RENEB - Running the European Network of biological dosimetry and physical retrospective dosimetry.

PURPOSE: A European network was initiated in 2012 by 23 partners from 16 European countries with the aim to significantly increase individualized dose reconstruction in case of large-scale radiological emergency scenarios. RESULTS: The network was built on three complementary pillars: (1) an operational basis with seven biological and physical dosimetric assays in ready-to-use mode, (2) a basis for education, training and quality assurance, and (3) a basis for further network development regarding new techniques and members. Techniques for individual dose estimation based on biological samples and/or inert personalized devices as mobile phones or smart phones were optimized to support rapid categorization of many potential victims according to the received dose to the blood or personal devices. Communication and cross-border collaboration were also standardized. To assure long-term sustainability of the network, cooperation with national and international emergency preparedness organizations was initiated and links to radiation protection and research platforms have been developed. A legal framework, based on a Memorandum of Understanding, was established and signed by 27 organizations by the end of 2015. CONCLUSIONS: RENEB is a European Network of biological and physical-retrospective dosimetry, with the capacity and capability to perform large-scale rapid individualized dose estimation. Specialized to handle large numbers of samples, RENEB is able to contribute to radiological emergency preparedness and wider large-scale research projects.

Hyperthermia adds to trabectedin effectiveness and thermal enhancement is associated with BRCA2 degradation and impairment of DNA homologous recombination repair.

The tetrahydroisoquinoline trabectedin is a marine compound with approved activity against human soft-tissue sarcoma. It exerts antiproliferative activity mainly by specific binding to the DNA and inducing DNA double-strand breaks (DSB). As homologous recombination repair (HRR)-deficient tumors are more susceptible to trabectedin, hyperthermia-mediated on-demand induction of HRR deficiency represents a novel and promising strategy to boost trabectedin treatment. For the first time, we demonstrate enhancement of trabectedin effectiveness in human sarcoma cell lines by heat and characterize cellular events and molecular mechanisms related to heat-induced effects. Hyperthermic temperatures (41.8 or 43°C) enhanced significantly trabectedin-related clonogenic cell death and G2/M cell cycle arrest followed by cell type-dependent induction of apoptosis or senescence. Heat combination increased accumulation of γH2AX foci as key marker of DSBs. Expression of BRCA2 protein, an integral protein of the HRR machinery, was significantly decreased by heat. Consequently, recruitment of downstream RAD51 to γH2AX-positive repair foci was almost abolished indicating relevant impairment of HRR by heat. Accordingly, enhancement of trabectedin effectiveness was significantly augmented in BRCA2-proficient cells by hyperthermia and alleviated in BRCA2 knockout or siRNA-transfected BRCA2 knockdown cells. In peripheral blood mononuclear cells isolated from sarcoma patients, increased numbers of nuclear γH2AX foci were detected after systemic treatment with trabectedin and hyperthermia of the tumor region. The findings establish BRCA2 degradation by heat as a key factor for a novel treatment strategy that allows targeted chemosensitization to trabectedin and other DNA damaging antitumor drugs by on-demand induction of HRR deficiency.

Radiation-induced alterations of histone post-translational modification levels in lymphoblastoid cell lines.

BACKGROUND: Radiation-induced alterations in posttranslational histone modifications (PTMs) may affect the cellular response to radiation damage in the DNA. If not reverted appropriately, altered PTM patterns may cause long-term alterations in gene expression regulation and thus lead to cancer. It is therefore important to characterize radiation-induced alterations in PTM patterns and the factors affecting them. METHODS: A lymphoblastoid cell line established from a normal donor was used to screen for alterations in methylation levels at H3K4, H3K9, H3K27, and H4K20, as well as acetylation at H3K9, H3K56, H4K5, and H4K16, by quantitative Western Blot analysis at 15 min, 1 h and 24 h after irradiation with 2 Gy and 10 Gy. The variability of alterations in acetylation marks was in addition investigated in a panel of lymphoblastoid cell lines with differing radiosensitivity established from lung cancer patients. RESULTS: The screening procedure demonstrated consistent hypomethylation at H3K4me3 and hypoacetylation at all acetylation marks tested. In the panel of lymphoblastoid cell lines, however, a high degree of inter-individual variability became apparent. Radiosensitive cell lines showed more pronounced and longer lasting H4K16 hypoacetylation than radioresistant lines, which correlates with higher levels of residual γ-H2AX foci after 24 h. CONCLUSION: So far, the factors affecting extent and duration of radiation-induced histone alterations are poorly defined. The present work hints at a high degree of inter-individual variability and a potential correlation of DNA damage repair capacity and alterations in PTM levels.

