women in radiation sciences a century after marie curie workshop may 8-20, 2009, münchen PROGRAM & ABSTRACTS www.helmholtz-muenchen.de/radiation2009
OVERVIEW OF THE CAMPUS GENERAL INFORMATION Scientific Program Committee Cafeteria Kerstin Borgmann, Hamburg Friederike Eckardt-Schupp, Neuherberg Hedda Eichholtz-Wirth, Neuherberg Claudia Fournier (GBS), Darmstadt Marlis Frankenberg-Schwager, Göttingen Anna A. Friedl, München Simone Mörtl, Neuherberg Lisa Wiesmüller (DGDR), Ulm Planing and Local Organisation Lecture Hall Bus Station and Entrance Kerstin Borgmann, Hamburg Friederike Eckardt-Schupp, Neuherberg Anna A. Friedl, München Simone Mörtl, Neuherberg Professional Congress Organisation Congress, Meeting & Event Management AG Elvira Dieringer Albert-Rosshaupter-Str. 65 D-8369 München Phone: +49 89 54823462 Fax: +49 89 54823443 E-Mail: radiation09@interplan.de Congress Venue Helmholtz Zentrum München Building 33 Ingolstädter Landstrasse D-85764 Neuherberg Phone during the workshop: +49 52 09209828 Contact Institut für Strahlenbiologie Helmholtz Zentrum München- Deutsches Forschungszentrum für Gesundheit und Umwelt GmbH Ingolstädter Landstrasse D-85764 Neuherberg Phone: +49 89 374 40/343 Fax: +49 89 374 338 eckardt-schupp@helmholtz-muenchen.de moertl@helmholtz-muenchen.de www.helmholtz-muenchen.de/radiation2009 2 3
SUPPORT PROGRAM THE ORGANIZERS GRATEFULLY ACKNOWLEDGE THE GENEROUS SUPPORT OF Helmholtz Zentrum München MONDAY 8.5.2009 Women in Radiation Sciences - A Century after Marie Curie May 8-20, 2009, Helmholtz Zentrum München, Neuherberg :00 Registration Bundesministerium für Umwelt, Naturschutz und Reaktorsicherheit (BMU) 3:30 Opening Friederike Eckardt-Schupp BMU: Axel Böttger GBS/HGF: Claudia Fournier DGDR: Lisa Wiesmüller 4:00 5:00 Keynote Lecture Chair: Friederike Eckardt-Schupp Ethel Moustacchi (Paris) The biological effects of low doses of ionizing radiation: the quest of the Holy Grail? Bundesamt für Strahlenschutz (BfS) Session I Cellular Responses to Radiation: DNA Repair and Disease Chair: Marlis Frankenberg-Schwager, Susanne Auer 5:00 5:35 Priscilla K. Cooper (Berkeley) Mechanistic studies of the adaptive response to ionizing radiation 5:35 6:00 Coffee break Deutsche Gesellschaft für DNA Reparaturforschung (DGDR) 6:00 6:35 Penny Jeggo (Brighton) DNA repair in heterochromatin 6:35 7:0 Lisa Wiesmüller (Ulm) Identification of increased breast cancer risk through the detection of DNA-double-strand break repair defects 7:0 7:30 Odilia Popanda (Heidelberg) Genetic variants in DNA repair and damage response genes and late normal tissue complications after radiotherapy in breast cancer patients Gesellschaft für Biologische Strahlenforschung (GBS) 7:30 7:50 Verena Bauer (Neuherberg) Chromosomal changes characterize head and neck cancer with poor prognosis 7:50 8:0 Katja Storch (Dresden) The role of histone deacetylases for the radiation response of 3D cultured cells Helmholtz Gemeinschaft (HGF) 8:0 8:30 Christiane Kuschal (Göttingen) Cyclosporin A inhibits nucleotide excision repair via downregulation of xeroderma pigmentosum group A protein but not other xeroderma pigmentosum proteins 8.30 Welcome reception Bayrische Brotzeit and poster session 4 5
PROGRAM PROGRAM TUESDAY 9.5.2009 Session II Cellular Responses to Radiation: Signalling, Cell Cycle and Apoptosis Chair: Hedda Eichholtz-Wirth, Sylvia Steininger 09:00 09:35 Eva Petermann (Oxford) Rad5 promotes the restart of stalled replication forks via a mechanism that does not result in recombination 09:35 0:0 Irene Dornreiter (Hamburg) Role of the p53 gene family in the intra-s-phase checkpoint 0:0 0:30 Petra Ohneseit (Tübingen) The phosphatase inhibitor suramin differentially modulates radiosensitivity of p53 wildtype and p53 mutant tumor cells and of normal fibroblasts 0:30 :05 Berit Jungnickel (München) Genetic flexibility in the adaptive immune system.05 :30 Coffee break :30 2:05 Irmela Jeremias (München) Cell death mechanisms and resistance to radiotherapy 2:05 2:40 Gabriele Multhoff (München) Radiation-induced stress proteins 2:40 3:00 Ulla Kasten-Pisula (Hamburg) Variation in EGFR protein expression is correlated with cellular and tumour radiosensitivity, except for tumours with extreme EGFR gene amplification 3:00 4:5 Lunch Session III Sex-Specific Reactions towards Genotoxic Agents? Chair: Anna A. Friedl; Iria Gonzales Vasconcellos 4:5 4:50 Denise Barlow (Vienna) Regulation of imprinted expression by macro non-coding RNAs 4:50 5:25 Janet Hall (Paris) From Marie Curie to SNP s: responses to radiation 5:25 6:00 Kerstin Borgmann (Hamburg) Sex-specific aspects of individual radiosensitivity 6:00 6:35 Ulrike Höller (Berlin) Do women survive longer? Sex and cancer therapy from a clinical perspective 6:35 7:00 Coffee break 7:00 7:35 Soile Tapio (Neuherberg) Proteomics approach in radiation-induced cardiovascular diseases 7:35 7:55 Dominik Schmaltz (Neuherberg) Increased oxidative stress in cardiac mitochondria even 4 weeks after heart-focused low dose ionizing radiation 8:00 9:00 Special Lecture Chair: Helga Schüßler (Erlangen) Friederike Eckardt-Schupp (Neuherberg) Women in Radiation Sciences Some glances at history 9:00 Drinks and dinner (Cafeteria) WEDNESDAY 20.5.2009 Session IV Hot Spicy Topics in Radiation Sciences Chair: Lisa Wiesmüller; Anne Krämer 09.00 09:35 Anna A. Friedl (München) Microbeam and single cell irradiation 09:35 09:55 Belinda Mazurek (München) DNA repair in artificially modified chromatin 09:55 0:30 Gisela Taucher-Scholz (Darmstadt) Clustered DNA damage: Spatiotemporal dynamics and cellular effects 0:30 :05 Barbara Röper (München) Advances in radiotherapy at the interphase between biology, diagnostics, physics and technology :05 :30 Coffee break :30 2:05 Beate Volkmer (Buxtehude) UV-radiation, epidermal stem cells and skin cancer 2:05 2:40 Claudia Fournier (Darmstadt) Irradiated human hematopoietic stem and progenitor cells show an altered differentiation pattern, apoptosis, but no genetic instability 2:40 3:5 Simone Mörtl (München) mirnas and radiosensitivity 3:5 M.A. Atkinson (ISB Neuherberg) Future perspectives for radiation sciences 3:25 Closing and farewell 6 7
ABSTRACTS ABSTRACTS Keynote Lecture Session I: Cellular Responses to Radiation: DNA Repair and Disease The biological effects of low doses of ionizing radiations: the quest of the Holy Grail? Ethel Moustacchi Institut Curie -Recherche /CNRS, 26 rue d ULM. Paris cedex5 France Institut Curie Recherche /CNRS. Abstract Text The issue of low dose risk (<00mGy) to human populations is still controversial although exposure to ionizing radiations is unavoidable. In relation to industrial uses and as part of modern medecine, over or under estimation could lead to unnecessary expenses or more dramatically to unsufficient protection. A number of molecular and cellular events are now detected and quantitated at doses as low as mgy. How such early events may determine the long term outcome in terms of carcinogenic and genetic consequences is not yet understood. Moreover the well established biological effects of high doses cannot be qualitatively equated to the observed effects at low doses. For instance different sets of genes are induced at low and high doses. Consequently the direct extrapolation according to the linear non threshold (LNT) relationship from high to low doses is at least questionable, especially if it may hamper certain medical applications of radiations. In the actual state of knowledge the prudent attitude in terms of regulatory policy is to maintain the use of the LNT relationship. Mechanistic studies actually in progress and which will be reported are likely to modify in the near future the methodology for risk assessment. Mechanistic studies of the adaptive response to ionizing radiation Priscilla K. Cooper Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA The radiation adaptive response is a phenomenon in which exposures to low doses of ionizing radiation (IR) induce protection against various genotoxic effects of subsequent higher doses. Such protection might well result in a non-linear dose response. Hence, understanding the mechanism, genetic control, and variability among individuals and across tissues and endpoints of the adaptive response is critical for an informed ability to estimate cancer risk from low dose exposures. We are investigating biochemical and genetic mechanisms that could confer adaptation, in particular the hypothesis that inducible repair of oxidative DNA base damage is critical for cellular responses to low IR doses, as well as being a key determinant of the effects of endogenous DNA damage. Removal of oxidized pyrimidines is inducible by low-dose IR, dependent on XPG, TFIIH, CSB, and PARP-. This result, together with transcription profiling and mechanistic studies, implicates scaffolding functions of XPG in base excision repair (BER) and transcription-coupled repair (TCR) in the adaptive response. The enzymatic activity of XPG is essential for incision in nucleotide excision repair, but XPG also coordinates and stimulates BER by functional interactions with multiple oxidative damage-specific glycosylases, AP endonuclease, and post-incision BER proteins, and such non-enzymatic function(s) are essential postnatally. Supporting its importance in repair of oxidative damage, cells lacking XPG are hypersensitive to IR and hydrogen peroxide. Extracts of these cells are deficient in incision at oxidative lesions and defective in long patch-ber. These functions suggest a likely mechanism for adaptive protection against chromosomal damage and IR-induced senescence. 8 9
ABSTRACTS ABSTRACTS Session I: Cellular Responses to Radiation: DNA Repair and Disease Session I: Cellular Responses to Radiation: DNA Repair and Disease DNA Repair in heterochromatin. Penny Jeggo Genome Damage and Stability Centre, University of Sussex, BN7TL. The pioneering work of Marie Curie already a century ago demonstrated that radiation is a powerful tool with a wide range of potential uses that have since been shown to include energy generation and exploitation for medical use including a range of diagnostic procedures as well as radiotherapy. But the life and death of Marie Curie and her husband also demonstrated that it can be a dangerous tool that must be handled with respect. So how do we Tame the Shrew to maximize the exploitation of radiation. Importantly, it is necessary to understand the harmful impacts of radiation on human cells and the processes that our cells exploit to handle these impacts their strengths and limitations. In 2009, we now have an intimate knowledge of DNA damage inflicted by radiation and we are gaining a good understanding of the damage response pathways that limit the cellular impact of these damages. Recent studies have demonstrated that the higher order structure of mammalian cells imparts a barrier to the damage response pathways and that a critical function of Ataxia telangiectasia mutated protein (ATM) is to overcome this barrier to enhance the efficacy of the evolutionary conserved damage response pathways. My talk will focus on how ATM overcomes the barrier to DNA double strand break (DSB) repair posed by heterochromatin and the clinical impact of deficiencies in the repair pathway. DNA non-homologous end-joining (NHEJ) is the major DSB repair pathway in G phase mammalian cells. ATM signaling represents the major signaling pathway in response to DSB formation. Whilst 85 % of X-ray induced DSBs require only the core NHEJ proteins for their repair, a subset of DSBs require additional components including ATM and the damage response mediator proteins. Recent studies have shown that ATM and the mediator proteins are specifically required for the repair of DSBs located within or close to heterochromatin and that KAP, an essential heterochromatin building factor, is a critical ATM substrate in this response. The available evidence suggests that ATM dependent phosphorylation of KAP diminishes its chromatin binding allowing localized chromatin relaxation without disruption of the heterochromatin superstructure. We have shown that heterochromatin is a barrier to γ H2AX foci expansion and to DSB repair, which can be overcome by phosphorylation of KAP. The role of the mediator proteins in this process will be discussed. Cells lacking core NHEJ proteins, ATM and the mediator proteins display significant radiosensitivity. Patients harbouring mutations in some of these components have been identified and, in some cases, when given radiotherapy have shown marked over responses demonstrating clinical radiosensitivity. The significance of these pathways in avoiding radiation damage is therefore important to understand as well as their limitation. Identification of increased breast cancer risk through the detection of DNA-double-strand break repair defects. Lisa Wiesmüller Gynaecological Oncology, Department of Obstetrics and Gynaecology of the University of Ulm Ten high-penetrance breast cancer susceptibility genes, among them BRCA and BRCA2, have been identified as components of DNA double strand break repair (DSBR). Furthermore, accumulation of subtle aberrations in DSBR due to polymorphisms in multiple modifier genes and/or epigenetic changes ultimately lead to breast cancerogenesis. We have developed a process for the sensitive detection of distinct DSBR pathways, and consequently analysed its potential to serve as an indicator for DSBR defects and hence enhanced breast cancer risk. Based on the results that we obtained so far (I) this test system is capable of detecting DSBR defects stemming from defined predisposing gene defects. Our data from epithelial cells derived from primary mammary carcinomas (II) support the hypothesis that women without mutations in BRCA or BRCA2, but still with a DSBR defect, carry enhanced breast cancer risk. Our systematic analyses of DSBR activities in peripheral blood lymphocytes from 78 patients with primary mammary carcinomas, 37 high risk family members and 26 healthy donors unravelled (III) an increase of the error prone Non Homologous End Joining and Single-Strand Annealing pathways in breast cancer patients and high risk individuals compared with the controls. These data suggest that the detection of distinct DSBR activities can serve as a powerful tool for the identification of breast cancer susceptibility beyond the limitations of genotyping. In conclusion, this DSBR assay system is indeed a potential method for breast cancer risk assessment, and, even more importantly, may predict responsiveness to classical and new therapeutics targeting DNA repair dysfunctional tumors. 0
ABSTRACTS ABSTRACTS Session I: Cellular Responses to Radiation: DNA Repair and Disease Session I: Cellular Responses to Radiation: DNA Repair and Disease Genetic variants in DNA repair and damage response genes and late normal tissue complications after radiotherapy in breast cancer patients O. Popanda, C. B. Ambrosone 2, C. Lilla 3, S. Kropp 3, I. Helmbold 3, D. von Fournier 4, W. Haase 5, M. Sautter-Bihl 6, F. Wenz 7, P. Schmezer, J. Chang-Claude 3 Epigenomics and Cancer Risk Factors, German Cancer Research Center (DKFZ), Heidelberg, Germany; 2 Roswell Park Cancer Institute, Buffalo, USA; 3 Cancer Epidemiology, DKFZ; 4 University Hospital Heidelberg; 5 St. Vincentius-Clinics Karlsruhe; 6 Municipal Hospital Karlsruhe; 7 University Hospital Mannheim Breast-conserving surgery followed by radiotherapy is very effective in reducing breast cancer recurrence; however, adverse side effects can occur, even years after therapy. Great inter-individual variation in clinical radiation reaction has been observed. Since radiation acts through producing DNA damage, we investigated whether genetic variation in DNA repair (APEX, XRCC, XRCC2, XRCC3, NBS, and XPD) and damage response genes (TP53 and P2) confers increased susceptibility to develop late normal skin complications. In a prospective study, 409 breast cancer patients who received radiotherapy after breast-conserving surgery were examined for late complications (median follow-up time 5 months). 3 patients presented with telangiectasia, and 28 patients with fibrosis. Patients with variant TP53 genotypes either for the Arg72Pro or the PIN3 polymorphism were at increased risk of telangiectasia. The odds ratios (OR) were.66 (95% confidence interval (CI).02-2.72) for 72Pro carriers and.95 (95% CI.3-3.35) for PIN3 A2 allele carriers compared to non-carriers. Further analysis for effect modification showed that the elevated risk of telangiectasia associated with the TP53 ProA2 haplotype was found in patients who did not present with acute toxicity (moist desquamation) during radiotherapy but not in those who experienced acute skin toxicity (P heterogeneity=0.06). No further significant associations between genotypes and telangiectasia were observed. In conclusion, the study showed that variants of the TP53 gene are associated with the risk to develop telangiectasia after radiotherapy. If replicated in other studies, this genetic marker may lead to better treatment choices and improve quality of life for breast cancer patients. Chromosomal changes characterize head and neck cancer with poor prognosis V. Bauer, H. Braselmann, M. Henke 2, D. Mattern 3, A. Walch 4, Kristian Unger 7, Michael Baudis 5, S. Lassmann 3, R. Huber, J. Wienberg 6, M. Werner 3, Horst Zitzelsberger Institute of Molecular Radiation Biology, Helmholtz Center Munich German Research Center for Environmental Health GmbH, Germany 2 Clinic for Radiation Therapy, University Clinic Freiburg, Germany 3 Institute of Pathology, University Clinic Freiburg, Germany 4 Institute of Pathology, Helmholtz Center Munich German Research Center for Environmental Health GmbH, Germany 5 Institute of Molecular Biology, University of Zurich, Switzerland 6 Department Biology II, Anthropology and Human Genetics, Ludwig-Maximilians-University, Grosshaderner Str.2, Germany 7 Department of Histopathology - Imperial College London Hammersmith Hospital, United Kingdom It is well established that genetic alterations may be associated to prognosis in tumour patients. We investigated chromosomal changes that predict the clinical outcome of head and neck squamous cell carcinoma (HNSCC) and correlate to characteristic clinico-pathological parameters. We applied comparative genomic hybridization (CGH) to tissue samples from 7 HNSCC patients scheduled for radiotherapy. Genomic aberrations occurring in more than five patients were studied for impact on locoregional-progression-(lpr)-free survival. P-values were adjusted by the Hochberg-Benjamini procedure and significant aberrations and clinical variables subjected to a stepwise backwards Cox proportional model. Significant alterations were further analyzed by array CGH and Fluorescence in situ Hybridization (FISH). In multivariate survival analysis gains on q and 6q predict reduced LPR-free survival after radiotherapy independently from known prognostic factors. Cluster analysis separated the patients into two clusters that are characterized by significant differences for imbalances in 3 chromosomal regions. Array-CGH pinpoints 6q24.3 to be the region of interest on chromosome 6 which was further verified by FISH analysis where an increased copy number of FANCA, a member of the FA/BRCA pathway, could be identified. Quantitative Real-Time-PCR confirmed increased FANCA expression on RNA-level. To analyze radio sensitivity of FANCA-over-expressing cells, two cell lines were stably transfected with the gene and tested for cell survival after irradiation with different doses of ionizing radiation. The findings demonstrate that chromosomal gains on q and 6q represent prognostic markers in HNSCC and that these alterations may explain to some extent the dismal course of a subgroup of patients after radiotherapy. Preliminary results of the cell survival experiments indicate reduced radiation sensitivity in FANCA-over-expressing cells. 2 3
