistance to radiation. The radiosensitising capacity of both lung cancer cell lines was not dependent on their platinum sensitivity. The formation of dsbs in DNA directly after radiation was not influenced by pretreatment of either drug in the sensitive or in the resistant cell line. Drug treatment resulted in decreased DNA extractability in control as well as in irradiated cells. Modest enhancement ratio for radiosensitisation by platinum drugs cannot be explained on the level of dsb formation in DNA in both cell lines. Interaction of radiation with the clinically less toxic carboplatin can be improved by prolonged low-dose carboplatin exposure before irradiation and is as potent as cisplatin in the resistant lung cancer cell line. This suggests an advantage in combining radiation and carboplatin in lung cancer patients.

Hyperthermia specifically inhibits the repair of UV-induced DNA photolesions in transcriptionally active genes, To define more precisely which mechanisms underlie the heat-induced inhibition of repair of active genes, removal of cyclobutane pyrimidine dimers (CPDs) was studied in human fibroblasts with different repair capacities and different transcriptional status of the adenosine deaminase gene, i.e. normal human cells, human cells carrying an inactive copy of the adenosine deaminase gene and xeroderma pigmentosum complementation group C fibroblasts, The results indicate that repair of active genes is impaired by inhibition of two repair pathways: (i) a global repair system involved in the repair of CPDs in potentially active genes; and (ii) the transcription-coupled repair pathway responsible for the accelerated repair of the transcribed strand. Since X-ray-induced DNA damage is also preferentially removed from the transcribed strand of active genes, selective inhibition of repair of radiation-induced DNA damage in active genes may play a key role in radiosensitization due to hyperthermia.

Multidrug-resistant (MDR) cell lines often have a compound phenotype, combining reduced drug accumulation with a decrease in topoisomerase II. We have analysed alterations in topoisomerase II in MDR derivatives of the human lung cancer cell line SW-1573. Selection with doxorubicin frequently resulted in reduced topo II alpha mRNA and protein levels, whereas clones selected with vincristine showed normal levels of topo II alpha. No alterations of topo II beta levels were detected. To determine the contribution of topo II alterations to drug resistance, topo II activity was analysed by the determination of DNA breaks induced by the topo II-inhibiting drug 4'-(9-acridinylamino)methane-sulphon-m-anisidide (m-AMSA) in living cells, as m-AMSA is not affected by the drug efflux mechanism in the SW-1573 cells. The number of m-AMSA-induced DNA breaks correlated well (r = 0.96) with in vitro m-AMSA sensitivity. Drug sensitivity, however, did not always correlate with reduced topo II mRNA or protein levels. In one of the five doxorubicin-selected clones m-AMSA resistance and a reduction in m-AMSA-induced DNA breaks were found in the absence of reduced topo II protein levels. Therefore, we assume that post-translational modifications of topo II also contribute to drug resistance in SW-1573 cells. These results suggest that methods that detect quantitative as well as qualitative alterations of topo II should be used to predict the responsiveness of tumours to cytotoxic agents. The assay we used, which measures DNA breaks as an end point of topo II activity, could be a good candidate.

The regeneration of the hematopoietic system following total body irradiation is supported by host-derived mesenchymal stromal cells (MSCs) within the bone marrow. The mechanisms used by MSCs to survive radiation doses that are lethal to the hematopoietic system are poorly understood. The DNA damage response (DDR) represents a cohort of signaling pathways that enable cells to execute biological responses to genotoxic stress. Here, we examine the role of the DDR in mediating the resistance of MSCs to ionizing radiation (IR) treatment using two authentic clonal mouse MSC lines, MS5 and ST2, and primary bulk mouse MSCs. We show that multiple DDR mechanisms contribute to the radio-resistance of MSCs: robust DDR activation via rapid γ-H2AX formation, activation of effective S and G2/M DNA damage checkpoints, and efficient repair of IR-induced DNA double-strand breaks. We show that MSCs are intrinsically programmed to maximize survival following IR treatment by expressing high levels of key DDR proteins including ATM, Chk2, and DNA Ligase IV; high levels of the anti-apoptotic, Bcl-2 and Bcl-XL; and low levels of the pro-apoptotic, Bim and Puma. As a result, we demonstrate that irradiated mouse MSCs withstand IR-induced genotoxic stress, continue to proliferate, and retain their capacity to differentiate long-term along mesenchymal-derived lineages. We have shown, for the first time, that the DDR plays key roles in mediating the radioresistance of mouse MSCs which may have important implications for the study and application of MSCs in allogeneic bone marrow transplantation, graft-versus-host disease, and cancer treatment.

Treatment of advanced-stage cervical cancers with (chemo)radiation causes cytotoxicity through induction of high levels of DNA damage. Tumour cells respond to DNA damage by activation of the DNA damage response (DDR), which induces DNA repair and may counteract chemoradiation efficacy. Here, we investigated DDR components as potential therapeutic targets and verified the predictive and prognostic value of DDR activation in patients with cervical cancer treated with (chemo)radiation. In a panel of cervical cancer cell lines, inactivation of ataxia telangiectasia mutated (ATM) or its substrate p53-binding protein-1 (53BP1) clearly gave rise to cell cycle defects in response to irradiation. Concordantly, clonogenic survival analysis revealed that ATM inhibition, but not 53BP1 depletion, strongly radiosensitised cervical cancer cells. In contrast, ATM inhibition did not radiosensitise non-transformed epithelial cells or non-transformed BJ fibroblasts. Interestingly, high levels of active ATM prior to irradiation were related with increased radioresistance. To test whether active ATM in tumours prior to treatment also resulted in resistance to therapy, immunohistochemistry was performed on tumour material of patients with advanced-stage cervical cancer (n = 375) treated with (chemo)radiation. High levels of phosphorylated (p-)ATM [p = 0.006, hazard ratio (HR) = 1.817] were related to poor locoregional disease-free survival. Furthermore, high levels of p-ATM predicted shorter disease-specific survival (p = 0.038, HR = 1.418). The presence of phosphorylated 53BP1 was associated with p-ATM (p = 0.001, odds ratio = 2.206) but was not related to any clinicopathological features or survival. In conclusion, both our in vitro and patient-related findings indicate a protective role for ATM in response to (chemo)radiation in cervical cancer and point at ATM inhibition as a possible means to improve the efficacy of (chemo)radiation.