Evaluation of different biomarkers to predict individual radiosensitivity in an inter-laboratory comparison--lessons for future studies.

Radiotherapy is a powerful cure for several types of solid tumours, but its application is often limited because of severe side effects in individual patients. With the aim to find biomarkers capable of predicting normal tissue side reactions we analysed the radiation responses of cells from individual head and neck tumour and breast cancer patients of different clinical radiosensitivity in a multicentric study. Multiple parameters of cellular radiosensitivity were analysed in coded samples of peripheral blood lymphocytes (PBLs) and derived lymphoblastoid cell lines (LCLs) from 15 clinical radio-hypersensitive tumour patients and compared to age- and sex-matched non-radiosensitive patient controls and 15 lymphoblastoid cell lines from age- and sex- matched healthy controls of the KORA study. Experimental parameters included ionizing radiation (IR)-induced cell death (AnnexinV), induction and repair of DNA strand breaks (Comet assay), induction of yH2AX foci (as a result of DNA double strand breaks), and whole genome expression analyses. Considerable inter-individual differences in IR-induced DNA strand breaks and their repair and/or cell death could be detected in primary and immortalised cells with the applied assays. The group of clinically radiosensitive patients was not unequivocally distinguishable from normal responding patients nor were individual overreacting patients in the test system unambiguously identified by two different laboratories. Thus, the in vitro test systems investigated here seem not to be appropriate for a general prediction of clinical reactions during or after radiotherapy due to the experimental variability compared to the small effect of radiation sensitivity. Genome-wide expression analysis however revealed a set of 67 marker genes which were differentially induced 6 h after in vitro-irradiation in lymphocytes from radio-hypersensitive and non-radiosensitive patients. These results warrant future validation in larger cohorts in order to determine parameters potentially predictive for clinical radiosensitivity.

MiRNA expression patterns predict survival in glioblastoma.

BACKGROUND: In order to define new prognostic subgroups in patients with glioblastoma a miRNA screen (> 1000 miRNAs) from paraffin tissues followed by a bio-mathematical analysis was performed. METHODS: 35 glioblastoma patients treated between 7/2005 - 8/2008 at a single institution with surgery and postoperative radio(chemo)therapy were included in this retrospective analysis. For microarray analysis the febit biochip "Geniom® Biochip MPEA homo-sapiens" was used. Total RNA was isolated from FFPE tissue sections and 1100 different miRNAs were analyzed. RESULTS: It was possible to define a distinct miRNA expression pattern allowing for a separation of distinct prognostic subgroups. The defined miRNA pattern was significantly associated with early death versus long-term survival (split at 450 days) (p = 0.01). The pattern and the prognostic power were both independent of the MGMT status. CONCLUSIONS: At present, this is the first dataset defining a prognostic role of miRNA expression patterns in patients with glioblastoma. Having defined such a pattern, a prospective validation of this observation is required.

Low-dose irradiation causes rapid alterations to the proteome of the human endothelial cell line EA.hy926.

High doses of ionising radiation damage the heart by an as yet unknown mechanism. A concern for radiological protection is the recent epidemiological data indicating that doses as low as 100-500 mGy may induce cardiac damage. The aim of this study was to identify potential molecular targets and/or mechanisms involved in the pathogenesis of low-dose radiation-induced cardiovascular disease. The vascular endothelium plays a pivotal role in the regulation of cardiac function and is therefore a potential target tissue. We report here that low-dose radiation induced rapid and time-dependent changes in the cytoplasmic proteome of the human endothelial cell line EA.hy926. The proteomes were investigated at 4 and 24 h after irradiation at two different dose rates (Co-60 gamma ray total dose 200 mGy; 20 mGy/min and 190 mGy/min) using 2D-DIGE technology. Differentially expressed proteins were identified, after in-gel trypsin digestion, by MALDI-TOF/TOF tandem mass spectrometry, and peptide mass fingerprint analyses. We identified 15 significantly differentially expressed proteins, of which 10 were up-regulated and 5 down-regulated, with more than ±1.5-fold difference compared with unexposed cells. Pathways influenced by the low-dose exposures included the Ran and RhoA pathways, fatty acid metabolism and stress response.

The DNA repair protein NBS1 influences the base excision repair pathway.