ABSTRACTS ABSTRACTS Session I: Cellular Responses to Radiation: DNA Repair and Disease Session I: Cellular Responses to Radiation: DNA Repair and Disease The role of histonedeacetylases for the radiation response of 3D cultured cells Katja Storch and Nils Cordes OncoRay - Center for Radiation Research in Oncology, Dresden University of Technology, Dresden, Germany Using three-dimensional (3D) cell culture models, cellular behaviour upon irradiation can be examined under more physiological conditions. Due to changes in cellular morphology in 3D, the nuclear structure shows a more condensed chromatin organization. This is reflected by elevated heterochromatin protein (HP) -alpha and decreased histone H3 acetylation. Histone acetylation is dynamically regulated by histone acetylases (HAT) and histone deacetylases (HDAC). The aim of this study was to identify the relevance of HDACs in 3D laminin-rich extracellular matrix grown, irradiated A549 human lung cancer cells by the means of two different approaches using HDAC sirna knockdown and pharmacological HDAC inhibition with LBH589 (Novartis). Upon single or combined sirna knockdown of HDAC, 2 or 4 or LBH589 treatment, clonogenic survival, number of residual DNA double-strand breaks (DSBs, foci assay) and protein expression (e.g. acetyl-h3, HP-alpha) were examined. For optimal visualisation of chromatin changes, cells were stably transfected with EGFP-HP-alpha. While the knockdown of HDACs showed no effect on radiosensitivity, LBH589 significantly enhanced the radiosensitivity and the number of residual DBSs in a dosedependent manner and caused a strong histone H3 hyperacetylation and a decrease of HP-alpha. These observations were confirmed in EGFP-HP-alpha transfectants. As a consequence of our findings using a genetic and pharmacological approach, the role of HDACs for the cellular radiation response is currently unclear. By inhibiting a broader spectrum of HDACs then only HDAC, 2 or 4, LBH589 presented as a potent radiosensitiser in our 3D cell culture model. Cyclosporin A inhibits nucleotide excision repair via downregulation of the xeroderma pigmentosum group A protein, but not other xeroderma pigmentosum proteins C. Kuschal, K.-M. Thoms, L. Boeckmann, P. Laspe, S. Emmert Department of Dermatology and Venerology, Georg-August-University, Goettingen, Germany Cyclosporin A (CsA) rather than other immunosuppressive drugs like everolimus enhances UV-induced skin carcinogenesis in organ transplant patients. Xeroderma pigmentosum (XP) patients also exhibit an increased skin cancer risk due to reduced nucleotide excision repair (NER). We have previously shown that CsA but not everolimus inhibits NER in human cells. In this study, we investigated the effects of CsA on expression and function of XP proteins. We measured XP protein levels in CsA-treated GM00637 fibroblasts. Western blot data revealed that the XPA protein was reduced in the cytosol of 0. µm and 0.5 µm CsA-treated cells, and no XPA-protein was detectable in the nucleus. In contrast, no other XP proteins were downregulated by CsA treatment. Using host cell reactivation (HCR) to measure functional NER, we found that the CsA-induced reduction of NER in GM00637 fibroblasts could be fully corrected by transfecting a XPA-cDNA-containing plasmid under the control of the SV40-promoter, resulting in overexpression of XPA protein. In contrast, overexpression of XPC protein by transfecting a XPC-cDNA containing plasmid could not complement the decreased NER induced by CsA. In addition, NER in XPA-deficient XP2OS fibroblasts was not influenced by CsA. Furthermore, XP2OSCAH9WS fully corrected XP2OS cells expressing XPA under the control of a CMVchicken promoter showed normal NER under CsA-treatment. Our data confirm that CsA downregulates NER and show that inhibition of NER through CsA is mediated by downregulation of the XPA protein, but not other XP proteins. This contributes to the increased UV-induced skin cancer risk in organ transplant patients. 4 5
ABSTRACTS ABSTRACTS Session II: Cellular Responses to Radiation: Signalling, Cell Cycle and Apoptosis Session II: Cellular Responses to Radiation: Signalling, Cell Cycle and Apoptosis Rad5 promotes the restart of stalled replication forks via a mechanism that does not result in recombination names & affiliations Eva Petermann Gray Institute for Radiation Oncology and Biology, University of Oxford Nilklas Schultz 2, Luis Orta-Vazquez,3, Natalia Issaeva 2, Cecilia Lundin, Thomas Helleday,2 Gray Institute for Radiation Oncology & Biology, University of Oxford, Oxford, United Kingdom/ 2 Department of Genetics, Microbiology and Toxicology, Stockholm University, Stockholm, Sweden/ 3 Department of Cell Biology, University of Seville, Seville, Spain Faithful DNA replication is essential for genomic integrity and survival. Inhibition of replication can lead to stalling and collapse of forks into lethal double strand breaks (DSB). Many cancer treatments interfere with replication, and mechanisms dealing with replication fork stalling are potentially important for treatment susceptibility and resistance. Replication inhibitors like hydroxyurea (HU) strongly induce homologous recombination (HR), suggesting that HR is important for replication fork restart and repair. If forks are stalled for many hours in HU, fork-associated DSBs start being generated by the endonuclease Mus8. This supports a model of fork restart via recombination initiated by a one-ended DSB. However Rad5 forms foci at stalled forks independently of DSB generation, suggesting that it might be involved in a fork restart mechanism avoiding DSB formation, especially after short replication blocks. Here we use the DNA fibre technique to analyze how replication forks restart after different lengths of HU blocks and the roles played by Rad5 and HR in this process. We find that with increasing lengths of replication blocks replication forks become increasingly incompetent to restart, coinciding with DSB formation and HR activation. Under these conditions replication restarts largely by firing of new origins, suggesting that HR does not restart forks but performs post-replication repair of collapsed forks. However after short replication blocks, Rad5 and XRCC3 promote fork restart in a process that does not result in recombination. Our data suggest that Rad5 has distinct early and late roles in fork restart and the repair of collapsed forks. Role of the p53 gene family in the intra-s-phase checkpoint Irena Dornreiter Heinrich-Pette-Institut, Hamburg Our investigations demonstrated that the UV-induced ATR/Chk signaling pathway causes transient delays in S-phase progression and reversible inhibition of DNA replication for six hours. The key players of this pathway, which allow extended S-phase attenuation and inhibition of origin-dependent replication initiation, are transcriptional active wt p53 and transcriptional impaired wtp53, respectively. The transcriptional activity of p53 leads to p2-mediated down regulation of cyclin A-Cdk2 activity, an activity that is required for S-phase progression and processive DNA synthesis performed by polymerases δ and ε. Down regulation of cyclin A-Cdk2 activity is a prerequisite for facilitating interaction of wtp53 with hypophosphorylated, origin-competent DNA polymerase α-primase (Pol α), an event that prevents origin loading of Pol α and consequently initiation of DNA replication. Therefore, the p53- and the p53-dependent sequential branches of the ATR-mediated intra-s checkpoint are essential to slow down S-phase progression and to prevent origin firing. Apart from replication inhibition, the intra-s checkpoint provides another critical function: error-free repair of damaged DNA before DNA replication is initiated. The assumption is based on the fact that inactivation of p53 or its gene product p2 abrogates extended S-phase attenuation, resulting in accelerated, faulty repair of mutagenic (6-4)PP lesions giving rise to secondary DNA damage. Moreover, UV-irradiated cells proficient in the ATR- p53-p2-cyclin A-Cdk2 pathway but deficient in functional wtp53 initiate origin-dependent DNA replication and acquire DNA double strand breaks in the absence of processive DNA replication. Although secondary DNA damages are repaired, these cells may have already missed their best opportunity to perform error-free repair before replication is initiated. 6 7
ABSTRACTS ABSTRACTS Session II: Cellular Responses to Radiation: Signalling, Cell Cycle and Apoptosis Session II: Cellular Responses to Radiation: Signalling, Cell Cycle and Apoptosis The phosphatase inhibitor suramin differentially modulates radiosensitivity of p53 wildtype and p53 mutant tumor cells and of normal fibroblasts Petra A. Ohneseit, Heidrun Löffler, Rainer Kehlbach 2, Mahmoud Toulany, H. Peter Rodemann Division of Radiobiology and Molecular Environmental Research, Department of Radiation Oncology, 2 Department of Diagnostic Radiology, University of Tübingen, Germany The purpose of this study was to investigate the influence of suramin, a non-specific growth factor antagonist and proteintyrosine phosphatase inhibitor, in modulating radiation-stimulated membrane receptor activities as well as clonogenic survival in different cell lines. To investigate radiosensitizing properties of suramin prostate carcinoma cell lines PC3 and DU45, bronchial carcinoma cell line A549, glioblastoma cell line U87MG, wildtype and syngenic K-Ras mutated (vector from K. Giehl, Ulm) head and neck cancer cell lines FaDu, as well as normal skin (HSF, HSF7) and fetal lung fibroblasts (HFL) have been pretreated with suramin (0.05; 0.; or 0.2 mm) prior to irradiation with single doses (; 2; 4; 6 Gy) and twice during colony formation assay. The possible influence of suramin on the cell cycle has been investigated by FACS analysis. Treatment with the phosphatase inhibitor suramin affected radiation response in a concentration-dependent manner. The different cell lines investigated reacted differentially, but independent of K-Ras status. Importantly, tumor cells harbouring a mutant p53 gene (PC3, DU45, FaDu) were radiosensitized by suramin, while those with wildtype p53 gene (A549, U87MG) remained unaffected. However, suramin treatment exerted a radioprotective effect on normal fibroblasts. Preliminary results of cell cycle analysis seem to support these findings. While A549 cells were almost not affected, suramin in combination with IR increased the amount of cycling FaDu cells, but strongly decreased the proportion of cycling HFL cells, inducing both G and G2/M arrest. The impact of p53 is under further investigation. Genetic flexibility in the adaptive immune system Berit Jungnickel Institute of Clinical Molecular Biology and Tumor Genetics, Helmholtz Zentrum München Maintenance of genetic integrity is a prerequisite for survival, and is thus ensured by a complex network of DNA repair mechanisms. In some instances, though, genetic changes may be advantageous for fitness. A prototypic example is the adaptive immune system of vertebrates, which uses genetic mechanisms to produce a vast array of antigen receptors that can fight all potential pathogens. After assembly of functional receptor genes by rearrangements, the antigen receptors produced by B lymphocytes (immunoglobulins, Ig) can be further diversified by targeted mutagenesis. This process, somatic hypermutation, is initiated by cytidine deamination by AID (activation induced cytidine deaminase) in the rearranged portion of the Ig genes, and the resultant lesions are repaired by multiple error-prone processes. Its is unclear to date how the activity of AID is targeted to the Ig genes, and how and why error-prone repair is used despite multiple errorfree repair processes in the cell. We are studying the regulation and interactions of AID as well as the participation of different repair processes in somatic hypermutation. Also, we analyse the regulation of the overall process by oncogenes and tumor suppressors to investigate how hypermutating B lymphocytes prevent aberrant genetic changes and hence lymphomagenesis. 8 9
ABSTRACTS ABSTRACTS Session II: Cellular Responses to Radiation: Signalling, Cell Cycle and Apoptosis Session II: Cellular Responses to Radiation: Signalling, Cell Cycle and Apoptosis Cell death mechanisms and resistance to radiotherapy Irmela Jeremias Junior Research Group Apoptosis, Helmholtz Zentrum München Cell death is a tightly regulated process aimed at maintaining organ size and function. Cell death balances cell proliferation, and alterations in cell death rates participate in the pathophysiology of numerous diseases. Increased cell death leads to degenerative diseases such as Parkinson disease or Diabetes, while decreased cell death enables abnormal cell formation in tumors. Therefore, cell death processes require tight regulation. Various cell death programs exist which are executed in a well organized, energy consuming and programmed manner. Of these, apoptosis is best characterized and involves activation of mitochondria, caspases and DNA degradation. Manipulation of cell death represents an important therapeutic option aiming at inhibition of cell death in degenerative diseases and induction of cell death in tumors. An important approach to induce cell death in tumor cells is irradiation which is used in many current anti-cancer protocols. The intracellular signaling pathway activated by irradiation in tumor cells is not yet completely characterized, but involves apoptosis sometimes accompanied by signs of autophagy, while high dose irradiation induces necrosis. Unfortunately, tumor cells might display constitutive or acquired resistance against irradiation-induced cell death disabling effective anti-tumor treatment by irradiation. Irradiation furthermore induces cell cycle arrest in tumor cells. In our lab, we found that irradiationinduced cell cycle arrest significantly influences the anti-apoptotic effectivity of cytotoxic drugs on tumor cells. Irradiation-induced cell death might be increased by using targeted radio-sensitizers. To increase the efficiency of anti-cancer treatment, research aims at characterizing and overcoming the mechanisms responsible for resistance against irradiation-induced cell death. Radiation induced stress proteins Daniela Schilling, Mathias Gehrmann, Claudia Steinem, Antonio De Maio, Allen G Pockley, Michael Abend, Michael Molls and Gabriele Multhoff Klinikum Rechts der Isar für Strahlentherapie und radiologische Onkologie Tumors frequently overexpress and secrete Hsp70, the major stress-inducible member of the HSP70 family of stress (heat shock) proteins. We and others have previously demonstrated that tumors in contrast to normal tissues present Hsp70 on their plasma membrane. Hypoxia, which is known to limit the curability of tumors by ionizing radiation, has been found to further increase the cell surface density of Hsp70 on the cell surface of tumor cells. In non-stressed tumor cells anchorage of Hsp70 in the plasma membrane is enabled by an interaction with the lipid component globoyltriaoslyceramide (Gb3; Gehrmann et al. 2008). Following hypoxic stress, Hsp70 co-localizes with phosphatidylserine (PS). In line with these findings an interaction of Hsp70 and PS was confirmed in vesicle aggregation assays using artificial unilamellar PS-containing liposomes. Depending on their subcellular localization Hsp70s play dual roles in cancer immunity. On the one hand membrane-bound Hsp70 acts as a recognition structure for the innate immune system, on the other hand a cytosolic overexpression of Hsp70 has been found to protect tumors from lethal damage mediated by environmental stress. Therefore, we asked the question about the effects of extracellular added Hsp70 protein on normoxic and hypoxic tumor cells. We demonstrated that the addition of Hsp70 protein to hypoxic tumor cells significantly reduces clonogenic cell survival in normoxic (EC50=85 µg/ml) and hypoxic (EC50=55 µg/ml) tumor cells and thus promotes a better tumor killing. These data might have further clinical relevance in the context of the treatment of radioresistant tumors. References Gehrmann et al. Plos One (4):e925,2008 Schilling et al. FASEB J, accepted 2009 20 2