Crohn's disease is a chronic, disabling disease that, over time, can lead to irreversible bowel damage. MRI can be used to diagnose and assess the activity, severity and complications of Crohn's disease; however, the role of MRI in therapeutic monitoring of changes in disease-related intestinal damage is still to be defined. Objective, validated MRI-based scores have been developed to assess the activity of Crohn's disease; these indices are based on the extent and severity of intestinal inflammation, postoperative recurrence and perianal disease. MRI is accurate, safe, reproducible and can allow repeated evaluations of patients without radiation exposure. Evidence that MRI might be valuable in the therapeutic monitoring of patients with Crohn's disease is increasing and, in combination with endoscopy and surgical history, this imaging technique could enable clinicians to assess Crohn's-disease-related intestinal damage. MRI could, therefore, have a crucial role in a future 'damage-driven' treatment paradigm--in which imaging is used to monitor intestinal damage and medication use is targeted to prevent the accumulation of further damage. This damage-driven therapeutic approach could potentially change the course of Crohn's disease.

The moss Physcomitrella patens is unique among plant models for the high frequency with which targeted transgene insertion occurs via homologous recombination. Transgene integration is believed to utilize existing machinery for the detection and repair of DNA double-strand breaks (DSBs). We undertook targeted knockout of the Physcomitrella genes encoding components of the principal sensor of DNA DSBs, the MRN complex. Loss of function of PpMRE11 or PpRAD50 strongly and specifically inhibited gene targeting, whilst rates of untargeted transgene integration were relatively unaffected. In contrast, disruption of the PpNBS1 gene retained the wild-type capacity to integrate transforming DNA efficiently at homologous loci. Analysis of the kinetics of DNA-DSB repair in wild-type and mutant plants by single-nucleus agarose gel electrophoresis revealed that bleomycin-induced fragmentation of genomic DNA was repaired at approximately equal rates in each genotype, although both the Ppmre11 and Pprad50 mutants exhibited severely restricted growth and development and enhanced sensitivity to UV-B and bleomycin-induced DNA damage, compared with wild-type and Ppnbs1 plants. This implies that while extensive DNA repair can occur in the absence of a functional MRN complex; this is unsupervised in nature and results in the accumulation of deleterious mutations incompatible with normal growth and development.

Following previous work, we investigated in more detail the relationship between apoptosis and delayed luminescence (DL) in human leukemia Jurkat T cells under a wide variety of treatments. We used menadione and hydrogen peroxide to induce oxidative stress and two flavonoids, quercetin, and epigallocatechin gallate, applied alone or in combination with menadione or H2O2. 62 MeV proton beams were used to irradiate cells under a uniform dose of 2 or 10 Gy, respectively. We assessed apoptosis, cell cycle distributions, and DL. Menadione, H2O2and quercetin were potent inducers of apoptosis and DL inhibitors. Quercetin decreased clonogenic survival and the NAD(P)H level in a dose-dependent manner. Proton irradiation with 2 Gy but not 10 Gy increased the apoptotic rate. However, both doses induced a substantial G2/M arrest. Quercetin reduced apoptosis and prolonged the G2/M arrest induced by radiation. DL spectroscopy indicated that proton irradiation disrupted the electron flow within Complex I of the mitochondrial respiratory chain, thus explaining the massive necrosis induced by 10 Gy of protons and also suggested an equivalent action of menadione and quercetin at the level of the Fe/S center N2, which may be mediated by their binding to a common site within Complex I, probably the rotenone-binding site.

Background Many prostate cancers demonstrate an increased expression of growth factor receptors such as vascular endothelial growth factor receptor (VEGFR) and platelet derived growth factor receptor (PDGFR) which have been correlated with increased resistance to radiotherapy and poor prognosis in other tumors. Therefore, response to radiation could potentially be improved by using inhibitors of these abnormally activated pathways. We have investigated the radiosensitizing effects of sunitinib, a potent, multi-tyrosine kinase inhibitor of the VEGFR and PDGFR receptors, on human prostate cancer cells. Methods The radiosensitizing effects of sunitinib were assessed on human prostate cancer cell lines DU145, PC3 and LNCaP by clonogenic assay. Sunitinib’s ability to inhibit the activities of its key targets was determined by immunoblot analysis. The radiosensitizing effects of sunitinib in vivo were tested on human tumor xenografts growing in nude mice where response was assessed by tumor growth delay. Results Clonogenic survival curve assays for both DU145 and PC3 cells showed that the surviving fraction at 2 Gy was reduced from 0.70 and 0.52 in controls to 0.44 and 0.38, respectively, by a 24 hr pretreatment with 100 nM sunitinib. LNCaP cells were not radiosensitized by sunitinib. Dose dependent decreases in VEGFR and PDGFR activation were also observed following sunitinib in both DU145 and PC3 cells. We assessed the ability of sunitinib to radiosensitize PC3 xenograft tumors growing in the hind limb of nude mice. Sunitinib given concurrently with radiation did not prolong tumor growth delay. However, when animals were treated with sunitinib commencing the day after fractionated radiation was complete, tumor growth delay was enhanced compared to radiation alone. Conclusions We conclude, based on the in vivo results, that sunitinib and radiation do not interact directly to radiosensitize the PC3 tumor cells in vivo as they did in vitro. The fact that tumor growth delay was enhanced when sunitinib was given after radiotherapy was completed suggests that sunitinib may be acting on the irradiated tumor stroma and suppressing its ability to sustain regrowth of the irradiated tumor. Based on these preclinical findings, we suggest that the combination of sunitinib and radiation for the treatment of prostate cancer deserves further development.