NBS1 fulfills important functions for the maintenance of genomic stability and cellular survival. Mutations in the NBS1 (Nijmegen Breakage Syndrome 1) gene are responsible for the Nijmegen breakage syndrome (NBS) in humans. The symptoms of this disease and the phenotypes of NBS1-defective cells, especially their enhanced radiosensitivity, can be explained by an impaired DNA double-strand break-induced signaling and a disturbed repair of these DNA lesions. We now provide evidence that NBS1 is also important for cellular survival after oxidative or alkylating stress where it is required for the proper initiation of base excision repair (BER). NBS1 downregulated cells show reduced activation of poly-(adenosine diphosphate-ribose)-polymerase-1 (PARP1) following genotoxic treatment with H(2)O(2) or methyl methanesulfonate, indicating impaired processing of damaged bases by BER as PARP1 activity is stimulated by the single-strand breaks intermediately generated during this repair pathway. Furthermore, extracts of these cells have a decreased capacity for the in vitro repair of a double-stranded oligonucleotide containing either uracil or 8-oxo-7,8-dihydroguanine to trigger BER. Our data presented here highlight for the first time a functional role for NBS1 in DNA maintenance by the BER pathway.

Enhanced CD95-mediated apoptosis contributes to radiation hypersensitivity of NBS lymphoblasts.

The molecular causes for enhanced radiosensitivity of Nijmegen Breakage Syndrome cells are unclear, especially as repair of DNA damage is hardly impeded in these cells. We clearly demonstrate that radiation hypersensitivity is accompanied by enhanced gamma-radiation-induced apoptosis in NBS1 deficient lymphoblastoid cell lines. Differences in the apoptotic behavior of NBS1 (-/-) and NBS1 (+/-) cells are not due to an altered p53 stabilization or phosphorylation in NBS1 (-/-) cells. gamma-radiation-induced caspase-8 activity is increased and visualization of CD95 clustering by laser scanning microscopy shows a significant higher activation of the death receptor in NBS1 (-/-) cells. Further investigation of the molecular mechanisms reveals a role for reactive oxygen species-triggered activation of CD95. These results demonstrate that NBS1 suppresses the CD95 death receptor-dependent apoptotic pathway after gamma-irradiation and evidence is given that this is achieved by regulation of the PI3-K/AKT survival pathway.

Biosynthesis of riboflavin in archaea. 6,7-dimethyl-8-ribityllumazine synthase of Methanococcus jannaschii.

Heterologous expression of the putative open reading frame MJ0303 of Methanococcus jannaschii provided a recombinant protein catalysing the formation of the riboflavin precursor, 6,7-dimethyl-8-ribityllumazine, by condensation of 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione and 3,4-dihydroxy-2-butanone 4-phosphate. Steady state kinetic analysis at 37 degrees C and pH 7.0 indicated a catalytic rate of 11 nmol.mg-1.min-1; Km values for 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione and 3,4-dihydroxybutanone 4-phosphate were 12.5 and 52 micro m, respectively. The enzyme sediments at an apparent velocity of about 12 S. Sedimentation equilibrium analysis indicated a molecular mass around 1 MDa but was hampered by nonideal solute behaviour. Negative-stained electron micrographs showed predominantly spherical particles with a diameter of about 150 A. The data suggest that the enzyme from M. jannaschii can form capsids with icosahedral 532 symmetry consisting of 60 subunits.

The 18-kDa cytoplasmic protein of Brucella species --an antigen useful for diagnosis--is a lumazine synthase.

Previous studies have shown that the detection of antibodies to an 18-kDa cytoplasmic protein of Brucella spp. is useful for the diagnosis of human and animal brucellosis. This protein has now been expressed in recombinant form in Escherichia coli. The recombinant protein is soluble only under reducing conditions, but alkylation with iodoacetamide renders it soluble in non-reducing media. As shown by gel exclusion chromatography, this soluble form arranges in pentamers of 90 kDa. The reactivity of human and animal sera against the recombinant protein was similar to that found with the native protein present in brucella cytoplasmic fraction, suggesting that the recombinant protein is correctly folded. The protein has low but significant homology (30%) with lumazine synthases involved in bacterial riboflavin biosynthesis, which also arrange as pentamers. Biological tests on the crude extract of the recombinant bacteria and on the purified recombinant protein showed that the biological activity of the Brucella spp. 18-kDa protein is that of lumazine synthase. Preliminary crystallographic analysis showed that the Brucella spp. lumazine synthase arranges in icosahedric capsids similar to those formed by the lumazine synthases of other bacteria. The high immunogenicity of this protein, potentially useful for the design of acellular vaccines, could be explained by this polymeric arrangement.