ABSTRACTS ABSTRACTS Session II: Cellular Responses to Radiation: Signalling, Cell Cycle and Apoptosisse Session III: Sex-Specific Reactions towards Genotoxic Agents? Variation in EGFR protein expression is correlated with cellular and tumour radiosensitivity, except for tumours with extreme EGFR gene amplification U. Kasten-Pisula, W. Eicheler 3, M. Krause 3, A. Yaromina 3, S. Meyer-Staeckling 2, J. Saker, B. Scherkl, B. Brandt 2, E. Dikomey and M. Baumann 3 Lab. of Radiobiology & Experimental Radiooncology / 2 Inst. for Tumourbiology, University Medical Center Hamburg-Eppendorf, 20246 Hamburg, Germany; 3 Dep. of Radiation Oncology, Medical Faculty and University Hospital Carl Gustav Carus, University of Technology, Dresden, Germany. Purpose: The epidermal growth factor receptor (EGFR) often is overexpressed in solid tumours, which is associated with a more aggressive phenotype. The prognostic value of the extent of EGFR expression for the outcome of radiotherapy is not clear. This project performed a systematic study with 0 squamous cell carcinoma cell lines using both, in vitro cultures as well as xenografts. Results: EGFR protein expression varies considerably between different tumour cell lines (factor of 80) with most cell lines showing a moderate variation (factor up to 45) and only some with an extreme up-regulation. Only for these latter cell lines, up-regulation of EGFR could be attributed to massive gene amplification. EGFR level was not correlated with the number of CA-repeats. For SCC cell lines, detailed analysis of EGFR distribution revealed that the main fraction of EGFR is localized in the membrane. With increasing amount of EGFR this fraction declines from 75 to 55% accompanied by a respective increase of cytoplasmic EGFR, while the nuclear amount (about 5%) remains constant. In case of moderate expression, EGFR level was found to be well correlated with both cellular (SF6Gy) and tumour radiosensitivity (TCD50). There was, however, no correlation when EGFR was characterised by an extreme up-regulation. Conclusions: Squamous cell carcinomas are characterised by a huge variation in EGFR protein expression with most cell lines showing a moderate and only few an extreme up-regulation. Only in case of moderate variation, the level of EGFR can be used as an indicator of tumour response after radiotherapy. This association does not hold for tumours with an extreme EGFR up-regulation, probably due to a deregulation of EGFR-dependent processes. Regulation of imprinted expression by macro non-coding RNAs Denise P. Barlow CeMM Center for Molecular Medicine of the Austrian Academy of Science. c/o Dr. Bohr-Gasse 9/4, Vienna Biocenter, A030 Vienna, Austria. denise.barlow@univie.ac.at Mammalian reproduction strictly requires a mother and a father who each donate a haploid chromosome set to the embryo. Classical genetics shows that most mutations are recessive which indicates that for most genes, both the maternal and paternal gene copy is equally expressed in all cells. However, a small percentage of mammalian genes (estimated at less than % of all genes) show parental-specific expression, such that a diploid cell will contain some genes expressed from the maternal chromosome and repressed on the paternal chromosome and vice versa. This arises from an epigenetic mechanism known as genomic imprinting. The existence of imprinted genes controlling essential developmental functions explains the mammalian embryo s strict requirement for both parents. In mammals, imprinted genes are clustered and at least one gene in each imprinted cluster is a long i.e., macro noncoding (nc) RNA. Most genes in an imprinted cluster show concordant parental-specific expression but the ncrna is the odd one out - and is expressed from the opposite parental chromosome. While reciprocal expression between imprinted macro non-coding RNAs and flanking mrna genes is indicative of a functional role, only two of three tested macro ncrnas, have been shown to induce imprinted gene expression. The two known functional imprinted macro non-coding RNAs (Airn and Kcnqot) are both atypical RNAPII transcripts that are very large (09kb and 9.5kb respectively), unspliced and nuclear localized. These unusual transcriptional properties may contribute to their regulatory function. I will describe our recent work on the Airn ncrna that induces parental-specific expression of three genes in the Igf2r imprinted cluster. In general our results show that expression of Airn is necessary to induce imprinted expression of flanking genes but they surprisingly indicate that transcription and not the ncrna itself, may be sufficient for its regulatory function. REFERENCES: Pauler FM, Koerner MV, Barlow DP. Silencing by imprinted noncoding RNAs: is transcription the answer? Trends Genet. 2007 Jun;23(6):284-92. Review. Stricker SH, Steenpass L, Pauler FM, Santoro F, Latos PA, Huang R, Koerner MV, Sloane MA, Warczok KE, Barlow DP. Silencing and transcriptional properties of the imprinted Airn ncrna are independent of the endogenous promoter. EMBO J. 2008 Dec 3;27(23):36-28. Latos PA, Stricker SH, Steenpass L, Pauler FM, Huang R, Senergin BH, Regha K, Koerner MV, Warczok KE, Unger C, Barlow DP. An In Vitro ES Imprinting Model Shows That Imprinted Expression Of The Igf2r Gene Arises From An Allele-Specific Expression Bias. Development 2009 Jan 3;36(3) in press. 22 23
ABSTRACTS ABSTRACTS Session III: Sex-Specific Reactions towards Genotoxic Agents? Session III: Sex-Specific Reactions towards Genotoxic Agents? From Marie Curie to SNPs: responses to radiation Janet Hall Institut Curie Research, INSERM U62, Orsay, France Women have a longer life expectancy than men and a better survival of chronic diseases like cardiovascular disease and cancer. The molecular and cellular basis of these sex-specific sensitivities are poorly understood, although sex hormone patterns probably play a role. DNA repair systems maintain the integrity of the human genome and cell cycle checkpoints are a critical component of the cellular response to DNA damage. Thus the presence of variants in genes involved in these pathways which modulate their activity might impact on cancer risk. Many molecular epidemiological studies have studied the association between variants, and in particular single nucleotide polymorphisms (SNPs), and cancer risk and responses to therapy. For example ATM SNPs have been associated with an increased risk of breast, prostate, leukaemia, colon and early onset lung cancer and OGG SNPs and the intron 3 6-bp repeat in TP53 with an increased risk of lung cancer. In contrast the variant allele of the rare CHEK2 missense variant (rs787996) was significantly associated with a lower incidence of lung and upper aero-digestive cancers. There is also a small but growing body of evidence that sequence variants within DNA damage response genes may contribute to therapeutic responses to chemotherapy and radiotherapy and normal tissue response to radiotherapy. The challenge is now to understand the molecular mechanisms underlying these associations and to assess whether sex-specific differences exist. Sex-specific aspects of individual radiosensitivity Kerstin Borgmann Laboratory of Radiobiology & Experimental Radiooncology, Center of Oncology, University Medical Center Hamburg-Eppendorf Kerstin Borgmann*, a, Ekkehard Dikomey a, Cordula Petersen b, Petra Feyer c, Ulrike Hoeller c a Laboratory of Radiobiology & Experimental Radiooncology, Clinic of Radiotherapy and Radiooncology, University Medical Center Hamburg-Eppendorf, Hamburg, b Practice for Radiotherapy Hamburg, c Vivantes Klinikum Neukoelln, Berlin, Germany There is increasing evidence that sex-specific differences in toxicity profiles and outcome after tumortherapy is accumulating in medical oncology and treatment strategies may be modified. Furthermore, the sex-specific sensitivity to genotoxic and therapeutical agents is of general concern for risk estimation. In this context biological endpoints with respect to sex-specific differences of tumour and normal tissue sensitivity after irradiation as well as the respective underlying molecular and cellular mechanisms were of great interest. Resuming the literature, it is obvious that there are sex-specific differences of radio sensitivity of both, tumor and normal tissue, as well as the respective underlying mechanisms. The data available so far are limited and, as a consequence, final conclusions are not yet possible. The impact of sex differences in effects of treatment should be considered in both experimental studies as well as clinical trials, e.g. sex should be stratified for in clinical trials and results reported sex-specifically. Ideally, the numerous questions, for instance toxicity of, access to and compliance with therapy and finally outcome of treatment, should be addressed in adequately designed and sufficiently sized trials. 24 25
ABSTRACTS ABSTRACTS Session III: Sex-Specific Reactions towards Genotoxic Agents? Session III: Sex-Specific Reactions towards Genotoxic Agents? Do women survive longer? Sex and cancer therapy from a clinical perspective Ulrike Hoeller, Cordula Petersen*, Kerstin Borgmann** Radiation Oncology, Charité Campus Mitte, Berlin * Praxis für Strahlentherapie, Hamburg, ** Laboratory of Radiobiology & Experimental Radiooncology, University Medical Center Hamburg-Eppendorf, Hamburg Outcome of tumor therapy depends on the biologic properties of the tumor itself and on a multitude of host factors. In medical oncology, evidence on sex specific differences in toxicity profiles and outcome has been gathered during the recent years. This overview is focussed on the specific effects of sex/gender on effectiveness and toxicity of radio(chemo)therapy from a clinical point of view. The literature was reviewed with respect to sex specific differences of tumour response and normal tissue sensitivity to irradiation. In clinical studies, sex is routinely tested as potentially prognostic factor. However, it is essential to analyse results in a sex specific way in order to establish or refute the impact of sex on the outcome of tumor therapy. Recently, sex specific, pooled analyses of large, comparable trials pointed to sex specific differences in the outcome of Hodgkin lymphoma and small cell lung cancer. The specific impact of sex on the observed differences in tumour control may be either truly sex related and/or linked to any of the underlying conditions pertinent to tumor control, e.g. age, comorbidities, tumor extent at first presentation. The interactions of prognosis relevant factors is complex and will be discussed for patients with rectal and lung cancer. Gender as opposed to sex encompasses the cultural and social role and personal identity attributed to man- or womanhood. Gender has an impact on health behaviour and may be related to therapy outcome. Therefore sex/gender specific aspects should be included in clinical studies in order to optimize treatment strategies. Proteomics approach in radiation-induced cardiovascular diseases F. Pluder, S. Steininger, R. Nylund 2, D. Leszczynski 2, M.J. Atkinson and S. Tapio Institute of Radiation Biology, Helmholtz Centre Munich German Research Center for Environmental Health (GmbH), Ingolstaedter Landstrasse, 85764 Neuherberg, Germany 2 BioNIR Research Group, STUK Radiation and Nuclear Safety Authority, Helsinki, Finland Cardiovascular diseases represent one of the main causes of morbidity and mortality in the world. Recent epidemiologic evidence suggests that medium and low radiation doses to the heart result in a moderate, but significant increase in cardiovascular mortality. The aim of this study is to identify via a proteomic approach new molecular targets involved in the pathogenesis of radiation induced cardiovascular disease. Since the vascular endothelium plays a pivotal role in the regulation of vascular function it is therefore addressed as the target tissue in this study. As model system we studied the human endothelial cell line Ea.hy926. To characterize the impact of low dose radiation induced changes we investigated different time points (4h and 24h) as well as dose rates of radiation (Co-60 gamma ray total dose 200mGy; 4 mgy*min- and 40 mgy*min- ). Subfractionation of the whole cell lysate was performed by differential detergent fractionation. The chosen proteomic strategy is based on two-dimensional gel electrophoresis enhanced by the use of the Difference gel electrophoresis (DIGE) technology. Differentially expressed proteins were identified by in-gel trypsin digestion, MALDI-TOF/TOF tandem mass spectrometry, and peptide mass fingerprint analyses. Out of more than fifty protein spots that showed significant alterations in their expression we were able to identify 22 proteins, of which were up-regulated and down-regulated; 7 proteins showed more than +.5-fold difference compared to the unexposed cells. Among the pathways affected by the low dose of ionising radiation are Ran and Rho/Rock pathways, stress response and glycolysis. The 2-way-Anova analysis shows that some protein alterations are time-, dose-rate-, or interaction-dependent whereas others occur in a general, independent manner. We are able to show that the relatively low dose of 200 mgy gamma radiation alters the endothelial cytoplasmic proteome significantly during the first 24 h. These results demonstrate the importance of proteomics in finding new biological target molecules of a low-dose radiation response in a critical tissue. 26 27
ABSTRACTS ABSTRACTS Session III: Sex-Specific Reactions towards Genotoxic Agents? Session III: Sex-Specific Reactions towards Genotoxic Agents? Increased oxidative stress in cardiac mitochondria even 4 weeks after heart-focused low dose ionizing radiation Dominik Schmaltz *, Hans Zischka 2, Julia Haagen 3, Wolfgang Dörr 3, Michael J. Atkinson, Soile Tapio Institute of Radiation Biology, 2 Institute of Toxicology, Helmholtz Center Munich, German Research Center for Enviromental Health; 3 Department of Radiotherapy and Radiooncology, Medical Faculty Carl Gustav Carus, University of Technology, Dresden, Germany Low doses of ionizing radiation induce cardiac disease, as demonstrated by epidemiological studies. We provide for the first time experimental evidences for this causality in form of increased oxidative stress in cardiac mitochondria even 4 weeks after low dose heart-focused ionizing radiation. Similar to aging-effects of the heart, it takes several decades for radiation-induced cardiac diseases to develop and to become clinically obvious in terms of myocardial infarction, ischemic heart disease, heart failure and coronary disease. Since mitochondrial dysfunctions in aged hearts have been often reported, we analysed possible functional alterations in mitochondria isolated from irradiated and sham-irradiated murine hearts 4 weeks after heart-focused low dose irradiation. The mitochondrial assays included respiratory coupling, production of ATP, oxidative stress and swelling of mitochondria. We found enhanced amounts of free radicals and indication of less effective consumption of oxygen to produce ATP in cardiac mitochondria after 2 Gy irradiation vs. sham-irradiation. No differences between non- and irradiated cardiac mitochondria were displayed in swelling and respiratory coupling. However, we could identify increased oxidative stress in cardiac mitochondria as a chronically manifested, pathologic alteration 4 weeks after low dose heart-focused ionizing radiation. This result underlines the disadvantageous side effect of radiotherapy causing low-dose exposure on heart and consequently the potential importance of co-application of antioxidants with the therapy. Women in radiation sciences some glances at history Friederike Eckardt-Schupp Institute of Radiation Biology, Helmholtz Zentrum München, The restrictions in the higher education of girls and in the academic career of female students and scientists at the end of the 9th and beginning of the 20th century were enormous. Despite that, some excellent female scientists were amongst the first to explore the effects of radiation, and I want to acknowledge their outstanding scientific work. Some were extremely successful like Marie Curie, her daughter Irene Joliot-Curie and Maria Goeppert-Mayer who won Nobel Prizes in Physics and Chemistry, or like Charlotte Auerbach and Paula Hertwig, who had remarkable academic careers as radiation biologists. Others suffered from the extreme hardship that they were not sufficiently acknowledged despite their extraordinary work like Lise Meitner and Rosalin Franklin, who both deserved the Nobel laureate due to their outstanding scientific creativity and did not obtain it due to an extremely hostile environment. Studying the curricula vitae of these women, some general features are obvious: All these women were absolutely dedicated to science. They were determined to cope with all problems which made them suffer and the social and political restrictions which were very hard especially for those of Jewish origin in the times of National Socialism in Europe. Looking into their biographies it is touching to discover their dedication as mothers, their warm relationship to friends, colleagues and their students, and their social and political engagements. In the middle of the 20th century, after the discovery that DNA is the hereditary material (944) and its double-helical structure by Rosalin Franklin, J. Watson and F. Crick (953), a few women participated in the exploration of DNA functions, in particular, of DNA repair. Ruth Hill and Evelyn Witkin isolated the first bacteria mutants which were radiation-resistant resp. radiation sensitive threatening target theory and supporting the notion of a complex cellular response to radiation damages. Radioactive labelling techniques were invented, and Alma Howard was the first to explore and describe the cell cycle in eukaryotic cells. And last not least, our honoured guest, Ethel Moustacchi contributed in many fields of modern research on DNA repair and many of its applied aspects. 28 29
ABSTRACTS ABSTRACTS Session IV: Hot Spicy Topics in Radiation Sciences Session IV: Hot Spicy Topics in Radiation Sciences Microbeam and single cell irradiation Anna A. Friedl University of Munich, Institute of Cell Biology By localized micro-irradiation of single cells or subcellular regions open questions in the field of cellular radiation response can be tackled. For example, the sites of protein accumulations in cells can be correlated with known damage sites induced by micro-irradiation. So far, mainly UV laser micro-irradiation has been used to demonstrate accumulation of a variety of proteins in so-called foci. Since DNA lesions induced by laser micro-irradiation are poorly characterized, it remained unclear to what extent these observations are transferable to damage induced by ionizing radiation. We compared foci obtained after UV laser irradiation and after irradiation with 00 MeV O, 55 MeV C or 35 MeV Li ions over a time course of 24 h using an object counting approach to characterize the fine structure of the foci independently of fluorescence intensity thresholds. The results hint at comparable foci fine structures, especially at later time points. As the ion microbeam SNAKE allows to target single ions with high spatial accuracy, geometric irradiation patterns can be generated. With this approach we investigate how the distribution of chromatin in the cell nucleus affects the distribution of foci. We observe γ-h2ax foci preferentially at the surface of chromatin domains, where also transcription and replication processes are known to be performed. By applying geometric irradiation patterns it is also possible to irradiate cells twice and differentiate foci generated after the first and the second irradiation experiment. Using this approach, we observe that the formation of 53BP and Rad5 foci is inhibited in pre-irradiated cells, while γ-h2ax, Mdc and Phospho-ATM foci are not affected. DNA repair in artificially modified chromatin B. Mazurek (p), D.M. Seiler, S. Breitkopf G.A. Drexler, C Greubel 2, V. Hable 2, G. Dollinger 2, S. Dietzel 2, T. Cremer 4, A.A. Friedl Ludwigs-Maximilians-Universität München Strahlenbiologisches Institut, D-80336 München, Germany 2 Universität der Bundeswehr München, Angewandte Physik und Messtechnik, LRT2, D85579 Neubiberg, Germany 4 Ludwigs-Maximilians-Universität München Anthropologie und Humangenetik, Dept. Biologie II, D-8252 Planegg-Martinsried, Germany Recent findings show that the organization of the chromatin plays not only an important role in biological processes like transcription, cell differentiation and X inactivation but also in the DNA-damage response. The ion-microbeam SNAKE (Superconducting Nanoscope for Applied Nuclear (=Kern-) Physics Experiments) at the Munich tandem accelerator facility is an excellent tool for addressing the questions of chromatin dynamics after DNA damage. After applying single ions, DNA damage response factors which accumulate at irradiation induced foci (IRIF) can be visualized in fixed cells using immunofluorescence techniques. Forcing the chromatin into hypercondensation by treatment with high salt concentration or hypocondensation by treatment with a low salt concentration or the application of Trichostatin A (histone deacetylase inhibitor), after ion-micro-irradiation leads to an inhibition of foci formation depending on the osmolarity of the medium. Treating the cells with a high salt concentration of 750 mosm leads to a total inhibition of foci formation for the DNA damage response factors 53BP, phospho-atm, phospho-nbs, BRCA, MDC and Rad5. Only the γ-h2ax foci formation is unaffected. Usage of a hypertonic medium with 570 mosm or hypotonic medium (~40 mosm) only restricts the Rad5 Foci formation. Incubation with Trichostatin A has no effect on any of these proteins. This is a good example of the importance of chromatin structure in the recruitment of DNA repair and signalling factors. In subsequent western blots, it was observed that this effect was not only caused by a general inaccessibility of the chromatin but also through variations in the amount of protein inside the nucleus. 30 3