Members of the early growth response (EGR) family of transcription factors play diverse functions in response to many cellular stimuli, including growth, stress, and inflammation. Egr3 has gone relatively unstudied, but here through use of the SPECS (Strategic Partners for the Evaluation of Predictive Signatures of Prostate Cancer) Affymetrix whole genome gene expression database we report that Egr3 mRNA is significantly over-expressed in prostate cancer compared to normal prostate tissue (5-fold). The Human Protein Atlas (http://www.proteinatlas.org), a database of tissue microarrays labeled with antibodies against over 11,000 human proteins, was utilized to quantify Egr3 protein expression in normal prostate and prostate cancer patients. In agreement with the SPECS data, we found that Egr3 protein is significantly increased in prostate cancer. The SPECS database has the benefit of extensive clinical follow up for the prostate cancer patients. Analysis of Egr3 mRNA expression in relation to the relapse status reveals that Egr3 mRNA expression is increased in tumor cells of non-relapsed samples (n = 63) compared to normal prostate cells, but is significantly lower in relapsed samples (n = 38) compared to non-relapse. The observations were confirmed using an independent data set. A list of genes correlating with this unique expression pattern was determined. These Egr3-correlated genes were enriched with Egr binding sites in their promoters. The gene list contains inflammatory genes such as IL-6, IL-8, IL1β and COX-2, which have extensive connections to prostate cancer. © 2013 Pio et al.

We have investigated the damage of synthetic plasmid pBR322 DNA in dilute aqueous solutions induced by fast carbon ions. The relative contribution of indirect damage and direct damage to the DNA itself is expected to vary with linear energy transfer along the ion track, with the direct damage contribution increasing towards the Bragg peak. Therefore, C-12 ions at the spread-out Bragg peak (dose averaged LETa = 189 +/- 15 keV/mu m) and in the plateau region of the Bragg curve (LET = 40 keV/mu m) were employed and the radical scavenger concentration in the plasmid solution was varied to quantify the indirect effect. In order to minimize the influence of C-12 break-up fragments, a relatively low initial energy of 90 MeV/nucleon was employed for the carbon ions. DNA damage has been quantified by subsequent electrophoresis on agarose gels. We find that strand breaks due to both indirect and direct effects are systematically higher in the plateau region as compared to the Bragg peak region with the difference being smallest at high scavenging capacities. In view of the fact that the relative biological effectiveness for many biological endpoints is maximum at the Bragg peak our findings imply that DNA damage at the Bragg peak is qualitatively most severe.

Background NEK1, the first mammalian ortholog of the fungal protein kinase never-in-mitosis A (NIMA), is involved early in the DNA damage sensing/repair pathway. A defect in DNA repair in NEK1-deficient cells is suggested by persistence of DNA double strand breaks after low dose ionizing radiation (IR). NEK1-deficient cells also fail to activate the checkpoint kinases CHK1 and CHK2, and fail to arrest properly at G1/S or G2/M-phase checkpoints after DNA damage. Results We show here that NEK1-deficient cells suffer major errors in mitotic chromosome segregation and cytokinesis, and become aneuploid. These NEK1-deficient cells transform, acquire the ability to grow in anchorage-independent conditions, and form tumors when injected into syngeneic mice. Genomic instability is also manifest in NEK1 +/- mice, which late in life develop lymphomas with a much higher incidence than wild type littermates. Conclusion NEK1 is required for the maintenance of genome stability by acting at multiple junctures, including control of chromosome stability.

Food-grade plasticized polyvinylidene chloride/polyvinyl chloride (PVDC/PVC) film (saran wrap) containing acetyl tributyl citrate (ATBC) plasticizer was used to wrap cod and herring fillets. The ratio of film surface to weight of food was ca. 89:1, in contrast to the generally agreed relationship of 6 dm(2) to 1 kg food (6:1). Wrapped fish samples were subjected to electron beam irradiation at doses equal to 5 and 10 kGy, stored at 4 A degrees C and analyzed for ATBC content at time intervals between 12 and 240 h of contact. Determination of the analyte was performed by ultrasonic-assisted solvent extraction followed by analysis on a gas chromatograph coupled with flame ionization detector. Final ATBC concentrations in cod fillets ranged from 11.1 to 12.8 mg/kg, while the corresponding values for herring samples were between 32.4 and 33.4 mg/kg. Data showed that e-beam radiation at pasteurizing doses did not significantly affect the copolymer's specific migration characteristics. On the contrary, fat content of the packaged fish fillets substantially affected the diffusion coefficient (D) values, as well as the extent to which migration of ATBC occurred. No violations of the tolerable daily intake (TDI) of 1.0 mg/kg body weight set by the EU for ATBC were found in the present study. However, food overwrapping or rewrapping with flexible films is often applied in household applications. Since in such cases unrealistically high plasticizer concentrations are determined experimentally, present specific migration limits (SML) should be redefined on a different basis. European Food Research and Technology

Background Radiotherapists are highly interested in optimizing doses especially for patients who tend to suffer from side effects of radiotherapy (RT). It seems to be helpful to identify radiosensitive individuals before RT. Thus we examined aberrations in FISH painted chromosomes in in vitro irradiated blood samples of a group of patients suffering from breast cancer. In parallel, a follow-up of side effects in these patients was registered and compared to detected chromosome aberrations. Methods Blood samples (taken before radiotherapy) were irradiated in vitro with 3 Gy X-rays and analysed by FISH-painting to obtain aberration frequencies of first cycle metaphases for each patient. Aberration frequencies were analysed statistically to identify individuals with an elevated or reduced radiation response. Clinical data of patients have been recorded in parallel to gain knowledge on acute side effects of radiotherapy. Results Eight patients with a significantly elevated or reduced aberration yield were identified by use of a t-test criterion. A comparison with clinical side effects revealed that among patients with elevated aberration yields one exhibited a higher degree of acute toxicity and two patients a premature onset of skin reaction already after a cumulative dose of only 10 Gy. A significant relationship existed between translocations in vitro and the time dependent occurrence of side effects of the skin during the therapy period. Conclusions The results suggest that translocations can be used as a test to identify individuals with a potentially elevated radiosensitivity.

How centrosomes amplify after DNA damage is unclear. Cell fusions demonstrated that only irradiated centrosomes duplicate when fused with untreated partners, suggesting a licensing signal that does not move from one centrosome to another. Our data indicate that centriole disengagement occurs after irradiation, suggesting this as the signal., DNA damage can induce centrosome overduplication in a manner that requires G2-to-M checkpoint function, suggesting that genotoxic stress can decouple the centrosome and chromosome cycles. How this happens is unclear. Using live-cell imaging of cells that express fluorescently tagged NEDD1/GCP-WD and proliferating cell nuclear antigen, we found that ionizing radiation (IR)-induced centrosome amplification can occur outside S phase. Analysis of synchronized populations showed that significantly more centrosome amplification occurred after irradiation of G2-enriched populations compared with G1-enriched or asynchronous cells, consistent with G2 phase centrosome amplification. Irradiated and control populations of G2 cells were then fused to test whether centrosome overduplication is allowed through a diffusible stimulatory signal, or the loss of a duplication-inhibiting signal. Irradiated G2/irradiated G2 cell fusions showed significantly higher centrosome amplification levels than irradiated G2/unirradiated G2 fusions. Chicken–human cell fusions demonstrated that centrosome amplification was limited to the irradiated partner. Our finding that only the irradiated centrosome can duplicate supports a model where a centrosome-autonomous inhibitory signal is lost upon irradiation of G2 cells. We observed centriole disengagement after irradiation. Although overexpression of dominant-negative securin did not affect IR-induced centrosome amplification, Plk1 inhibition reduced radiation-induced amplification. Together, our data support centriole disengagement as a licensing signal for DNA damage-induced centrosome amplification.

Biocompatible Au nanoparticles with surfaces modified by PEG (polyethylene glycol) were developed in view of possible applications for the enhancement of radiotherapy. Such nanoparticles exhibit preferential deposition at tumor sites due to the enhanced permeation and retention (EPR) effect. Here, we systematically studied their effects on EMT-6 and CT26 cell survival rates during irradiation for a dose up to 10 Gy with a commercial biological irradiator (E(average) = 73 keV), a Cu-Kalpha(1) x-ray source (8.048 keV), a monochromatized synchrotron source (6.5 keV), a radio-oncology linear accelerator (6 MeV) and a proton source (3 MeV). The percentage of surviving cells after irradiation was found to decrease by approximately 2-45% in the presence of PEG-Au nanoparticles ([Au] = 400, 500 or 1000 microM). The cell survival rates decreased as a function of the dose for all sources and nanoparticle concentrations. These results could open the way to more effective cancer irradiation therapies by using nanoparticles with optimized surface treatment. Difficulties in applying MTT assays were also brought to light, showing that this approach is not suitable for radiobiology.

In the present paper, analysis of solid cancer mortality and incidence risk after radiation exposure in the Techa River Cohort in the Southern Urals region of Russia is described. Residents along the Techa River received protracted exposure to ionizing radiation in the 1950s due to the releases of radioactive materials from the Mayak Production Association. The current follow-up through December 2003 includes individuals exposed on the Techa riverside within the Chelyabinsk and Kurgan oblasts using mortality data, and within the Chelyabinsk oblast using incidence data. The analysis was performed by means of the biologically based two-stage clonal expansion (TSCE) model and conventional excess relative risk models. For the mortality and incidence cohorts, central estimates of the excess relative risk per dose of 0.85 Gy(-1) (95% CI 0.36; 1.38) and 0.91 Gy(-1) (95% CI 0.35; 1.52) were found, respectively. For both the mortality and incidence cohorts, the best description of the radiation risk was achieved with the same TSCE model including a lifelong radiation effect on the promotion rate of initiated cells. An increase in the excess risk with attained age was observed, whereas no significant change of risk with age at exposure was seen. Direct comparison of the mortality and incidence cohorts showed that the excess relative risk estimates agreed very well in both cohorts, as did the excess absolute risk and the hazard after correction for the different background rates.