ABSTRACTS ABSTRACTS Session IV: Hot Spicy Topics in Radiation Sciences Session IV: Hot Spicy Topics in Radiation Sciences Clustered DNA damage: Spatiotemporal dynamics and cellular effects Gisela Taucher-Scholz GSI Biophysik, Darmstadt, Germany The spatial distribution (e.g. clustering) of double-strand breaks (DBSs) is crucial regarding their repair capability and biological effects. On simple DNA plasmid molecules, the formation of multiple nearby DSBs was detected by Atomic Force Microscopy. In mammalian cells, upon DSB immunostaining ion irradiation leads to streaks of spatially defined damaged chromatin domains across the nuclei. Loss of γh2ax foci over time indicates efficient DSB repair after carbon ion irradiation, but repair was less pronounced with increasing ionizing density. Repair impairment and an increased DSB production by stopping carbon ions are exploited in heavy ion radiation therapy, combining a tumor conform dose deposition with an enhanced cell killing effectiveness in the tumor region. Taking advantage of fast foci formation and delayed repair, the motional activity of DSBs was also monitored in live cells up to 2 hours post-irradiation. A fast small-scale Brownian motion of GFP-tagged repair protein foci was observed independent of irradiation. A superimposed slow mobility of damaged domains (mean square displacement about 0.6 µm2/h), most likely driven by normal chromatin diffusion, was additionally observed independent of the radiation type. Only occasionally, larger translational motion connected to morphological changes of the nucleus or a (transient) formation of multi-centered repair clusters could be observed. In conclusion, damaged chromatin shows a restricted mobility independent of lesion density and irradiation, supporting the idea that the spatial proximity of DNA breaks is required for the formation of radiation-induced chromosomal exchanges. Advances in radiotherapy at the interface between biology, diagnostics, physics and technology Barbara Röper Klinikum rechts der Isar, Department of Radiation Oncology, Technische Universitaet Muenchen, Muenchen, Germany All advances in radiotherapy (RT) aim for improving the therapeutic ratio, i.e. better tumor control, less treatment-related toxicity or both. i) Modern diagnostics do not only rely on morphological tools and image fusion between them (US, CT, MRI), but also on functional imaging (PET), providing complementary data about the extent of the tumor, its spread and metabolism. Thus, the choice of appropriate treatment elements and target volume definition including a biological target volume can be based on the patient s individual situation. ii) Advances in technology brought up tools for the optimization of dose distribution to a target volume of any shape. Along with the availability of intensity-modulated radiotherapy (IMRT), concepts of deliberate inhomogenous dose distributions have emerged that are presently under clinical evaluation (simultaneous integrated boost=sib). iii) The precision of dose delivery to the target volume is enhanced by stereotactic techniques and / or daily image-guidance in the treatment position (cone-beam CT, MV-CT, tomotherapy). Even internal organ movements or shrinkage of tumor can be accounted for (e.g. breathing control / adaptive RT). All three aspects contribute to the precision of targeting the stem cell-like tumor cells and sparing normal tissue. Thus, dose escalation is possible with a higher percentage of uncomplicated cure. Further steps, like developing coherent X-rays for diagnostics which visualize tissues in vivo on a histological level or promoting the availability of hadrons by laser-based technology as pursued in the framework of the excellence cluster Munich-Centre for Advanced Photonics (MAP), may lead to RT concepts of the future. 32 33
ABSTRACTS ABSTRACTS Session IV: Hot Spicy Topics in Radiation Sciences Session IV: Hot Spicy Topics in Radiation Sciences UV-radiation, epidermal stem cells and skin cancer Beate Volkmer Krankenhaus Buxtehude, Dermatologie There is increasing evidence that adult stem cells play an important role in cancer development. This is especially true for highly proliferating tissues like skin. Due to special attributes like long persistence, rare cycling activity in-vivo, high proliferation capacity (in-vitro) and damage retainment, epidermal stem cells are the ideal target for the carcinogenic effectiveness of UV-irradiation. In UV-exposed skin areas we found cells accumulating UV-induced DNA-damage like cyclobutane-pryrimidine-dimers (CPD) called CPD retaining cells (CRBC). The cells are located interfolicular in the basal layer predominantly in the deep rete ridges and show several attributes of adult stem cells like damage retainment and long persistence. Since numerous CRBCs are induced in every UV-exposed skin area CRBCs may characterize cancer precursor cells in the way that some of the damaged cells will develop into cancer (stem) cells in dependence of genetic and epigenetic changes in the daughter cells after proliferation. In-vitro investigations showed that damaged cells will repair their genome if they start to proliferate including possibilities of induction of genetic mutations and epigenetic modifications. In skin sections it could be shown that the epigenetic pattern of CRBCs differs from that of undamaged basal cells. Since stem cells as well as cancer (stem) cells seem to be characterized by a specific epigenetic pattern, we started to investigate whether UV induces epigenetic changes which can be found at a later time point in cancer (stem) cells. This may increase our knowledge about mechanisms of (skin) cancer development. Irradiated human hematopoietic stem and progenitor cells show an altered differentiation pattern, apoptosis, but no genetic instability Daniela Becker,2, Sylvia Ritter, Thilo Elsässer, Erhard Seifried 2, Torsten Tonn 2, Marco Durante, Claudia Fournier GSI, Darmstadt, Germany; 2 Institute for Transfusion Medicine and Immune Haematology, Frankfurt, Germany Human hematopietic stem and progenitor cells (HSPC) potentially contribute to acute and late effects during radiation treatment when located in the exposed part of the normal and tumour tissue. In particular the response to densely ionizing irradiation is becoming increasingly interesting, especially for carbon-ion cancer therapy and radiation protection (deep space). CD34+ enriched HSPCs, isolated from the peripheral or cord blood of healthy donors were exposed to X-rays and carbon ions as used in particle therapy (SIS, GSI). After exposure, the cells were cultured in semisolid methylcellulose, supplemented with cytokines (CFU assay). Two weeks later, the colony forming capacity and the differentiation potential of progenitors and lineage restricted progenitors of the erythroid, granulocytic and monocyte-macrophage pathways was assessed by morphological features. In parallel, cell death, progression in the cell cycle, and the occurrence of DNA and chromosomal damage were measured in mass cultures and in colonies grown in methylcellulose. During the first days after exposure, part of the HSPCs underwent apoptosis, but no pronounced cell cycle arrest. The progeny of irradiated cells displayed clonal expansion of cytogenetic damage, but in contrast to mature lymphocytes, no genetic instability was observed. The CFU assay reveals a high radiosensitivity for HSPCs and a low relative biological effectiveness of carbon ions for inactivation (<2). Interestingly, after 2 weeks an enhanced fraction of mature cells for all lineages and enrichment in erythroid lineage cells were observed, suggesting that after irradiation the blood cell type distribution will be altered. (F&E 78; BMBF 02 S 8497). 34 35
ABSTRACTS LIST OF POSTERS Session IV: Hot Spicy Topics in Radiation Sciences Modulation of mirna expression after ionizing radiation Simone Moertl, Anne Krämer, Natasa Anastasov and Friederike Eckardt-Schupp Institute of Radiation Biology, Helmholtz Center Munich German Research Center for Environmental Health GmbH, Germany MicroRNAs (mirnas) are a large family of non-coding, small RNAs, which regulate gene expression through base pairing with target mrnas at the 3'-terminal untranslated regions (3'-UTRs), leading to mrna cleavage or translational repression. Disease related deregulation in mirna expression has most notably been identified in cancer. In fact, mirna expression profiles accurately classify various tumours, and thus provide new diagnostic biomarkers. Recently, mirnas have been implicated in cellular stress response such cytotoxic treatment. However, the role of micro RNAs and their involvement into the radiation signaling pathways is largely unknown. We analysed the mirna response in human T-lymphocytes induced by 37 Cs-gamma radiation. To obtain a general pattern radiation induced mirnas we analysed samples from several donors. For our analysis we used highly multiplexed RT primers for cdna synthesis and a low density mirna array, which allows the simultaneous evaluation of 384 mirnas. MiRNA expression profiling was done 24h after 2Gy irradiation. Relative quantification of mirna expression revealed that about 00 mirnas are expressed in T-lymphocytes, at which the levels of about 30 mirnas significantly changed after irradiation. Comparison of different donors suggest about 0 common mirnas which are upregulated in response to ionizing radiation and 5 common mirnas downregulated in all donors. In summary our experiments should enable us to identify a universal panel of mirnas, which are specifically altered by ionizing radiation. In future these mirnas could be used as biomarkers or biodosimeter in medical applications of ionizing radiation. A Rad6 Group Members influence plasmidial Gap Repair Fred Ahne A 2 A study of DNA repair gene polymorphisms in relation to G2 chromosomal radiosensitivity Gillian Curwen and cancer predisposition A 3 Role of genetic susceptibility in the incidence of radiation induced thyroid cancer in mice Claudia Dalke A 4 Histological evaluation and elucidation of functional aspects concerning osteosarcoma Tanja Domke development in a murine model: Th227-induced osteosarcomas in Rb- conditional and p6 constitutive knockout-mice A 5 Chromosomal instability induced by 0.5 Gy mammography X-rays in cells from biopsies of BRCA Marlis Frankenberg-Schwager and BRCA2 mutation carriers A 6 Induction of alt pathway in radiation induced osteosarcomas Iria Gonzalez Vasconcellos A 7 Structural and functional studies of smc proteins: how is dna trapped inside the ring? Julia Griese A 8 Cytogenetic characterisation of transgenic TRK-T and RET/PTC3 mouse cases Kathrin Heiliger A 9 Copy number aberrations in post-chernobyl childhood papillary thyroid carcinoma (PTC) Julia Hess detected by array-cgh A 0 The role of the MRN (Mre, Rad50, Nbs)- complex in the base excision repair Carina Kröger A Testing Flavopiridol and ionizing radiation as a possible treatment for lung cancer in a murine model Sophie Lindemaier A 2 Effects of histone acetyl transferase Tip60 recruitment to complex DNA lesions Barbara Meyer A 3 Development of an alkaline Comet Assay in the EpidermFT TM 3D human skin model for detecting Dörte Michalski DNA single- and double-strand breaks A 4 Influence of Single Nucleotid Polymorphisms (SNPs) in ATM, GSTP, SOD2, TGFß, XPD and XRCC Annette Raabe on cellular and clinical radiosensitivity A 5 Mapping of Allelic Imballances in Radiogenic Murine Osteosarcoma using Whole Genome MD SNP Christine Rümenapp Arrays reveales a new potential Tumor-Suppressor-Locus on Mmu 3 A 6 Genetic and biochemical analysis of base excision repair complexes Ulrike Schötz A 7 Analysis of radiation-induced changes in epigenetic information Doris Seiler A 8 A new role of the MRX complex in base excision repair Sylvia Steininger B Analysis of molecular mechanisms of radiation-induced apoptosis Marita Angermeier B 2 An assessment of the effects of radiation quality on the interplay between the PI3K/Akt signalling May Eid pathway and DNA repair B 3 Uptake of caesium and strontium via roots and leaves in Arabidopsis thaliana Ulrike Kanter B 4 Are differentially regulated let-7 mirnas responsible for increased radiosensitivity in EA.hy926 Anne Krämer compared to A549 cell lines? B 5 RAD5 overexpression reduces replication fork progression Ann-Christin Parplys C Changes in mirna expression profiling after ionizing radiation in cardiac endothelial cell line Natasa Anastasov C 2 Radiobiological effects of pulsed versus continous proton beams Susanne Auer C 3 e.era - The electronic European Radiobiological Archives database reanimates old data Mandy Birschwilks C 4 STORE Sustaining access to Tissues and data from Radiobiological Experiments Mandy Birschwilks (2. Abstract) C 5 Investigation of radiopharmaceutical potential of radiolabeled bevacizumab Ilkmur Demir C 6 Development of a biophysical treatment planning system for the FRM II neutron therapy beamline Sylvia Garny C 7 The German Uranium Miners Biobank GUMB Maria Gomolka C 8 Real time assessment of individual radon exposure Eva Gruber C 9 Thermoluminescence Dosimetry of 90 Sr in teeth of the population living in the Techa River area, Natalia Semioschkina Southern Ural, Russia C 0 Analyzing the acute responses of cells to low dose irradiation through proteomic studies Arundhathi Sriharshan C Analysis of gamma-h2ax foci formation after irradiation with pulsed and continuous proton beams Olga Zlobinskaya 36 37
POSTER POSTER Session A: DNA Repair and Disease Poster A2 Poster A Rad6 group members influence plasmidial gap repair F. Ahne, G. Karras*, F. Eckardt-Schupp and S. Moertl Institute of Radiation Biology, Helmholtz-Zentrum München, German Research Center for Environmental Health, Post Box 29, Ingolstaedter Landstrasse, D-85764 Neuherberg, Germany * Department of Molecular Cell Biology, Max-Planck Institute of Biochemistry, Am Klopferspitz 8, D-8252 Martinsried, Germany The RAD6 pathway allows replication across DNA lesions by either an error-prone or error-free mode. Error-prone replication involves translesion polymerases and requires mono-ubiquitylation at lysine (K) 64 of PCNA by the Rad6 and Rad8 enzymes. By contrast, the error-free bypass is triggered by modification of PCNA by K63-linked polyubiquitin chains, a reaction that requires in addition to Rad6 and Rad8 the enzymes Rad5 and Ubc3-Mms2. Here, we show that the RAD6 pathway is also critical for controlling repair pathways that act on double-strand breaks. By using gapped plasmids as substrates, we found that repair in wild-type cells proceeds almost exclusively by homology-dependent repair (HDR) using chromosomal DNA as a template, whereas non-homologous end-joining (NHEJ) is suppressed. In contrast, in cells deficient in PCNA polyubiquitylation, plasmid repair occurs largely by NHEJ. Mutant cells that are completely deficient in PCNA ubiquitylation, repair plasmids by HDR similar to wild-type cells. These findings are consistent with a model in which unmodified PCNA supports HDR, whereas PCNA monoubiquitylation diverts repair to NHEJ, which is suppressed by PCNA polyubiquitylation. More generally, our data suggest that the balance between HDR and NHEJ pathways is crucially controlled by genes of the RAD6 pathway through modifications of PCNA. A study of DNA repair gene polymorphisms in relation to G2 chromosomal radiosensitivity and cancer predisposition Curwen Gillian B, Murphy Samantha 2, Tawn E Janet 2, Winther Jeanette F 3, Boice Jr John D 4,5 Westlakes Research Institute, Cumbria, UK 2 University of Central Lancashire, Cumbria, UK 3 Institute of Cancer Epidemiology, Copenhagen, Denmark 4 International Epidemiology Institute, Rockville, USA 5 School of Medicine, Vanderbilt University, Nashville, USA Increased G2 chromosomal radiosensitivity has been reported for a range of cancers and family studies have indicated heritability of the radiosensitivity phenotype. Polymorphisms of DNA repair genes may explain the genetic basis of chromosomal radiosensitivity. We have previously examined polymorphisms in DNA repair genes in a group of 23 Danish childhood and adolescent cancer survivors, their 23 partners and 37 offspring who were being studied for G2 chromosomal radiosensitivity. A statistically significant difference between survivors and partners was observed for the APEX Asp48Glu site and associations with G2 chromosomal radiosensitivity were suggestive, but not significant for the XRCC3 Thr24Met and hogg Ser326Cys SNP sites (Wilding et al, Environ Mol Mutagen 48:48-57, 2007). This study has been extended with an additional 29 cancer survivors, their 29 partners and 53 offspring. G2 chromosomal radiosensitivity of peripheral blood lymphocytes was determined using a standard assay (Smart et al, Mutat Res 528:05-0, 2003). The three previously studied SNPs in XRCC3, APEX and hogg plus hogg Arg46Gln were investigated using PCR restriction fragment length polymorphism analysis. Genotype frequencies at the four polymorphic sites did not differ between survivors and partners, nor between parents and offspring. No statistically significant associations were found between G2 radiosensitivity scores and the polymorphisms studied. A combined analysis of data from the two studies also failed to reveal any significant associations. This study failed to confirm suggestions arising from the earlier study of an association of these particular DNA repair gene polymorphisms with either cancer susceptibility or individual variation in radiosensitivity. This work was funded by a grant from the National Institutes of Health, U.S.A. (Grant Number RO CA 04666) through Vanderbilt Medical Centre, USA and a student internship from the University of Central Lancashire, UK. 38 39