Crit. Rev. Food Sci. Nutr. ISI Document Delivery No.: 685EP Times Cited: 3 Cited Reference Count: 148 Guillard, V. Mauricio-Iglesias, M. Gontard, N. Commission of the European Communities [Q FP6-CT-2006-015710] This study has been carried out with the financial support from the Commission of the European Communities, Framework 6, Priority 5, "Food Quality and Safety," Integrated Project Novel Q FP6-CT-2006-015710. Although the project's information is considered accurate, no responsibility will be accepted for any subsequent use thereof. The European Community accepts no responsibility or liability whatsoever with regard to the presented material, and the work hereby presented does not anticipate the Commission's future policy in this area. Taylor & francis inc Philadelphia; International audience; Classical stabilization techniques (thermal treatments) usually involve food to be packed after being processed. On the contrary and increasingly, novel food processing methods, such as high pressure or microwaves, imply that both packaging and foodstuff undergo the stabilization treatment. Moreover, novel treatments (UV light, irradiation, ozone, cold plasma) are specifically used for disinfection and sterilization of the packaging material itself. Therefore, in the last several years a number of papers have focused on the effects of these new treatments on food-packaging interactions with a special emphasis on chemical migration and safety concerns. New packaging materials merged on the market with specific interest regarding the environment (i.e. bio-sourced materials) or mechanical and barrier properties (i.e. nanocomposites packaging materials). It is time to evaluate the knowledge about how these in-package food technologies affect food/packaging interactions, and especially for novel biodegradable and/or active materials. This article presents the effect of high pressure treatment, microwave heating, irradiation, UV-light, ozone and, cold plasma treatment on food/packaging interactions.

We present simulations of the 21-cm signal during the epoch of reionization. We focus on properly modeling the absorption regime in the presence of inhomogeneous Wouthuysen-Field effect and X-ray heating. We ran radiative transfer simulations for three bands in the source spectrum (Lyman, UV, and X-ray) to fully account for these processes. We find that the brightness temperature fluctuation of the 21 cm signal has an amplitude greater than 100 mK during the early reionization, up to 10 times greater than the typical amplitude of a few 10 mK obtained during the later emission phase. More importantly, we find that even a rather high contribution from QSO-like sources only damps the absorption regime without erasing it. Heating the IGM with X-ray takes time. Our results show that observations of the early reionization will probably benefit from a higher signal-to-noise value than during later stages. After analyzing the statistical properties of the signal (power spectrum and PDF) we find three diagnostics to constrain the level of X-ray, hence the nature of the first sources., Comment: 15 pages, 7 figures, 2 tables, accepted for publication in A&A. High resolution version available at http://homepage.sns.it/baek/quasar.pdf

The aim was to study the effect of electron-beam irradiation on the production of radiolysis products and sensory changes in experimental high-barrier packaging films composed of polyamide (PA), ethylene-vinyl alcohol (EVOH) and low-density polyethylene (LDPE). Films contained a middle buried layer of recycled LDPE, while films containing 100% virgin LDPE as the middle buried layer were taken as controls. Irradiation doses ranged between zero and 60 kGy. Generally, a large number of radiolysis products were produced during electron-beam irradiation, even at the lower absorbed doses of 5 and 10 kGy (approved doses for food 'cold pasteurization'). The quantity of radiolysis products increased with irradiation dose. There were no significant differences in radiolysis products identified between samples containing a recycled layer of LDPE and those containing virgin LDPE (all absorbed doses), indicating the 'functional barrier' properties of external virgin polymer layers. Sensory properties (mainly taste) of potable water were affected after contact with irradiated as low as 5 kGy packaging films. This effect increased with increasing irradiation dose. Food Additives and Contaminants Part a-Chemistry Analysis Control Exposure & Risk Assessment

The DNA replication process represents a source of DNA stress that causes potentially spontaneous genome damage. This effect might be strengthened by mutations in crucial replication factors, requiring the activation of DNA damage checkpoints to enable DNA repair before anaphase onset. Here, we demonstrate that depletion of the evolutionarily conserved minichromosome maintenance helicase-binding protein ETG1 of Arabidopsis thaliana resulted in a stringent late G2 cell cycle arrest. This arrest correlated with a partial loss of sister chromatid cohesion. The lack-of-cohesion phenotype was intensified in plants without functional CTF18, a replication fork factor needed for cohesion establishment. The synergistic effect of the etg1 and ctf18 mutants on sister chromatid cohesion strengthened the impact on plant growth of the replication stress caused by ETG1 deficiency because of inefficient DNA repair. We conclude that the ETG1 replication factor is required for efficient cohesion and that cohesion establishment is essential for proper development of plants suffering from endogenous DNA stress. Cohesion defects observed upon knockdown of its human counterpart suggest an equally important developmental role for the orthologous mammalian ETG1 protein., Author Summary DNA replication is a highly complex process and the source of potential DNA damage. It is of utmost importance that the damaged DNA is repaired before cells proceed through mitosis, because the genome holds all the information required for correct development. DNA replication results in two identical sister chromatids. A trick applied by cells to overcome damaged DNA is homologous recombination, using the undamaged copy of the sister chromatid as a template to repair the damaged one. This process is aided by keeping the two sister chromatids in close proximity after the replication process by the deposition of a molecular glue, called cohesin. In the present work, we identified the Arabidopsis thaliana ETG1 protein as a novel evolutionarily conserved replication factor that is needed for maintaining the sister chromatids physically aligned. In plants without ETG1, DNA damage builds up due to inefficient DNA repair. As a consequence, cell division is impaired with a huge impact on plant growth, highlighting the importance of cohesin for the correct development of eukaryotic organisms. Cohesion phenotypes observed upon the depletion of the orthologous human ETG1 protein indicate equally prominent roles for this particular factor during mammalian development.

Int J Epidemiol

We studied the DNA fragmentation induced in human fibroblasts by iron-ion beams of two different energies: 115 MeV/nucleon and 414 MeV/nucleon. Experimental data were obtained in the fragment size range 1-5700 kbp; Monte Carlo simulations were performed with the PARTRAC code; data analysis was also performed through the Generalized Broken Stick (GBS) model. The comparison between experimental and simulated data for the number of fragments produced in two different size ranges, 1-23 kbp and 23-5700 kbp, gives a satisfactory agreement for both radiation qualities. The Monte Carlo simulations also allow the counting of fragments outside the experimental range: The number of fragments smaller than 1 kbp is large for both beams, although with a strong difference between the two cases. As a consequence, we can compute different RBEs depending on the size range considered for the fragment counting. The PARTRAC evaluation takes into account fragments of all sizes, while the evaluation from the experimental data considers only the fragments in the range of 1-5700 kbp. When the PARTRAC evaluation is restricted to this range, the agreement between experimental and computed RBE values is again good. When fragments smaller than 1 kbp are also considered, the RBE increases considerably, since gamma rays produce a small number of such fragments. The analysis performed with the GBS model proved to be quite sensitive to showing, with a phenomenological single parameter, variations in double-strand break (DSB) correlation.

The expression of ENTPD1 (ecto-nucleoside triphosphate diphosphohydrolase) along the glomerular microvasculature of the kidney is downregulated in ischemic conditions, in contrast to E5NT (ecto-5'-nucleotidase), which may explain the increased tendency for intraglomerular microthrombus formation in vivo. It has been suggested that in ischemia, reactive oxygen species (ROS) affect glomerular ENTPD1, whereas E5NT seems less sensitive to oxidant stress. To test this hypothesis, a soluble ATP and ADP hydrolyzing enzyme solution (apyrase) [0.4 U/ml] or 5'-nucleotidase solution [0.33 U/ml] as well renal tissue were exposed to ROS, generated by gamma-irradiation in vitro. The enzymes diluted in distilled water or cryostat rat kidney sections were exposed to gamma-irradiation (0.037 Gy/s) for 0, 2, 5, 10, or 15 min, with or without supplementation of the ROS scavenger dimethylsulfoxide (DMSO). The enzyme activity of the samples was biochemically tested using standard methods, before and after irradiation. The reaction product of irradiated versus nonirradiated kidney sections was semiquantitatively evaluated after histochemical staining for either glomerular ENTPD1 or glomerular E5NT expression. The results show that the enzyme activity in samples of soluble apyrase was significantly decreased after irradiation. This effect was inhibited by DMSO. In contrast, 5'-nucleotidase activity showed only a limited decline of the activity curve after irradiation, which could also be restored following supplementation of DMSO. Glomerular ENTPD1 expression showed significant decrease after irradiation of kidney sections; again, this was inhibitable by DMSO. Glomerular E5NT activity was not altered by irradiation and DMSO supplementation did not affect its activity. It is concluded that soluble apyrase as well as the glomerular ENTPD1 are sensitive to oxidant stress, which may explain their down-regulation in the ischemic condition in vivo. However, soluble 5'-nucleotidase and E5NT seem much less sensitive to ROS. This relative insensitivity of E5NT to oxidant injury may counteract ischemic injury by promoting local generation of adenosine in the ischemic micro-environment. Copyright (C) 2008 S. Karger AG, Basel

Background The ubiquitous Rad50 and Mre11 proteins play a key role in many processes involved in the maintenance of genome integrity in Bacteria and Eucarya, but their function in the Archaea is presently unknown. We showed previously that in most hyperthermophilic archaea, rad50-mre11 genes are linked to nurA encoding both a single-strand endonuclease and a 5' to 3' exonuclease, and herA, encoding a bipolar DNA helicase which suggests the involvement of the four proteins in common molecular pathway(s). Since genetic tools for hyperthermophilic archaea are just emerging, we utilized immuno-detection approaches to get the first in vivo data on the role(s) of these proteins in the hyperthermophilic crenarchaeon Sulfolobus acidocaldarius. Results We first showed that S. acidocaldarius can repair DNA damage induced by high doses of gamma rays, and we performed a time course analysis of the total levels and sub-cellular partitioning of Rad50, Mre11, HerA and NurA along with the RadA recombinase in both control and irradiated cells. We found that during the exponential phase, all proteins are synthesized and display constant levels, but that all of them exhibit a different sub-cellular partitioning. Following gamma irradiation, both Mre11 and RadA are immediately recruited to DNA and remain DNA-bound in the course of DNA repair. Furthermore, we show by immuno-precipitation assays that Rad50, Mre11 and the HerA helicase interact altogether. Conclusion Our analyses strongly support that in Sulfolobus acidocaldarius, the Mre11 protein and the RadA recombinase might play an active role in the repair of DNA damage introduced by gamma rays and/or may act as DNA damage sensors. Moreover, our results demonstrate the functional interaction between Mre11, Rad50 and the HerA helicase and suggest that each protein play different roles when acting on its own or in association with its partners. This report provides the first in vivo evidence supporting the implication of the Mre11 protein in DNA repair processes in the Archaea and showing its interaction with both Rad50 and the HerA bipolar helicase. Further studies on the functional interactions between these proteins, the NurA nuclease and the RadA recombinase, will allow us to define their roles and mechanism of action.