POSTER POSTER Poster A3 Poster A4 Role of genetic susceptibility in the incidence of radiation induced thyroid cancer in mice Claudia Dalke, Gabriele Hölzlwimmer 2, Michael Rosemann, Michael Atkinson Helmholtz Zentrum München German Research Center for Environmental Health, Institute of Radiation Biology, 2 Institute of Pathology, Neuherberg, Germany According to the human situation, mice develop thyroid tumours at a high rate after incorporation of Iodine3 at a young age. Genes frequently affected in thyroid carcinogenesis were analysed for mutations. Furthermore the radiation susceptibility of different mouse strains was evaluated. To induce thyroid tumours in mice, females were put on an iodine-deficient diet six weeks before mating. The dams were injected after two weeks of pregnancy and one week after birth with I3 leading to an incorporated activity between four and 8 kbq in the offspring. Mice that developed thyroid aberrations were killed and the thyroids were analysed for morphologic and genetic alterations. Genomic DNA was extracted from 43 irradiated thyroids showing follicular thyroid carcinoma or adenoma, nodular or simple hyperplasia or Goiter and three unaffected thyroids. Four unirradiated thyroids served as controls. Additionally DNA was extracted from six follicular thyroid carcinomas and one hyperplasia from Thorium227 injected mice. Sequencing of the hot spot regions of Braf, ras and kit revealed no mutations in these genes. Following I3 treatment, JF x C3H mice showed significantly less carcinoma, adenoma or complex hyperplasia of the thyroid as compared to BALB/c x C3H or C3H x C57BL/6 mice. This implies that JF strain has a suppressor allele for radiation-induced thyroid cancer. This work was supported by an EU Euratom grant (GENRISK-T and GENRAD-T) Histological evaluation and elucidation of functional aspects concerning osteosarcoma development in a murine model: Th227-induced osteosarcomas in Rb- conditional and p6 constitutive knockout-mice Tanja Domke, Bahar Sanli-Bonazzi, Irene Esposito 2, Mike J. Atkinson and Michael Rosemann HMGU Institute for Radiation Biology and HMGU Institute for Pathology 2 Introduction: Th227-induced murine osteosarcomas in variable sensitive mouse strains showed an increased mutation in the areas of tumour suppressor genes Rb- and p6 in former LOH-studies. This leads to the presumption of predisposed germline mutations. Methods: A mouse model with 350 Th227-injected mice and about 20 control mice was established and was divided into three groups. The first group was represented by bone specific Rb- conditional knockout mice (RT), the second one by p6 constitutive mice (PT) and the third group a crossbred between C3H and a Japanese wildtype formed the third group (JT). Following injection of the bone-seeking alpha-emitter Th227 at the age of approximately 3 month, the animals were monitored for the development for bone- or other malignancies. Osteosarcoma were diagnosed radiologically and validated by histopathology. Results: Proportionately, most osteosarcomas were developed by the RT mice, followed by the JT and the PT mice. Apart from the different bone-tumour incidences in the three mouse lines, the tumors exhibit significant histological differences. Whereas the mice of the PT group (bearing a P6 knowck-out alelle) developed well-structured typical osteosarcoma with plenty of osteoid, the bone-tumours of the other two groups (conditional Rb-knockout and C3HxJF hybrids) were more variable in morphology and even showed either an anaplastic type or were high in spindle-type cells. C3H x JF-hybrid mice (JT) appeared resistant for the development of other tumour types, whereas PT mice (carrying a P6 knockout-allele) developed in addition to osteosarcoma different kinds of lymphomas. Conclusion: The above mentioned study is an ongoing experiment but preliminary we conclude that there must be differences in the origin of the osteosarcomas in the different groups, which has to be proved by the examination of functional aspects like differentiation, genome stability, cell-cycle regulation or senescence. To allow detection of osteosarcoma-specific tumor markers we currently use paraffin-embedded blocks of mouse osteosarcoma cell-lines to validate the usability of a panell of antibodies, which can later be used for immunohistochemistry of the tumour tissue. Proteomics will follow to get the most precise protein status within the osteosarcomas. 40 4
POSTER POSTER Poster A5 Poster A6 Chromosomal instability induced by 0.5 Gy mammography X-rays in cells from biopsies of BRCA and BRCA2 mutation carriers M. Frankenberg-Schwager, A. Gregus Dep. of Nuclear Medicine, University of Göttingen, Germany Women who are carriers of a BRCA mutation have a high risk to develop breast cancer and are advised to undergo early and frequent mammography screening. BRCA/2 proteins are partners in a complex required for the error-free repair of radiationinduced DNA double-strand breaks by homologous recombination. Since an un- or misrepaired double-strand break may lead to genomic instability and finally to cancer we asked the question whether mammography is a suitable screening method for familial predisposed women. Fibroblasts of three BRCA+/- carriers with different terminal truncation of the protein, three BRCA2+/- carriers with the same truncation, one Fanconi anaemia patient with a biallelic mutation (BRCA2-/-) and three controls were exposed to 29 kv mammography X-rays. Chromosomal anomalies (Giemsa staining and chromosome X-, -, 9-, 3- and 7-sepecific FISH) were scored at. mitosis and several population doublings post-irradiation. Aneuploidy with great variation of chromosome numbers as typical for cancer cells was observed for BRCA+/- cells with the smallest truncation (C 2860), for one (C 2902) among three BRCA2+/- fibroblast lines and for the negative control FA 45 (BRCA2-/-) but only several population doublings after exposure to the lowest dose applied (0.5 Gy). The relative yield of dicentrics was enhanced 70-fold (FA 45) to 790-fold (C 2860), while total fragment formation was only increased 53-fold (C 2860) to 27-fold (FA 45), and signalling for chromatid breaks was similar in the three cell lines (23- to 39-fold). Chromosome-specific Fish-painting revealed that the smallest chromosome 7, which carries BRCA, shows the highest number of rearrangements. Induction of ALT pathway in radiation induced osteosarcomas Iria González Vasconcellos, Bahar Sanli-Bonazzi, Michael J. Atkinson, Michael Rosemann Institute of Radiation Biology, Helmholtz Zentrum Munich, Germany Introduction: Human osteosarcomas are prototype malignancies exhibiting alternative lengthening of telomeres (ALT). We have investigated the role of ALT in the etiology of radiation-induced osteosarcoma using a murine model. Methods: Cell lines were established from primary murine osteoblasts and from murine osteosarcoma following tumorinduction with a bone-seeking alpha-emitter. Primary osteoblast-cultures (derived from 2-3 month old animals) were analyzed in-vitro between passage 4 and 5. Four permanent osteosarcoma cell lines were shown to be immortal in-vitro, but were also analyzed at passage numbers not later than 5. Telomere length was measured by hybridization with a fluorescence-labelled PNA probe and FACS. Immunofluorescence for the protein PML was used as a marker of ALT. Results: Normal osteoblasts exhibit a passage-dependent decrease in the telomere-length and a PML signal, indicative of a normal age-dependent telomere-erosion without any sign of ALT activation. Osteosarcoma cell lines, in contrast, show a significantly increased telomere length (between.5 and 3.5 fold) with considerable cell-to-cell heterogeneity. Osteosarcoma cell lines, but not normal osteoblast cultures, were composed of two distinct sub-populations, which differed in their telomeresignal. PML signals were significantly increased in osteosarcoma as compared to primary osteoblasts, with clear co localisation of PML and telomeres in the former. Conclusion: Radiation-induced murine osteosarcoma show clear signs of ALT mechanisms. As compared to normal primary osteoblasts, their cells show extended and more variable telomeres length.. It is thus concluded that radiation-induced murine osteosarcoma, which can be induced in larger number and in mouse-models with various germ-line defects represent a valid in-vitro model to study the ALT mechanism. A low dose (0.5 Gy) of mammography X-rays has the potential to induce chromosomal instability in unirradiated descendents of irradiated fibroblasts of BRCA2-deficient Fanconi anaemia FA 45 and of some (C 2860, C 2902) but not all BRCA/2 fibroblasts derived from mutation carriers. These results may initiate to think about an individualization of screening methods for BRCA mutation carriers. This study was supported by the Wilhelm Sander-Stiftung project 2003.4./2 42 43
POSTER POSTER Poster A7 Poster A8 Structural and functional studies of SMC proteins: how is DNA trapped inside the ring? Julia J. Griese and Karl-Peter Hopfner Gene Center and Munich Center for Integrated Protein Science (CIPSM), University of Munich, Germany Structural maintenance of chromosomes (SMC) proteins are the major components of large ring-shaped protein complexes like cohesin and condensin that are essential for the faithful inheritance of genetic information in all kingdoms of life. They consist of a long antiparallel coiled-coil region with globular domains at both ends. Their N and C terminus fold into an ABC-type ATPase head domain. The central hinge domain mediates dimerization of SMC proteins. ATP binding to the heads dimerizes them to close the ring. SMC complexes bind DNA by topologically entrapping it. However, it is unclear how they are loaded onto DNA. One model proposes that SMC proteins initially bind DNA via their hinge domains. This interaction stimulates ATP hydrolysis, and the ring would open to close again after encircling the DNA. To test this hypothesis, we investigate the role of the SMC hinge domain during DNA loading by functional assays combined with x-ray crystallography and small-angle x-ray scattering. Preliminary results suggest significant differences in the DNA binding mode of cohesin and condensin which are mediated by their hinge domains. In our future work we want to define the DNA binding interface of the SMC hinge domain accurately. This would be a significant step towards a better understanding of SMC protein mechanism. Cytogenetic characterisation of transgenic TRK-T and RET/PTC3 mouse cases Katrin-Janine Heiliger*, Donatella Vitagliano**, Natalie A Conte***, Allan Bradley***, Kristian Unger****, Gerry Thomas****, Ludwig Hieber*, Martin Müller*, Massimo Santoro**, Horst Zitzelsberger* * Institute of Molecular Radiobiology, HelmholtzZentrum münchen, German Research Center for Environmental Health, Neuherberg/Munich, Germany ** Instituto di Endocrinologia ed Oncologia Sperimentale del CNR `G. Salvatore`, University `Federico II`, Naples, Italy *** T82, Mouse Genomics, The Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton/Cambridge, United Kingdom **** Institute of Molecular Pathology, Imperial College London, United Kingdom Rearrangements of genes along the MAP-kinase-pathway are frequent alterations in papillary thyroid carcinomas. To investigate whether additional gene alterations are present in these tumours we are analysing tumours from RET/PTC3 and TRK-T transgenic mice for chromosomal imbalances. So far, 7 TRK-T and 7 RET/PTC thyroid tumours derived from transgenic mice were analysed by conventional CGH in order to identify chromosomal imbalances that may have impact on the development of thyroid cancer. The most common aberrations were deletions on chromosomes 3, 4, 2 and 4 and amplifications on chromosomes, and 5. The deletions on chromosomes 3 and 4 as well as the amplification on chromosome could be verified by interphase FISH. In one RET/PTC3 and two TRK-T tumours a deletion on 4E5 (RET/PTC) and 4E4-E5 (TRK-T) has been detected by conventional CGH. This deletion has been further characterized by positional cloning using 26 BAC clones that confirmed a deletion of.6 Mb. Within this region SLITRK5 could be identified. Additionally, in three different TRK-T cases a heterozygous deletion of the gene was detected. Preliminary results indicate a down-regulation of SLITRK5 also in human tissue samples. Further studies, like FISH with BAC clones and RT-PCR are in progress. In addition, array CGH on 25 TRK-T and RET/PTC3 cases have been performed on mouse Mb BAC arrays provided by the Sanger Institute in Cambridge. The most frequent aberrations on TRK-T cases were amplifications on chromosome 5, 7, 0 and 5 as well as deletions on chromosome 4, 5 and. Numerical aberrations of chromosome 6 could be detected on RET/PTC3 cases. The findings indicate that additional gene alterations are present in RET/PTC3 and TRK-T positive tumours. Thus, it appears that multiple genetic changes are necessary for malignant transformation of epithelial thyroid cells. 44 45
POSTER POSTER Poster A9 Poster A0 Copy number aberrations in post-chernobyl childhood papillary thyroid carcinoma (PTC) detected by array-cgh Julia Hess, Kristian Unger 2, Herbert Braselmann, Verena Bauer, Gerry Thomas 2, Horst Zitzelsberger Institute of Molecular Radiation Biology, Helmholtz Center Munich - German Research Center for Environmental Health GmbH, Germany 2 Department of Histopathology - Imperial College London Hammersmith Hospital, United Kingdom 52 childhood PTC from the Chernobyl Tissue Bank were analysed. The cohort consisted of patients both exposed and not exposed to radioiodine fallout. In order to detect copy number aberrations (CNA) we applied array-cgh using -Mb BAC-Arrays. Cases with similar aberration patterns were identified by hierarchical cluster analysis (HCA). FISH analysis on FFPE tissue sections was applied to validate frequent CNA. HCA revealed two distinct tumour subgroups that correlate with the presence of RET/PTC rearrangements and tumour size. However, there was no correlation with radiation exposure. Statistical analysis identified significant differences in aberration patterns between the tumour subgroups which include DNA gains on chromosomes p and 4q and DNA losses on chromosomes 2p, 2q, 7q, 8p, 8q, p, q, 3q and 8q. Overall, we identified 98 tumour related candidate genes that are located in commonly or cluster-specific altered regions and are involved in various molecular pathways (e.g. MAP-kinase-, PI3K-pathway). FISH confirmed amplifications of candidate genes HRAS, WNT, CDK5 and CTTN. Our findings on pre- and postfallout tumours do not support the hypothesis of radiation-specific CNA. We detected distinct aberration patterns in RET/PTC-positive and -negative cases indicating different mechanisms of tumour development. The identification of new candidate genes in thyroid tumourigenesis and their involvement in different pathways may serve as a starting point for further expression and functional studies. The role of the MRN (Mre, Rad50, Nbs)- complex in the base excision repair C. Kröger, F. Eckardt-Schupp, S. Mörtl Institute for Radiation Biology, Helmholtz Center Munich for Environment and Health GmbH Neuherberg, Post Box 29, Ingolstaedter Landstrasse, D-85764 Neuherberg, Germany Nijmegen Breakage Syndrome and ataxia telangiectasia-like disorder are recessive genetic disorders which are characterized e.g. by immune deficiency, high sensitivity to ionizing radiation, chromosome instability and abnormal cell-cycle checkpoints. The NBS/MRE genes affected in these diseases encode for the nuclear Nbs/Mre proteins which are components of the trimeric MRN (Mre, Rad50 Nbs) complex. The MRN complex has well known functions in recognizing DNA double strand breaks. But recent publications suggest further functions for Nbs in pathway unrelated to double strand break processing, like apoptosis, growth factor signaling and base excision repair (BER). As Nbs was recently shown to be essential for the BER by several in vitro and in vivo experiments, the aim of this study was to analyse the function of Mre and Rad50 during BER. The knockdown of either MRE or RAD50 in htert-rpe cells led to a decreased BER efficiency of oligonucleotides including Uracil or 8-oxoGuanin, an increased sensitivity against methyl-methanosulfonate and a lower Parp- activity in Mre deficient cells. To investigate the mechanistic background of the BER-deficiency further experiments were done which showed that the Nbs, Mre and Rad50 stabilise each other within the complex and even influences the stability of some other BER proteins. Since defective/absent Nbs reduces the BER efficiency in both short patch and long patch pathway it was determined by coimmunoprecipitation if the defect is caused by a lacking interaction between Nbs and the DNA polymerase β. This enzyme is involved in both subpathways and is therefore an adequate candidate to be investigated. The experiment gave us a hint for the interaction between these two BER proteins. The enforced analyses in htert-rpe cells showed that a complex network exists for the repair of base damages and that Mre and Rad50 like Nbs are also involved in the BER. 46 47