Volatile and non-volatile radiolysis products and sensory changes of five-layer food packaging films have been determined after gamma irradiation (5-60 kGy). Barrier films were based on polyamide (PA) and low-density polyethylene (LDPE). Each film contained a middle buried layer of recycled LDPE or 100% virgin LDPE (control samples). Data showed that a large number of radiolysis products were produced such as hydrocarbons, alcohols, carbonyl compounds, carboxylic acid. These compounds were detected in the food simulant after contact with all films even at the lower absorbed doses of 5 and 10 kGy. The type and concentration of radiolysis products increased progressively with radiation dose, while no new compounds were detected as a result of the presence of recycled LDPE. In addition, irradiation dose appears to influence the sensory properties of table water in contact with films. (C) 2008 Elsevier Ltd. All rights reserved. Radiation Physics and Chemistry

Gamma radiation is used to sterilize diets for specific pathogen-free (SPF) animals. Because a gamma-irradiated diet was linked to leukoencephalomyelopathy in SPF cats, we investigated the effects of 'typical' (28.9-34.3 kGy) and 'high-end' (38.4-48.7 kGy) doses of gamma irradiation and of pasteurization (at 107 degrees C for 15 min) on the amounts of fat; protein; carbohydrate (and taurine in cat diet); vitamins A, E, B, B-1, B-2, and B-12; and peroxide in commercially available dry cat, dog, and rodent diets. The only treatment-related changes occurred with vitamin A and peroxide. The typical and high-end doses of gamma irradiation reduced the vitamin A level of the cat diet to 42% and 30% of the untreated value, respectively-levels below recommended allowances for growth and reproduction. Only the higher irradiation dose reduced vitamin A in the rodent diet, and neither dose altered the canine diet. Pasteurization reduced the vitamin A content of the cat diet to 50% of its original level, which was within the recommended level for this species. Irradiation increased the peroxide content of all 3 animal diets: by approximately 11-fold with the typical dose and by 14- to 25-fold with the high-end dose. Therefore gamma irradiation can have profound, selective effects on the vitamin A and peroxide contents of dry diets, and caution is advised when feeding such diets long-term and exclusively to SPF animals, particularly cats. Furthermore, pasteurization (with its fewer deleterious effects) may represent an alternative method of decontaminating diets for rodents, dogs, and cats. peer-reviewed

A pooled analysis of five biomonitoring studies was performed to assess the influence of hOGG1(326), XRCC1(399) and XRCC3(241) gene polymorphisms on micronuclei (MN) frequency in human peripheral blood lymphocytes, as measured by the ex vivo/in vitro cytokinesis-block micronucleus (CBMN) assay. Each study addressed a type of occupational exposure potentially able to induce DNA strand breakage (styrene, ionising radiation, cobalt/hard metal, welding fumes and inorganic arsenite compounds), and therefore MN, as a result of base excision repair and double-strand break repair deficiencies. The effect of genotype, age, exposure to genotoxic agents and smoking habit on MN induction was determined using Poisson regression analysis in 171 occupationally exposed male workers and in 132 non-exposed male referents. The analysis of genotype-genotype, genotype-smoking and genotype-exposure interactions by linear combinations of parameters showed significantly higher MN frequencies in the following subsets: (i) occupationally exposed workers carrying either the Thr/Thr or the Thr/Met XRCC3(241) genotypes compared to their referent counterparts (P < 0.001) and (ii) carriers of the Met/Met XRCC3(241) genotype compared to Thr/Thr XRCC3(241) carriers, as far as they are non-exposed and bear the variant (Ser/Cys or Cys/Cys) hOGG1(326) genotype (P < 0.01). Significantly lower MN frequencies were observed in carriers of the variant hOGG1(326) genotype compared to Ser/Ser hOGG1(326) carriers in the subgroup of non-smokers with Thr/Thr XRCC3(241) genotype (P < 0.01). Stratified analysis by occupational exposure showed a significant MN increase with smoking in occupationally exposed carriers of the Arg/Gln XRCC1(399)genotype (P < 0.001). In contrast, a significant MN decrease with smoking was observed in referents carrying the Ser/Ser hOGG1(326) genotype (P < 0.01). These findings provide evidence that the combination of different DNA repair genes and their interaction with environmental genotoxic agents may modulate MN induction. Understanding the complexity of the relationships between exposure, DNA repair and MN frequencies require larger scale studies and complementary biomarkers. Belgian Offices for Scientific, Technical and Cultural Affairs of the Prime Minister’s Office (contract PS/03/35); European Union project Cancer Risk Biomarkers (contract no QLK4- 2000-00628); Belgian Defense (Department RS&TD and DOVO) (VUB contract : WDGO251, study MS 03/01); the Associazione Italiana per la Ricerca sul Cancro; the Italian Space Agency (Project Mo.Ma., contract no I/014/06/); Environmental Cancer Risk, Nutrition and Individual Susceptibility, a network of excellence operating within the European Union 6th Framework Program, Priority 5: ‘‘Food Quality and Safety’’ (contract no 513943).

Purpose: To investigate the potential of irradiation in combination with drugs targeting the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) death receptor (DR)4 and DR5 and their mechanism of action in a cervical cancer cell line.Methods and Materials: Recombinant human TRAIL (rhTRAIL) and the agonistic antibodies against DR4 and DR5 were added to irradiated HeLa cells. The effect was evaluated with apoptosis and cytotoxicity assays and at the protein level. Membrane receptor expression was measured with flow cytometry. Small-interfering RNA against p53, DR4, and DR5 was used to investigate their function on the combined effect.Results: rhTRAIL and the agonistic DR4 and DR5 antibodies strongly enhanced 10-Gy-induced apoptosis. This extra effect was 22%, 23%, and 29% for rhTRAIL, DR4, and DR5, respectively. Irradiation increased p53 expression and increased the membrane expression of DR5 and DR4. p53 suppression, as well as small-interfering RNA against DR5, resulted in a significant downregulation of DR5 membrane expression but did not affect apoptosis induced by irradiation and rhTRAIL. After small-interfering RNA against DR4, rhTRAIL-induced apoptosis and the additive effect of irradiation on rhTRAIL-induced apoptosis were abrogated, implicating an important role for DR4 in apoptosis induced through irradiation in combination with rhTRAIL.Conclusion: Irradiation-induced apoptosis is strongly enhanced by targeting the pro-apoptotic TRAIL receptors DR4 or DR5. Irradiation results in a p53-dependent increase in DR5 membrane expression. The sensitizing effect of rhTRAIL on irradiation in the HeLa cell line is, however especially mediated through the DR4 receptor. (C) 2008 Elsevier Inc.

In the present study, the effect of electron-beam irradiation on physicochemical and mechanical properties of polypropylene (PP) syringes was studied. Three irradiation doses (30, 60 and 120 kGy) were applied to all samples. Non-irradiated PP syringes were used as control samples. Electron-beam irradiation caused an increase in the degree of yellowness and in the extractable radiolysis products. A decrease in compression strength and extension at break was the result of electron-beam irradiation on mechanical properties of PP syringes. Minor differences were observed in FTIR spectra, mainly in the region of 1720 cm(-1), corresponding to the absorption of carbonyl compounds. Gas chromatography/mass spectrometry (GC/MS) analysis indicated the formation of a number of radiolysis compounds while a number of compounds initially present in non-irradiated syringes were destroyed by the irradiation. The degradation on polymer properties caused by electron-beam irradiation was less severe than that caused by gamma irradiation. (C) 2006 Elsevier Ltd. All rights reserved. Radiation Physics and Chemistry

H4(D10S170) gene has been identified upon its frequent rearrangement with RET in papillary thyroid tumours (RET/PTC1). The kinase ataxia telangectasia mutated (ATM) phosphorylates a limited number of downstream protein targets in response to DNA damage. We investigated the potential role of H4(D10S170) in DNA damage signaling pathways. We found that in cells treated with etoposide or ionizing radiation (IR), H4(D10S170) underwent ATM-mediated phosphorylation at Thr 434, stabilizing nuclear H4. In ataxia telangectasia cells (A-T), endogenous H4(D10S170) was localized to cytoplasm and was excluded from the nucleus. Moreover, H4(D10S170) was not phosphorylated in ATM-deficient lymphoblasts after ionizing irradiation. Inhibition of ATM kinase interfered with H4(D10S170) apoptotic activity, and expression of H4 with threonine 434 mutated in Alanine, H4T434A, protected the cells from genotoxic stress-induced apoptosis. Most importantly, after exposure to IR we found that silencing of H4(D10S170) in mammalian cells increased cell survival, as shown by clonogenic assay, allows for DNA synthesis as evaluated by bromodeoxyuridine incorporation and permits cells to progress into mitosis as demonstrated by phosphorylation on Histone H3. Our results suggest that H4(D10S170) is involved in cellular response to DNA damage ATM-mediated, and that the impairment of H4(D10S170) gene function might have a role in thyroid carcinogenesis. © 2007 Nature Publishing Group All rights reserved.

The effect of gamma radiation on the migration of both di-(2-ethylhexyl)adipate (DEHA) and acetyltributyl citrate (ATBC) plasticizers from PVC film into the food simulant isooctane was studied as a function of time (0-48 h) at 20 degrees C. Food-grade PVC cling-film used contained 5.3% (w/w) DEHA, 3.0% (w/w) ATBC and polyadipate polymeric plasticizer. Irradiation of the films was carried out at doses of 5, 10 and 25 kGy using a [(CO)-C-60] gamma-radiation source. Determination of both plasticizers was performed using a direct gas chromatographic method. No radiation-induced transformation of the two plasticizers was observed after absorbed doses of 5-25 kGy. DEHA migrated rapidly into isooctane in contrast with ATBC. ATBC migrating amounts at equilibrium were approximately three times lower than the corresponding amounts of DEHA. Irradiation at doses 10 and 25 kGy had a small but statistically significant (P < 0.05) effect on the migration of both DEHA and ATBC into isooctane. Migration amount increased with increasing irradiation dose and contact time. The irradiation-induced increase of ATBC migration was significantly higher than the corresponding increase of DEHA migration. Results are discussed in relation to EU proposed upper limit for DEHA specific migration (18 mg/L). Diffusion coefficients for both plasticizers calculated, showed differences between irradiated and control samples. (c) 2005 Elsevier Ltd. All rights reserved. Journal of Food Engineering

Hypoxia through HRE (hypoxia-responsive element) activity in MG-63 human osteosarcoma cells grown in monolayer and as very small, three-dimensional tumor spheroids was investigated using molecular imaging techniques. MG-63 cells were stablv transfected with a vector constructed with multiple copies of ibe HRE sequence of the human vascular endothelial growth factor (VEGF) gene and with the enhanced green fluorescent protein (EGFP) coding sequence. During hypoxia when HIF-1 alpha (hypoxia-inducible factor-1 alpha) is stabilized, the binding of HIEF-1 to the HRE sequences of the vector allows the transcription of EGFP and the appearance of fluorescence. Transfecled monolayer cells were characterized by flow cytometric analysis in response to various hypoxic conditions and HIF-1 alpha expression in these cells was assessed by Western blotting. Two-photon excitation (TPE) microscopy was then used to examine both MG-63-transfected monolayer cells and spheroids at 2 and 5 days of growth in normoxic conditions. Monolayer cells reveal almost no fluorescence, whereas even very small spheroids (< 100 mu m) after 2 days of growth contain regions of high fluorescence. For the first time in the literature, at least to our knowledge, it is demonstrated, using highly sensitive and non-perturbing molecular imaging techniques, that three-dimensional cell organization leads to almost immediate HRE activation. This activation of the HRE sequences, which control a wide variety of genes, suggests that monolayer cells and spheroids of the MG-63 cell line have different genes activated and thus diverse functional activities. (c) 2007 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.