POSTER POSTER Poster A Testing Flavopiridol and ionizing radiation as a possible treatment for lung cancer in a murine model Sophie Lindemaier*, James F. Guenther**, Patricia F. Williams****, Daniel Herleth**, Cindy A. Morris***, Gilbert F. Morris** *Radiobiological Institute, University of Munich, Germany; **Department of Pathology, New Orleans, Louisiana; ***Department of Microbiology, New Orleans, Louisiana; ****VCU Medical Center, Richmond, Virginia Flavopiridol (FP) is a synthetic flavone that was initially identified as a selective anti-cancer drug in a screen of tumor cell lines conducted by the National Cancer Institute. Subsequent analyses in multiple laboratories have shown that FP selectively inhibits members of the cyclin-dependent kinase (CDK) family. Among the CDKs, FP more potently inhibits CDK9 (Ki=3-9nM), a component of positive-acting transcription elongation factor b, P-TEFb which promotes the processivity of RNA polymerase II. If it could be demonstrated that a tumor required CDK9 activity for survival, the high selectivity of FP for CDK9 would be an effective anti-tumor therapy. However, use of FP as a single agent to treat solid tumors produced limited results. Previous cell culture and in vivo assays in the laboratory of C. Morris showed that inhibition of CDK9 by FP in vascular endothelial cells disrupts pro-survival pathways initiated by angiogenic cytokines such as vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bfgf). In response to injury, solid tumors secrete these angiogenic cytokines to maintain the integrity of the tumor-associated vasculature. The hypothesis investigated here is that inhibition of CDK9 by FP will selectively injure the tumor-associated vasculature by preventing the pro-survival effects of tumor-derived angiogenic cytokines after radiation injury. Consistent with this hypothesis, FP inhibits expression of the pro-survival gene, Bcl-2, in vascular endothelial cells but does not inhibit activation of inhibitors of growth, such as p2/waf and p53 in these cells. The purpose of the experiments described here is to test this hypothesis in a clinically relevant mouse lung tumor model. Data retrieved in initial experiments suggest that FP and IR combination therapy causes increased weight loss and associated elevated inflammatory cytokines (KC and MCP-) in the lungs of tumor-bearing mice 4 days after the first treatment relative to mice lacking lung tumors that were treated simultaneously. Additional experiments will determine if these positive results of the FP/IR therapy at the early time point are amplified 8 days after the initial treatment. Poster A2 Effects of histone acetyl transferase Tip60 recruitment to complex DNA lesions Barbara Meyer, Nicole Averbeck, Kerstin Knoop and Gisela Taucher-Scholz GSI Helmholtzzentrum für Schwerionenforschung, Planckstraße, 6429 Darmstadt, Germany Chromatin remodeling is an early response to radiation-induced DNA damage aimed at providing accessibility of damaged DNA to repair proteins. We now raise the question whether chromatin remodeling is relevant for the initiation of DNA repair pathways, especially on complex lesions induced by heavy ion irradiation. We first analyzed the influence of Tip60 on double-strand break (DSB) repair following carbon ion irradiation. Tip60 is a histone acetyl transferase which is part of the NuA4 complex that acetylates e.g. histone H4. In addition Tip60 forms a stable complex with the inactive ATM dimer, acetylating and thereby activating ATM in response to DNA damage. We show that following heavy ion irradiation Tip60 is recruited to DNA damage sites and colocalizes with γh2ax in distinct foci. This recruitment occurs in an ATM-dependent manner. However, at the analyzed timepoints an irradiation-dependent acetylation of H4 was not reproducibly observed. Using the Tip60 inhibitor anacardic acid we show that the inhibition does not influence the formation of γh2ax foci after ion irradiation nor does it affect DSB repair. Interestingly, the distribution of phosphorylated ATM was influenced by anacardic acid-induced condensation of chromatin. The inhibition of acetylation yet needs to be verified independently. When chromatin was kept in a decondensed state using histone deacetylase inhibitor TSA there was no effect on Tip60 recruitment. Thus Tip60 might also play a role in later steps of DNA repair. 48 49
POSTER POSTER Poster A3 Poster A4 Development of an alkaline Comet Assay in the EpidermFT TM 3D human skin model for detecting DNA single- and double-strand breaks D. Michalski, T.E. Schmid, O. Zlobinskaya, M. Molls, B. Röper Klinikum rechts der Isar, Department of Radiation Oncology, Technische Universitaet Muenchen, Muenchen, Germany The Comet Assay, also known as single cell gel electrophoresis (SCGE), is a sensitive method to detect DNA damage at the level of individual cells. EpidermFT TM tissue is a 3D human skin model containing human keratinocytes and dermal fibroblasts. This tissue has a human skin-like structure with differentiated cells of epidermis and dermis, which preserves the three-dimensional geometric arrangement and communication of cells present in vivo. To investigate this 3D skin tissue with the Comet Assay, it is mandatory to prepare a single cell suspension. In the past, several methods for isolating cells from tissue were published, however, very little is known about isolating cells from skin tissue. With our proposed method the epidermis and dermis were processed differently after manual separation by using forceps. For isolating keratinocytes, a trypsin (0.25%) treatment (37 C, 20 minutes) worked successfully. Preparing fibroblasts from the dermis was more complicated because of adherence to collagen fibers. A mixture of collagenase (0.25%) and trypsin (0. 25%) was needed (37 C, 5 minutes) to degrade collagen and to isolate fibroblasts. The resulting suspensions contained in >90% isolated keratinocytes and fibroblasts, respectively, which could successfully be used in the Comet Assay. In conclusion, single cells can be obtained from EpiDermFT TM cultures at satisfactory levels. These are initial steps in developing a routine in vivo-like assay for DNA strand damage in human tissue after irradiation. Supported by the DFG Cluster of Excellence: Munich-Centre for Advanced Photonics, by the EU-project EuroDyna and by the Maier Leibnitz Laboratory Munich. Influence of Single Nucleotid Polymorphisms (SNPs) in ATM, GSTP, SOD2, TGFß, XPD and XRCC on cellular and clinical radiosensitivity Annette Raabe, Katharina Derda, Ulrike Höller 2, Dirk Rades 3, Silke Szymzak 4, Andreas Ziegler 4, Ekkehard Dikomey, Kerstin Bormann Laboratory of Radiobiology & Experimental Radiooncology, University Medical Center Hamburg-Eppendorf, 2 Clinic of Radiotherapy, Radiation Oncology and Nuclear Medicine, Vivantes Klinikum Neukölln, Berlin, 3 Department of Radiation Oncology, University Hospital Schleswig-Holstein, Lübeck, 4 Institute of Medical Biometry and Statistics, University of Lübeck. Previous studies revealed that individual cellular radiosensitivity is mainly determined by genetic factors and correlates with both acute and late clinical radiosensitivity. Therefore, the variations of normal tissue toxicity observed in patients treated with identical radiation protocols must be discussed as consequences of genetic variations such as single nucleotid polymorphism (SNPs). Two studies were performed on breast cancer patients receiving breast conserving therapy applying 54-55 Gy. Clinical radiosensitivity determined either as acute erythema (n= 89) or late fibrosis (n=69) was correlated with the cellular radiosensitivity, determined using the G0/ assay, but and with the presence of socalled risk-alleles determined via genotyping using MALDI- TOF or RFLP, for SNPs in genes involved either in signal transduction (TGFß), the induction (SOD2, GSTP) or repair (ATM, XPD, XRCC) of DNA-damage, respectively. Whereas SNPs in TGFß, XRCC, ATM and GSTP determined the risk of fibrosis in an allele-number dependent manner, the risk of severe erythema were solely influenced by the polymorphic TGFß-allel. However, none of the polymorphisms studied was correlated with cellular radiosensitivity. The results indicate that clinical radiosensitivity might result from interactions of multiple risk-alleles, therefore future studies must focus on identification of gene networks or genes linked in corresponding physiological pathways, respectively, whereas SNPs mediating cellular radiosensitivity still need to be identified. 50 5
POSTER POSTER Poster A5 Poster A6 Mapping of allelic imballances in radiogenic murine osteosarcoma using whole genome MD SNP Arrays reveales a new potential tumor-suppressor-locus on Mmu 3 Christine Ruemenapp, Jan Smida, Michaela Nathrath, Leticia Quintanilla-Fend, Mike J. Atkinson and Michael Rosemann HMGU Center for Environmental Health, Institute for Pathology (J.S, L.Q-F.) and Institute for Radiation Biology ( M.J.A., M.R), Neuherberg, Germany and Technical University Munich, Clinic Schwabing, Dept of Pediatric Oncology, Munich, Germany (M.N.), Central Institute for Medical Engineering (C.M.) and The Clinical Cooperation Group Osteosarcoma (J.S., M.N., M.J.A., M.R) Murine Osteosarcoma can easily be induced by injection with the osteotropic Alpha-Emitter Thorium227. Using F hybrid mice from different inbred strains allows mapping of tumor-specific allelic losses and imballances by comparative genotyping of normal tissue DNA versus tumor DNA. In order to use this approach with whole-genome coverage, we applied Illumina mouse MD SNP Arrays (Golden Gate) on a set of roughly 80 radiation-induced osteosarcoma induced in BALBxB6 and BALBxCBA mice. Chromosomal regions with potential allelic losses were identified using the LOH-module (part of Illumina Bead Studio) that is based on discrete genotypes and compared with an procedure developed by our self that uses a continous LOH-probability and adaptive thresholding. We show, that by masking non-infomative SNP markers, followed by calculation of moving average and adaptive thresholding it is possible, to detect new chromsomal loci with osteosarcoma specific allelic losses. As proof-ofprinciple, we could verify frequent alterations at the chromosome 4 locus (P5/P6/P9), that has be mapped in an earlier study using microsatellite markers. The most recurrent LOH locus in the present study was found on chromosome 3. Genetic and biochemical analysis of base excision repair complexes Ulrike Schötz, Sabine Schmidl, Petra Fiedler, Steffen Heuer and Randolph B Caldwell Helmholtz Center Munich, Institute of Molecular Radiobiology The project aims to understand how vertebrate cells cope with oxidative DNA damage induced by radiation. The work is of relevance for humans exposed to various forms and levels of radiation during environmental and occupational exposure and for many forms of human disease related to defects in DNA damage repair pathways. This work is also of relevance to cancer treatment as we work to understand the key overlapping repair pathway components that effectively work as antagonists to radiation-therapy. Oxidative DNA damage is mainly repaired by a family of DNA glycosylases which specifically recognize and excise oxidized bases. Although these glycosylases are highly conserved during evolution, it has been difficult to study their function in higher eukaryotes due to redundancies and the existence of backup repair pathways. This problem is further amplified in higher eukaryotes by the difficulty in creating gene deficient models and the lack of sensitive phenotypic read-out assays. Using the homologous recombination proficient DT40 cell line, we shall inactivate single and multiple genes encoding key repair factors. The marker that we have specifically developed for this purpose is a positive (green fluorescent protein)/negative (nitroreductase) dual selection system. This marker can easily be inserted into the genome of DT40 and functions as a site-specific DNA damage trap allowing us to isolate even extremely rare events leading to inactivation of gene function. The mutant clones will then be analyzed for damage induced by ionizing radiation with respect to i) genetic analysis of damage leading to inactivation of the negative selection marker, ii) protein/dna repair complex formation, and iii) phenotypic and morphological alterations. Supported by EU Euratom grant RISKRAD. 52 53
POSTER POSTER Poster A7 Poster A8 Analysis of radiation-induced changes in epigenetic information D.M. Seiler, G.A. Drexler, B. Mazurek, C. Greubel 2, V. Hable 2, G. Dollinger 2, H. Strickfaden 3, T. Cremer 3, A.A. Friedl Ludwigs-Maximilians-Universität München, Strahlenbiologisches Institut, D-80336 München, Germany 2 Universität der Bundeswehr München, Angewandte Physik und Messtechnik, LRT2, D85579 Neubiberg, Germany 3 Ludwigs-Maximilians-Universität München Anthropologie und Humangenetik, Dept. Biologie II, D-8252 Planegg-Martinsried, Germany Epigenetic information is a key regulator of gene expression as well as in defining chromatin architecture. The importance of epigenetics in processes like transcription, cell differentiation and x-inactivation became evident in the past years. Changes of epigenetic information in the course of radiation-induced DNA damage response is a growing field of research. The best known modification is the phosphorylation of the histone variant H2AX into gh2ax. Recent findings hint at global changes in different histone modifications post-irradiation. With the ion-microbeam SNAKE (Superconducting Nanoprobe for Applied Nuclear (=Kern-) Physics Experiments) at the munich tandem accelerator facility, single ions can be applied in geometrical patterns to living cells. Thereby, DNA damage induced by ionizing radiation is directed with a targeting accuracy of less than µm to a certain subset of cells or specific areas in single cells. The advantage of ion-microirradiation over laser microirradiation is that the distribution- and amount of damage-types thus induced is well characterized. With microirradiation, DNA double strand breaks are induced in a well defined manner and the formation of irradiation induced foci (IRIF) can be visualized in fixed cells by using immunofluorescence techniques. So local and even slight changes of histone modifications and other candidate proteins appearing directly at the site of DNA double strand breaks can be determined. At present we investigate local as well as global changes of modifications which mark either euchromatic or heterochromatic regions. A new role of the MRX complex in base excision repair Sylvia Steininger, Fred Ahne, Friederike Eckardt-Schupp, Simone Moertl Institute of Radiation Biology, Helmholtz Centre Munich - German Research Centre for Environmental Health, 85764 Neuherberg, Germany Purine and pyrimidine moieties are continuously damaged by endogenous as well as exogenous factors, such as alkylating and oxidizing agents. Repair of damaged bases is primarily carried out by the base excision repair (BER) pathway. The main steps are the removal of the damaged base by a DNA-N-glycosylase resulting in an apurinic/apyrimidinic (AP) site, the action of an AP-endonuclease or AP-lyase producing a single strand gap, gap filling by a DNA polymerase and finally, sealing by a DNA ligase. According to the number of replaced nucleotides BER includes long patch and short patch subpathways. The Mre/Rad50Xrs2 (MRX) complex in S. cerevisiae has manifold functions in DNA double strand break sensing and processing, checkpoint activation, telomere length maintenance and meiosis. To analyse a potential involvement of the MRX complex in the BER pathway we employed different assays. First, we studied the repair of MMS-induced heat-labile sites in chromosomal DNA in vivo. Detection of secondary DSB by pulse-field gel electrophoresis revealed a lower repair capacity in the xrs2 mutant compared to wildtype. In cell-free extracts we monitored a reduced in vitro BER capacity of uracil- and 8-oxoG-containing oligonucleotides in MRX deficient extracts as compared to wildtype extracts in both, long and short patch BER. Specific analysis of the repair mechanism showed reduced gap filling activity for the xrs2 deletion mutant, which suggests impaired polymerase activity, while base recognition, strand incision and ligation seems to be not affected. Epistasis analyses suggest a role of the MRX complex downstream of the AP-lyases Ntg/Ntg2. 54 55
POSTER POSTER Session B: Signalling, Cell Cycle and Apoptosis Poster B2 Poster B Analysis of molecular mechanisms of radiation-induced apoptosis M. Angermeier, H. Eichholtz-Wirth, T. Blunder, S. Hornhardt, F. Eckardt-Schupp, S. Mörtl Helmholtzzentrum München, German Research Center for Environmental Health, Ingolstädter Landstr., D-85764 Neuherberg, Germany 2 Bundesamt für Strahlenschutz, Ingolstädter Landstr., D-85764 Neuherberg, Germany The identification of individuals with increased radiosensitivity is important for tumour therapy and radiation protection. Apoptosis is an important genetic parameter for radiosensitivity. Therefore we want to obtain extensive knowledge of the different molecular mechanisms of radiation-induced apoptosis. The different apoptotic behaviour of lymphoblastoid patient cell lines was mechanistically characterised. In these cell lines radiation-induced apoptosis was analysed by different molecular methods and additionally the characterisation of different pro- and anti-apoptotic processes was investigated by protein array. 23 cell lines were explored by caspase3 activity, subg population and PARP cleavage as apoptoic markers and were classified as high (8), middle (7) and low (8) apoptotic. To analyse the pathways involved in radiation-induced apoptosis in these cell lines an array, containing 35 apoptosis-relevant proteins was performed. We found good correlations between the analysed proteins of the pro-apoptotic activation processes in the array and the apoptotic induction after irradiation, e.g. cleavage of caspase3. Furthermore we found p53-mediated apoptosis is the main pathway of apoptosis induction in most of the cell lines. Otherwise some cell lines showed significant differences in receptor activation pointing to a prominent role of receptor triggered apoptosis after irradiation Summing up all the data of radiation-induced apoptosis in lymphoblastoid patient cell lines we can classify these cell lines by their different apoptotic behaviour. Moreover our results strengthen our mechanistic understanding of radiation-induced apoptosis and after completion of data analysis our array data allow us to assess the importance of the various pro- and antiapoptotic processes after radiation exposure. An assessment of the effects of radiation quality on the interplay between the PI3K/Akt signalling pathway and DNA repair. M.M.Eid*, J.Harper**, Peter O Neil** *NRC, Cairo, Egypt ** Gray institute for radiation oncology and Biology, university of Oxford The insulin-like growth factor-i receptor (IGF-R) is a tyrosine kinase receptor that is important in both normal and pathological cell growth which is mediated by mitogenic and anti-apoptotic signalling. It is also involved in the regulation of cell differentiation. IGF-R is over-expressed in a number of human cancers and signalling through this receptor is known to mediate resistance to anti-cancer therapies including radiation (low-let). The relationship between increased IGF-R expression and radioresistance is unclear. However, increasing evidence exists to suggest a role for IGF-R in modulating DNA repair through effects on ATM signalling and also homologous recombination (HR). Indeed we have previously shown that pharmacological inhibition of PI3K leads to a persistence of radiation-induced RAD5 foci at later times post irradiation (Prevo et al, Cancer Research, 2008). We therefore intend to assess the interplay between the IGF-R/PI3K/Akt signalling axis and DNA repair. We aim to investigate the effects of inhibition of this extracellular signalling pathway on DNA double strand break (DSB) repair following mammalian cell exposure to low-let γ-rays or high-let α-particles. This will provide insight into the effects of high- vs. low-let on any interplay between extracellular signalling pathways and DNA DSB repair. Initially we aim to pharmacologically inhibit IGF-R in breast cancer and normal cells and investigate the effects on cell survival following exposure to low- or high-let radiation. We will also assess the effects on DSB repair using γh2ax immunostaining as a marker of DSBs and the effects on HR using immunostaining for RAD5, a key protein involved in this pathway. It is becoming increasingly evident that therapies targeting IGF-R signalling in combination with radiation therapy may yield increased anti-cancer efficacy; therefore, investigations into the modulation of DNA repair by extracellular signalling pathways are increasingly of special importance and will contribute to the further advancement of cancer treatment strategies. 56 57
POSTER POSTER Poster B3 Poster B4 Uptake of caesium and strontium via roots and leaves in Arabidopsis thaliana Ulrike Kanter, Monika Röhmuß and Wolfgang Schultz Helmholtz-Zentrum München In areas contaminated by nuclear accidents, radioactive isotopes of caesium and strontium (34Cs, 37Cs, 90Sr, 85Sr) are taken up by plants and may enter the human food chain meaning a serious hazard. To assess the genetic potential of plants for uptake regulation the natural variation of Arabidopsis thaliana was tested in 86 ecotypes. For root uptake plants were supplemented with radioactive isotopes in hydroponic culture, and caesium and strontium concentrations were measured in rosette leaves after a defined time point. To study uptake via the aerial plant part a radioactive solution was applied to the leaf surface, and the radionuclide concentration was measured in the uncontaminated tissue after a defined time. In both screens values showed near-normal distribution indicating the polygenic inheritance of the traits. High and low uptaking ecotypes were found to vary by a factor of about two to three, but the extreme ecotypes in both screens were not identical indicating organ specific factors for radionuclide transfer. As caesium and strontium share many physico-chemical properties with potassium and calcium, respectively, the extreme ecotypes identified in the root screen and the F3 progeny of their cross breeding were analysed for the concentration of these essential minerals as well. A strong correlation between strontium and calcium was found indicating a strong link to plant nutrition and mineral homeostasis. A QTL analysis was conducted to reveal genomic regions underlying the traits of radionuclide and mineral concentration. Are differentially regulated let-7 mirnas responsible for increased radiosensitivity in EA.hy926 co pared to A549 cell lines? A. Krämer, F. Eckardt-Schupp, N. Anastasov, S. Mörtl Institute of Radiation Biology, Helmholtz Center Munich German Research Center for Environmental Health (GmbH) Neuherberg, Ingolstädter Landstrasse, D-85764 Neuherberg, Germany MicroRNAs (mirnas) are small noncoding RNAs identified in a variety of organisms that control gene expression by binding to complementary sites on target mrna transcripts. Recently, mirnas have been implicated in cellular stress response such cytotoxic treatment. Especially, let-7 mirnas were shown to be regulated after ionizing radiation. Moreover, these mirnas have been identified to regulate the human RAS oncogene which was proven to be critical for protection from radiation-induced reproductive cell death, the primary form of radiation-induced target cell death. The aim of this study was to compare the mirna response to ionizing radiation in the cell lines A549 and EA.hy926 and correlating these data with cellular survival, apoptosis and induction of MAP signalling cascades which are regulated by RAS. EA.hy926 cells are derived from the fusion of human umbilical vein endothelial cells (HUVEC) with A549 lung carcinoma cells. Our results indicate a differential radiation response in let-7 mirna expression patterns in EA.hy926 cell line compared to A549 and we found that survival after irradiation is decreased. The survival after radiation was analysed via colony formation, the metabolic activity was dissected by a cell proliferation assay (MTT). Furthermore caspase-3 activity and subg population suggest increased apoptosis in Ea.hy926. Currently, we study the activation of Erk/2 and Akt signalling cascades, triggered by changes in mirna expression. The understanding of these mechanisms could help to improve radiation therapy for different cell types. 58 59
POSTER POSTER Poster B5 Session C: Hot Spicy Topics Poster C RAD5 overexpression reduces replication fork progression AC. Parplys, HW. Stürzbecher 2, E. Dikomey and K. Borgmann Lab of Radiobiology & Experimental Radiooncology, University Medical Center Hamburg; 2 Institute of Human Genetics, University Hospital Lübeck; Germany Purpose: Homologous recombination (HR) comprises a series of interrelated pathways that function in the repair of DNA-DSBS and ICLs. In addition, recombination provides critical support for DNA replication in the recovery of stalled replication forks, contributing to tolerance of DNA damage. RAD5 catalyzes the key reactions that typify HR: homology search and DNA strand invasion. The inability to properly repair complex DNA damage and resolve DNA replication stress leads to genomic instability and contributes to cancer. The presented project is focussed on the effect of Rad5 overexpression on replication fork progression in the response to endogenous and exogenous stress. Materials and Methods: Experiments were carried out with U2OS cells transfect with Rad5 under control of a ponasteroneinducible promoter. The replication fork progression was measured by the fibre-labeling assay and DSBs by H2AX-Foci assay. Results: Overexpression of Rad5 was found to result in a reduced fork progression with 0,59+/- 0,20 kb/min compared to 0,69 +/- 0,20 kb/min seen in controls. This difference was even more pronounced when cells were treated with H2O2. In normal cells treatment with H2O2 (0 and 200µM) results only in a modest reduction of fork progression, in contrast to a strong reduction in Rad5 overexpressing cells (6 to 50%), respectively. This higher sensitivity was also reflected by a higher number of DSBs. Conclusion: Overexpression of Rad5 leads to a sensitivity to endogenous and exogenous stress, which is evident in a decreased replication fork progression, perturbing DSB-repair. Changes in mirna expression profiling after ionizing radiation in cardiac endothelial cell line Natas a Anastasov, Anne Krämer, Simone Mörtl, Jan Smida, Michael Rosemann, Michael Atkinson and Soile Tapio Institute of Radiation Biology, Helmholtz Center Munich German Research Center for Environmental Health, Neuherberg, Germany Ionizing radiation (IR) is an important diagnostic and treatment modality. Conversely, it is a potent DNA damaging and cancer inducing agent. Recent epidemiologic evidence suggests that moderate and low radiation doses to the heart may result in a moderate, but significant increase in cardiovascular mortality. However, the role of micro RNAs and how they integrate into the radiation signaling pathways is largely unknown. Micro RNAs (mirnas) are a novel class of genes that regulate gene expression by translational repression or mrna decay. A specific looped-primer RT-PCR quantification method was used to quantify the expression of mature mirna in a global screening of the cardiac endothelial cell line (Ea.hy 96) after exposure to low radiation doses (0,2 Gy) in comparison to moderate radiation doses (2,5 Gy). From 378 mirnas analyzed, 76 were detectable, of which 64 mirnas showed significant changes 4 and 24 hours after irradiation. Most of the affected mirnas showed relatively minor changes (2 fold) in their expression, but a set of 22 mirnas presented 3 to 0 fold changes. Of these the most highly changed (mir-25, mir-58b and mir- 58d-5p) will be analyzed and validated under different conditions and times after exposure to radiation. Functional elucidation and mirna target prediction, according to mir-base software, will be used to compare mirna responses to the gobal proteomic change analysis in endothelial cells. This strategy will allow us to predict potential targets for cardiovascular disease development after irradiation. Supported by the EURATOM Radiation protection programme project CARDIORISK 60 6
POSTER POSTER Poster C2 Poster C3 Radiobiological effects of pulsed versus continous proton beams S. Auer, S. Lindemaier, G.A. Drexler, C. Greubel 2, V. Hable 2, G. Dollinger 2, A. Hauptner 3, T.E. Schmid 4, B. Röper 4, A.A. Friedl LMU-München, Strahlenbiologisches Institut, Germany 2 UniBw-München, Angewandte Physik und Messtechnik LRT2, Germany 3 TU-München, Physik Department E2, Germany 4 TU-München, Klinikum Rechts der Isar, Germany Within the framework of the excellence cluster MAP (Munich-Centre for Advanced Photonics) high-intensity lasers for particle acceleration are developed with the aim to apply them for cost-effective proton therapy. When irradiating tissue or cells with proton beams generated by laser-driven accelerators (LDA), the dose to a target voxel would be applied in ion pulses of nanosecond duration, while using conventional irradiation the same dose is delivered within milliseconds or seconds. It is unknown so far, if this extremely pulsed irradiation induces different radiobiological effects compared to conventional irradiation. To answer this question, we investigate the short-pulse effects with pulsed proton beams generated at the microprobe SNAKE at the Munich tandem accelerator, where bunching of 06 particles into a nanosecond pulse can be achieved for 20 MeV protons. By focusing these pulses to beam spots in the 00 µm diameter range, the ion density is comparable to that expected for laser-driven pulses. With this approach possible differences between pulsed and continuous irradiation can be examined on a single-cell level. We investigate a variety of endpoints. So far, analysis of G2-phase-arrest by immunofluorescence detection of cell cycle markers (cyclin B, CenP-F) did not reveal significant differences between pulsed and continuous proton-irradiation. Preliminary evidence of different effects was, however, observed concerning induction of apoptosis. Recently, a larger pulsed particle field was successfully implemented at SNAKE. With this setup, classical cell biology experiments such as investigations of clonogenic survival have become possible. In addition, we plan to perform gene expression analyses. e.era - The electronic European Radiobiological Archives database reanimates old data Mandy Birschwilks, Clemens Adelmann, Soile Tapio,2, Michael Gruenberger 2, Paul Schofield 2 and Bernd Grosche Federal Office for Radiation Protection, Department of Radiation Protection and Health, Ingolstaedter Landstrasse, 85764 Neuherberg, Germany 2 Institute of Radiation Biology, Helmholtz Centre Munich, German Research Centre for Environmental Health, 85764 Neuherberg, Germany 3 Department of Physiology, Development and Neuroscience, University of Cambridge, Downing Street, Cambridge CB2 3DY, UK Objectives In the mid 980s the European Radiobiological Archives (ERA) were founded. ERA holds information on 330 radiobiological animal experiments from 50 institutes. Together with the respective archives from the US (NRA) and Japan (JRA) they form the International Radiobiological Archives (IRA). Within the EC funded project ERA-PRO, the database e.era is developed to make information available on former high quality animal experiments for validation of radiation effects. e.era will be accessible on the internet. Methods Updating: Transfer the existing database to a relational one with a web-based graphical user interface. Data quality: A first validation and quality check of the existing database was conducted by re-entering randomly selected data. Further, a continuous quality check is crucial. Standardization: For rodents, the original pathology nomenclature Disease Rodent (DIS-ROD) was replaced by the formalism of Mouse Pathology (MPATH) and Mouse Anatomy (MA). A pathology panel checked histopathological slide material and compared the original diagnoses with currently accepted diagnostic criteria. Links: Links to other relevant databases will be created. Results Updating: The legacy system has been converted to a relational database. Data quality: The accuracy of the primary data input was assessed and improved. Standardization: The majority of the original rodent pathology nomenclature was successfully recoded. Links: Links were created. Conclusion e.era has the potential to become a scientific tool for research groups world-wide. The data can be used in research areas such as the risk assessment and biology based modelling as well as a resource for planning future animal studies. 62 63
POSTER POSTER Poster C4 Poster C5 STORE Sustaining access to Tissues and data from Radiobiological Experiments Mandy Birschwilks and The STORE-Consortium Federal Office for Radiation Protection, Department of Radiation Protection and Health, Ingolstaedter Landstrasse, 85764 Neuherberg, Germany Objectives Sharing data and biomaterials from publicly funded experimental radiation science will yield substantial scientific rewards through re-analysis and new investigations, adding enormous value to the original investment. STORE will generate a platform that allows the storage and retrieval of both data and the corresponding biological material from past, current and future radiobiological studies. Impacts on: A) Understanding mechanisms of low-dose radiation effects Through re-analysis and re-use of data and material, contribution on a better understanding and risk assessment (quantification and mechanisms) of low doses of radiation. B) Data and biomaterial mobilization and accessibility Providing a one-stop-shop on the www which will provide ready access to data and biomaterials from both legacy and current and future radiobiological experiments. C) Impact on models for long-term financial sustainability of archive resources STORE will critically assess improvements in cost efficiency and business models for financial sustainability relevant for the project itself and for the development of other research infrastructures. Methods A) Establishing SOPs on how best to evaluate, store and use biological-material B) STORE will sustain, curate and disseminate valuable existing information by making it available to the community ( Data- Warehouse ) C) Provide a foundation for the creation of both real and virtual stores of biological-material. Assess potential financial models for long term support of a bioresource and Data-Warehouse for radiobiology. Investigation of radiopharmaceutical potential of radiolabeled bevacizumab Ilknur Demir, F. Zumrut Biber Muftuler, P. Unak Ege University, Institute of Nuclear Sciences, Department of Nuclear Applications 3500 Bornova Izmir Turkey Angiogenesis plays a crucial role in many physiological and pathologic processes including tumor growth. Vascular endothelial growth factor (VEGF), released by tumor cells, is an important growth factor in tumor angiogenesis. Bevacizumab (BevMab), a humanized monoclonal antibody against VEGF, binds to soluble VEGF, preventing receptor binding and inhibiting endothelial cell proliferation and vessel formation. It is the first antiangiogenic treatment in various solid tumors approved by the American Food and Drug Administration. The goal of this study is to develope an antiangiogenic agent radiolabeled with 99m Tc and to be used for in vivo imaging of VEGF. For this purpose, BevMab was conjugated with the hydrophilic group diethylenetriamine pentaacetic acid (DTPA) and the product was labeled with 99m Tc using stannous chloride reducing method. The quality control studies of radiolabeled compound ( 99m Tc-BevMab-DTPA) were done with Thin Layer Radio Chromatography (TLRC) and High Performance Liquid Radio Chromatography (HPLRC) methods (% 95 ) to confirm labeling efficiency. The biodistribution studies are performed on female and male Albino Wistar rats. The injected dose per gram tissue is calculated and time-activity curves are examined. All the experimental data are evaluated using statistical SPSS 3 program. 99m Tc-BevMab-DTPA may be used for in vivo VEGF imaging agent as an antiangiogenic congugate. Keywords: Angiogenesis VEGF, Bevacizumab, 99m Tc, 99m Tc-BevMab-DTPA. Conclusion STORE will provide a mechanism for ensuring the future survival of invaluable and irreplaceable resources and provide a platform for the sharing of new data across the radiobiology community. 64 65
POSTER POSTER Poster C6 Poster C7 Development of a biophysical treatment planning system for the FRM II neutron therapy beamline S. Garny, V. Mares, W. Rühm, F.M. Wagner 2, M. Zankl, H.G. Paretzke ; HMGU/ISS, 2 TUM/FRM Since June 2007, neutron irradiation treatments are performed again at the new MEDical APPlication facility (MEDAPP) at the Research Neutron Source Hans-Maier Leibnitz (FRM II). To improve therapy planning, which is at the moment done with water phantom depth dose curves, a treatment planning system is developed using the Monte Carlo Code GEANT4. For this purpose, the neutron spectrum at the patient treatment position was measured with a Bonner sphere spectrometer applying gold foils as thermal neutron detectors inside. This spectrum was implemented into the GEANT4 code together with a calculated preliminary photon spectrum. GEANT4 was thoroughly tested for correctness of neutron transport beforehand, calculating the photon and neutron ambient dose equivalent. The dose distribution in a water phantom was simulated and the calculated depth dose curves compared to measured ones, testing also the used voxelisation algorithm. As a first test case, a salivary gland treatment beam was simulated in a voxel phantom. The German Uranium Miners Biobank GUMB Gomolka M, Danescu-Mayer J, Otten H 2, Pesch B 3, Johnen G 3, Lehnert M 3, Oestreicher U, Roessler U, Hornhardt S, Kulka U, Bruening T 3 and Kreuzer M Federal Office for Radiation Protection (BfS), Department Radiation Protection and Health, Ingolstädter Landstr., 85764 Oberschleissheim, Germany 2 German Social Accident Insurance (DGUV), Alte Heerstraße, 53757 Sankt Augustin, Germany 3 BGFA - Research Institute of Occupational Medicine, German Social Accident Insurance, Bürkle-de-la-Camp Platz, 44789 Bochum, Germany Objectives: The German uranium miners of the former Wismut mining company with about 400.000 employees are the largest radiation-exposed population of miners worldwide. 59,000 of them are included in the prospective Wismut Cohort Study. Yet, biobanks from occupationally radiation-exposed individuals do not exist or only on a small scale. A pilot study was initiated to collect blood samples and data on exposure and other information from 400 former miners. In addition, DNA and RNA from lung tissue of Wismut miner lung cancer cases will be isolated from the Wismut Autopsy Archive. Methods: In the next two years 300 uranium miners exposed to cumulative radon levels of >750 Working Level Months (WLM) and 00 age-matched uranium miners with exposures <50 WLM will be recruited. Blood samples are collected for isolation and cryoconservation of DNA, RNA, lymphocytes, and plasma proteins. Chromosome analysis will be performed for 50 highly exposed and 25 low exposed workers. Data on occupational exposure to radiation (radon, long-lived radionuclides, gamma radiation), quartz dust and arsenic as well as health data and smoking information are available. Results: A first feasibility study of 40 workers showed a high response rate of 63%. The isolated RNA and proteins were of high quality, which was demonstrated by successful cdna array and 2-D-proteome analysis. Conclusions: A biobank for uranium miners will be a powerful tool to analyse radiation risk with biological materials at various exposure levels, including low-dose exposure, individual susceptibility to radiation, long-term radiation exposure markers and co-exposure with quartz dust and other factors. 66 67
POSTER POSTER Poster C8 Poster C9 Real time assessment of individual radon exposure E. Gruber, F. Karinda, H. Paretzke, M. Wielunski, W. Rühm HMGU-ISS Radon in Germany depends on uranium in soil and ranges from outdoor concentrations (~0 Bq/m3) to very high concentrations (up to 0000 Bq/m3) with a mean indoor value of about 50 Bq/m3. To measure in real time the individual radon concentration of a person, the new HMGU electronic radon exposure meter was developed. Up to now, 26 prototypes were built and calibrated. The calibration was done in an airproof aluminium box with a radium-containing source and an AlphaGUARD-device for reference. Using these prototypes, the individual radon concentration was measured for 29 persons over a period of one week. To compare these measurements with radon assessment used in epidemiological studies, the concentration in the living room and bedroom was measured, too. For high radon concentration measurements, the exposure meters were tested in Bad Gastein, which is a radon spa with very high radon concentrations, high temperature and high humidity, used for therapy. Results of these measurements are discussed. Thermoluminescence Dosimetry of 90 Sr in teeth of the population living in the Techa River area, Southern Ural, Russia N. Semioshkina, E. Shishkina 2 HMGU, Institute of Radiation Protection, Neuherberg, Germany 2 Ural Research Center of Radiation Medicine, Chelyabinsk, Russia The Mayak Production Association (MPA) located in the Southern Urals was the first Russian nuclear fuel reprocessing plant. The Techa River was contaminated as a result of radioactive release from Mayak plutonium facilities. As a result, people living on the banks of the Techa River incorporated 90 Sr (in average of 3000 Bq) in their bones and teeth. Owing to the long physical and biological half-life of the strontium, Techa River population, exposed to considerably high internal dose has been the subject of much investigation since 960. The highly sensitive beta dosemeters (α-al2o3: C) developed in recent years has opened a new era in TL dosimetry, due to their simple emission spectrum, wide dose range and relatively low atomic number. All the properties make the thin-layer α-al2o3: C good detectors of low concentration of 90 Sr without chemical separation. More than 00 teeth samples of exposed population of Techa River area were measured using thin layer α-al2o3 :C beta dosimeters and current dose rate in teeth due 90 Sr intake of river water and local foodstuffs were estimated. 68 69
POSTER POSTER Poster C0 Poster C Analyzing the acute responses of cells to low dose irradiation through proteomic studies Arundhathi Sriharshan, Ludwig Hieber, Soile Tapio, Hakan Sarioglu, Mike Atkinson, Horst Zitzelsberger Introduction: Analysis of cells response to low dose irradiation is relatively complex when compared to the analysis of cells response to higher doses of irradiation. Most of the changes to lower doses are temporary and can be identified at protein levels only. In this project we are irradiating a human epithelial mammary cell line (B42-8) at low to moderate doses ranging from 0.2 Gy to 2 Gy to study the biological effects at the proteome level One of the most effective methods in the proteome analysis is SILAC (Stable Isotope Labeling with Amino acids in cell culture). SILAC is a technique that allows an incorporation of isotopic labels ( light vs. heavy ) into proteins of growing cells. The quantification of protein alterations by comparison of the relative amounts of the two isotopes is measured by mass spectrometry. Thus, two cell populations, in our case the irradiated vs. sham-irradiated cells, are grown in otherwise identical culture media, the only difference being the presence of a light amino acid in one and a heavy form of the same amino acid in the other. When the labeled analog of an amino acid is supplied to cells in culture instead of the natural amino acid, it is incorporated into all newly synthesized proteins. After about five cell divisions, every occurrence of this particular amino acid will be replaced by its isotope labeled analog. Due to almost no difference between the labeled amino acid and the natural amino acid isotope, the cells behave identically to the control cell population grown in the presence of normal ( light ) amino acid. SILAC approach is efficient and reproducible as the incorporation of the isotope label is 00%. Methods: Basic principle of SILAC is to incorporate stable isotope containing amino acids (e.g: arginine or lysine or both containing 3C) into the entire proteome of cells. A population of human epithelial mammary cell line (B42-8) were divided into two and grown in two separate media, the light medium containing the amino acid with the natural isotope (L-Lysine-2HCl, L-Arginine-HCl) and the heavy medium containing the SILAC amino acids (3C6 L-Lysine-2HCl and 3C6 5N4 L-Arginine). The cells were cultured in heavy medium for 8 cell doublings to allow the incorporation of the heavy amino acids. Usually after five cell doublings, 97% of the proteins are expected to be labeled. The protein extracts of the unlabelled and the labeled samples were mixed and analyzed with mass spectrometry (MS). Analysis of gamma-h2ax foci formation after irradiation with pulsed and continuous proton beams O. Zlobinskaya, T.E. Schmid, G. Dollinger 2, V. Hable 2, C. Greubel 2, J.Wilkens, D. Michalski, G. Du 3, M. Molls and B. Röper Klinikum rechts der Isar, Department of Radiation Oncology, Technische Universitaet Muenchen, Muenchen, Germany, 2 Institute for Applied Physics and Metrology, Universitaet der Bundeswehr Muenchen, Neubiberg, Germany, 3 Technische Universitaet München, Physik Department E2, Garching, Germany Introduction: Laser acceleration promises innovation in particle beam therapy of cancer. The unique feature of laser accelerated protons is the ultashort pulse duration (typically nanoseconds instead of the milliseconds common for a conventional accelerator). Very little is known about the relative biological effectiveness (RBE) of this new beam quality. In our present study we examine possible differences in RBE based on quantitative analysis of gamma-h2ax fluorescence. Methods: HeLa cells were irradiated with Gy of 20MeV protons at the Munich tandem accelerator, either at continuous mode (00 ms), or at pulsed mode with a single pulse of ns duration. The total number of gamma-h2ax foci per cell was determined semi-automatically. Results: Quantitative analysis of gamma-h2ax fluorescence revealed no significant difference (p=0.6) in yield of foci formation after irradiation with pulsed or continuous proton beams. gamma-h2ax data for cell samples irradiated with Gy of pulsed or continuous proton beams were 23.58 ± 2.8 and 26.54 ± 2.54 foci per cell, respectively. The corresponding RBE values for 20 MeV protons were 0.97 ± 0.9 and.3 ± 0.2 (p=0.5) for pulsed and continuous irradiation modes. However, the percentage of foci smaller than 5-0 pixels was decreased and foci were more clustered after irradiation with pulsed protons. Conclusions: Based on gamma-h2ax foci formation no significant difference in the RBE between pulsed and continuous proton irradiation beams has been detected so far. These results are well in line with our data on micronucleus induction in HeLa cells and 3D-skin model. Results: The MS results confirmed that the cells grown in the heavy media were completely labeled. After confirming the labeling of heavy cells, 8 petridishes containing heavy cells were exposed to a moderate dose of 2 Gy of 37 CS- γ-rays, simultaneously sham-irradiating 8 petridishes of light cells. Cell were harvested at four time intervals(hr, 4hrs, 2hrs, 24hrs) after irradiation and cell extracts were shock frozen in liquid N2. At all four time point s one petridish of heavy cells and one of the light cells were trypsinized and counted in order to estimate the cell growth during incubation period. The identification of the proteins that are altered by the irradiation is in progress 70 7
LOCATION MAP LIST OF AUTHORS AND PARTICIPANTS 8 By train By plane Airport Munich Neuherberg: S to station Feldmoching, with taxi or U2 to station Am Hart. From there bus 294 (direction Garching-Hochbrück, runs every 20 min hourly between 0 a.m. and 3 p.m.) to station Neuherberg, Forschungszentrum. Or: S8 to main railway station (Hauptbahnhof), from there U2 to station Am Hart, then bus 294 (direction Garching-Hochbrück, runs every 20 min hourly between 0 a.m. and 3 p.m.) to station Neuherberg, Forschungszentrum. Please note: The bus 294 runs hourly between 0 a.m. and 3 p.m.! It would be easier to take a taxi from Feldmoching to the Helmholtz Zentrum München. Taxi via Highway Deggendorf-München, direction Stuttgart, exit Neuherberg, 30 min to the Helmholtz Zentrum München (approx. 40.- to 50.- EUR). Main railway station Neuherberg: U2 to station Am Hart, then bus 294 (Garching-Hochbrück, runs every 20 min hourly between 0 a.m. and 3 p.m.) to station Neuherberg, Forschungszentrum. Please note: The bus 294 runs hourly between 0 a.m. and 3 p.m.! It would be easier to take a taxi from Feldmoching to the Helmholtz Zentrum München. By car 96 STUTTGART LINDAU 8 96 Hämatologikum Großhadern Das Hämatologikum ist mit der U6 ab Marienplatz bis Großhadern (ca. 30 Minuten) zu erreichen. A96-Ausfahrt Blumenau/Großhadern Klinikum Großhadern U6 Bus 56 Marchioninistraße 95 99 92 Feldmoching U2 S Taxi Am Hart U2 Bus 294 3 Landstr. Ingolstädter Garching- Hochbrück U6 Bus 294 Bus 294 MÜNCHEN HBF S/S8 U2/U6 GARMISCH- PARTENKIRCHEN DEGGENDORF A99-Ausfahrt Neuherberg Forschungscampus Neuherberg City centre Neuherberg Ludwigstrasse - Leopoldstrasse - Ingolstädter Strasse - continuation Ingolstädter Landstrasse, the Helmholtz Zentrum München on the right, approximately 800 m after city boundary, total distance from city centre about 2 km. Taxi from city centre to the Helmholtz Zentrum München about 30 min (fare approx. 20.- EUR). Highways Nürnberg - München: Northern ring, direction Stuttgart, first exit Neuherberg, from here follow signs (400 m to the Helmholtz Zentrum München). Stuttgart - München: Route A 99, direction Salzburg, exit Neuherberg, at traffic light turn right (300 m to the Helmholtz Zentrum München). Salzburg - München: Northern ring, direction Stuttgart, exit Neuherberg, from here follow signs 400 m to the Helmholtz Zentrum München). 9 94 PASSAU S/S8 Taxi NÜRNBERG 99 92 8 SALZBURG Adedapo, Moruf Adesola +23 480564955 poly.ibadan@yahoo.com Ahne, Fred +49 893872276 ahne@helmholtz-muenchen.de Anastasov, Natas a +49 893873798 natasa.anastasov@helmholtz-muenchen.de Angermeier, Marita +49 893872647 marita.angermeier@helmholtz-muenchen.de Atkinson, Michael - J. +49 893872983 atkinson@helmholtz-muenchen.de Auer, Susanne +49 8928075598 susanne.auer@lrz.uni-muenchen.de Barjaktarovic, Zarko +49 709743648 zarko.barjaktarovic@helmholtz-muenchen.de Barlow, Denise +43 42775460 denise.barlow@univie.ac.at Bauer, Verena +49 89387436 verena.bauer@helmholtz-muenchen.de Birschwilks, Mandy +49 3083332263 mbirschwilks@bfs.de Blum, Nikolaus blum@helmholtz-muenchen.de Blunder, Thomas +49 893872647 Thomas.Blunder@helmholtz-muenchen.de Borgmann, Kerstin +49 40428033596 borgmann@uke.uni-hamburg.de Böttger +49 228993052960 boettger@bmu.de Broggini-Tenzer, Angela +4 442553534 angela.broggini@usz.ch Brunner, Claudia +49 89387388 claudia.brunner@helmholtz-muenchen.de Caldwell, Randolph +49 89387262 randolph.caldwell@helmholtz-muenchen.de Cooper, Priscilla + 504867346 pkcooper@lbl.gov;nischwartz@lbl.gov Curwen, Gillian +44 9465402 gillian.curwen@westlakes.ac.uk Dahm-Daphi, Jochen +49 4074053930 dahm@uke.uni-hamburg.de Dalke, Claudia +49 89387290 dalke@helmholtz-muenchen.de Danescu-Mayer, Joana Jdanescu-mayer@bfs.de Dehos, Anne +49 893603243 adehos@bfs.de Domke, Tanja +49 893873854 tanja.domke@helmholtz-muenchen.de Dornreiter, Irene +49 404805224 irene.dornreiter@hpi.uni-hamburg.de Dorsch, Tatiana +49 8940734 tatiana.dorsch@lrz.tu-muenchen.de Doß, Brigitte +49 89440445 brigitte.doss@lrz.tu-muenchen.de Dräxl, Stephan +49 893872359 stephan.draexl@helmholtz-muenchen.de Eckardt-Schupp, Friederike eckardt-schupp@helmholtz-muenchen.de Eichholtz-Wirth, Hedda +49 893872538 Eichholtz@helmholtz-muenchen.de Eichinger, Teresa +49 84449824 teresa.eichinger@helmholtz-muenchen.de Eid, May +49 86567356 may.eid@rob.ox.ac.uk Favor, Jack +49 893872395 favor@helmholtz-muenchen.de Fournier, Claudia +49 659 72692 c.fournier@gsi.de Frankenberg-Schwager, Marlis +49 55398622 dfrank@med.uni-goettingen.de Frenzel, Nicole Nicole.Frenzel@usz.ch Friedl, Anna +49 8928075807 anna.friedl@lrz.uni-muenchen.de Garny, Sylvia +49 893874005 sylvia.garny@helmholtz-muenchen.de Gomolka, Maria +49 888333222 mgomolka@bfs.de Gonzalez Vasconcellos, Iria +49 893873888 iria.gonzalez@helmholtz-muenchen.de Griese, Julia Johanna +49 892807698 griese@lmb.uni-muenchen.de Gruber, Eva +49 89378308 Eva.Gruber@helmholtz-muenchen.de Hall, Janet +33 6986306 janet.hall@curie.u-psud.fr Heiliger, Katrin-Janine +49 893874436 katrin.heiliger@helmholtz-muenchen.de Hess, Julia +49 893872872 julia.hess@helmholtz-muenchen.de Hieber, Ludwig +49 8938748 ludwig.hieber@helmholtz-muenchen.de Höller, Ulrike +49 3045650205 ulrike.hoeller@charite.de Hornhardt, Sabine +49 888333222 shornhardt@bfs.de Janzen, Tilman +49 893872682 tilman.janzen@helmholtz-muenchen.de Jeggo, Penny +44 273678482 p.a.jeggo@sussex.ac.uk; j.devenyi-williams@sussex.ac.uk Jeremias, Irmela +49 897099424 Irmela.Jeremias@helmholtz-muenchen.de Jungnickel, Berit jungnickel@helmholtz-muenchen.de Kanter, Ulrike +49 89-3872370 ulrike.kanter@helmholtz-muenchen.de Kasten-Pisula, Ulla +49 4074052358 kasten@uke.uni-hamburg.de Kiefer, Jürgen +49 6406 587 juergen.kiefer@strz.uni-giessen.de 72 73
LIST OF AUTHORS AND PARTICIPANTS Krämer, Anne +49 89387330 anne.kraemer@helmholtz-muenchen.de Kröger, Carina +49 89387330 carina.kroeger@helmholtz-muenchen.de Kulka, Ulrike +49 888333222 ukulka@bfs.de Kuschal, Christiane +49 55399578 ckuscha@gwdg.de Lindemaier, Sophie +49 89280806 Sophie_Lindemaier@gmx.net Mazurek, Belinda +49 8928075838 Belinda.Mazurek@lrz.uni-muenchen.de Meyer, Barbara +49 6597352 B.Meyer@gsi.de Michalski, Dörte +49 8944064 Doerte.Michalski@lrz.tu-muenchen.de Mörtl, Simone +49 89387343 moertl@helmholtz-muenchen.de Mosconi, Marita +49 535926889 marita.mosconi@ptb.de Moustacchi, Ethel +33 5624670 ethel.moustacchi@curie.fr Mueller, Martin +49 893873356 martin.mueller@helmholtz-muenchen.de Müller-Laue, Edith +49 888333222 emueller-laue@bfs.de Multhoff, Gabriele +49 89440454 Gabriele.Multhoff@lrz.tu-muenchen.de Mund, Andreas +49 404805220 andreas.mund@hpi.uni-hamburg.de Nunez, Alejandro +49 404508224 alejandro.mena-nunez@hpi.uni-hamburg.de Oestreicher, Ursula +49 888333222 uoestreicher@bfs.de Ohneseit, Petra +49 707-298746 petra.ohneseit@uni-tuebingen.de Paretzke, Herwig +49 893874006 Paretzke@helmholtz-muenchen.de Parplys, Ann Christin +49 4074052352 a.parplys@uke.uni-hamburg.de Petermann, Eva +44 86567354 eva.petermann@rob.ox.ac.uk Poller, Manuel +49 8938744 manuel.poller@helmholtz-muenchen.de Popanda, Odilia +49 62242335 o.popanda@dkfz.de Raabe, Annette +49 4074054705 araabe@uke.uni-hamburg.de Rickinger, Astrid +49 893873255 Astrid.Rickinger@helmholtz-muenchen.de Romm, Horst +49 888333222 hromm@bfs.de Röper, Barbara +49 894405373 barbara.roeper@lrz.tum.de Rößler, Ute +49 888333222 uroessler@bfs.de Schilling, Daniela +49 894404302 daniela.schilling@lrz.tum.de Schmaltz, Dominik +49 893873887 dominik.schmaltz@helmholtz-muenchen.de Schmid, Thomas +49 894406350 t.e.schmid@lrz.tum.de Schoen, Annemarie +49 89-302969 amschoen@yahoo.de Schötz, Ulrike +49 89387262 ulrike.schoetz@helmholtz-muenchen.de Schüßler, Helga +49 93602638 he.schuessler@t-online.de Seiler, Doris Mirjam +49 8928075838 doris.seiler@lrz.uni-muenchen.de Semioshkina, Natalia +49 893872904 semi@helmholtz-muenchen.de Shi, Kuangyu +49 894406457 shi@lrz.tum.de Smida, Jan +49 893873797 smida@helmholtz-muenchen.de Sriharshan, Arundhathi +49 89387425 a.sriharshan@helmholtz-muenchen.de Steininger, Sylvia +49 893873255 sylvia.steininger@helmholtz-muenchen.de Storch, Katja +49 354587402 Katja.Storch@oncoray.de Tapio, Soile +49 893873445 soile.tapio@helmholtz-muenchen.de Taucher-Scholz Gisela +49 65972606 G.Taucher-Scholz@gsi.de Urban, Maria +49 89387355 maria-urban@t-online.de Volkmer, Beate +49 467036990 beate.volkmer@elbekliniken.de Wess, Günther guenther.wess@helmholtz-muenchen.de Wiesmüller, Lisa +49 7350058800 lisa.wiesmueller@uni-ulm.de Zankl, Maria +49 8938748 zankl@helmholtz-muenchen.de Zitzelsberger, Horst +49 89387342 zitzelsberger@helmholtz-muenchen.de Zlobinskaya, Olga +49 894406350 olga.zlobinskaya@lrz.tum.de 74 75
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