The Group

The Group

The Dark.Risk group consists of 5 participating institutions based in 4 different EU member states.

The consortium is made up of internationally recognised leaders from relevant fields of radiation research. Not only are the partners individually excellent, but through the synergy obtained by combining epidemiologists, clinicians, radiation biologists, cell biologists and molecular biologists, Dark.Risk has aconsortium that as a whole is excellent.


The Scientific Advisory Board (SAB, namly Tom Hei [Columbia University, USA],Tanja Paunescu [Northwestern University, USA] and Siegal Sadtezki [Gertner Institute, Israel]) of Dark.Risk is chartered to mainly:

  • ensure a high standard of research,
  • monitor the progress of the project by taking part in the annual Meetings,
  • consult the consortium and make recommendations as to improve their performance.

HMGU

Participant 1

HMGU - Helmholtz Zentrum München, Germany   

Brief description of the HMGU organisation and summary of experience relevant to the Dark.Risk project

Helmholtz Zentrum München is a German Research Center for Environmental Health. The HMGU undertakes basic and applied research into common diseases influenced by environmental exposures. The HMGU has over 120 scientists engaged on projects ranging from radiation ecology to clinical radiation therapy. The Institute of Radiation Biology (ISB) is led by Prof. Michael Atkinson and has 4 Principal Investigator-led research groups studying the mechanisms of radiation-associated chronic diseases, including cancer, cardiovascular and neurodegenerative diseases. We are a member of MELODI and a partner in the DoReMi network. The HMGU staff has a long tradition of contributing to EURATOM programmes, and our experience in training activities is demonstrated by our long-standing participation in the European MSc in Radiation Biology and in teaching radiation sciences at both Munich Universities.

 

Prof. Atkinson holds the chair of radiation biology at the Technical University of Munich where he is a member of the medical faculty. Prof. Atkinson is currently project coordinator of two FP7 EURATOM projects (ProCardio and Dark.Risk) and has coordinated previous EURATOM projects (CARDIORISK, GENRISK-T and GENRAD).

Dr. N. Anastasov is the Principal Investigator (PI) of Personalized Radiation Therapy group at the ISB. She currently serves as the assistant coordinator for the FP7 EURATOM project Dark.Risk and participates in the FP7 project ProCardio. She has a long standing experience with lentiviral transgene technology and profound know-how with Sirion Biotech cooperation in getting ZIM-Projects from 2010-2014, financed by the German Federal Ministry of Economics and Technology (BMWi).

We have been instrumental in indicating a regulatory role for non-coding RNAs in the radiation response. We have identified circulating microRNAs that change during therapeutic application of radiation, and that they are required for the survival of cells after radiation exposure. We have most recently shown that the retinoblastoma gene (Rb1) modifies susceptibility to radiation-induced osteosarcoma by regulating the transcription of the TERRA family of sub-telomeric RNAs that are required for maintaining telomere integrity. Loss or reduced expression of Rb1 leads to shortened telomeres and an increased genomic instability after radiation exposure. HMGU has a long tradition in radiation epidemiology and risk analysis. Our expertise in this field will be available to ROSA to assist in carrying out Tasks in WP1.

 

HMGU "key tasks" in Dark.Risk

Within Dark.Risk, HMGU will contribute to all work packages as coordinator.

HMGU particularly works in WP2 under the objective: Radiation-induced expression of non-coding RNAs in 3D mammosphere cultures (Task 2.1). As the non-coding genome is implicated in individual susceptibility, we will examine the response through profiling of the microRNA and long non-coding RNA transcripts in both 3 dimensional monotypic mammary epithelial and mixed fibroblast-mammary epithelial co-cultures. These will be compared with the expression profile in standard monotypic monolayers. The effects of low (0.01 to 0.1Gy) and moderate (0.1 to 2Gy) X-ray exposures will be examined. HMGU will collaborate with ENEA and UAB to validate functional importance of radiation-regulated non-coding RNAs (Task 2.3) and with SERGAS to establish a functional assay to investigate specific long non coding RNAs (lncRNAs) expression. In WP3 under the Task 3.2, HMGU will study the contribution of non-coding RNA transcriptome to individual susceptibility. Those ncRNAs showing significant differences within the analysis from WP2 will be used for biomarker analysis on samples taken in the feasibility study of the SRTCC in collaboration with ROSA (participant 2). Proving its success, the basic knowledge will be translated using biomaterials from SRTCC to determine the use of long non-coding RNAs as potential biomarkers.

HMGU staff involved in the project

Prof. Dr. Atkinson, J. Michael

Director of the Institute of Radiation Biology

Dr. Anastasov, Nataša

Principal investigator with expertise in radiation oncology, lentiviral technology and epigenetics

Dr. O’Leary, Valery

Research Scientist with a PhD since 1997 in molecular genetics and a wide range of scientific expertise that includes genome wide association studies into birth defects and the role of exercise physiology in diabetes prevention.

Her recent work in neurodegenerative research has focused on toxicological approaches and lentiviral technology for in vivo tissue specific gene targeting

Radulović, Vanja

PhD Student

Dr. Birschwilks, Mandy

Project Management Office

 

ROSA

Participant 2

 ROSA- Zenska Asocijacija ROSA , Serbia

                                 

Description of the organization: Zenska Asocijacija ROSA Zenska Asocijacija ROSA (The Women's Association Rosa)

ROSA was established in December 2005 as an NGO and non-profit research organization. Its primary goal is fostering research practices which could lead to improvements in health of the elderly and other vulnerable population groups, promotion of healthy ageing, as well as identification of risk factors associated to various health disorders. ROSA's two senior scientists are closely affiliated with the Institute for Gerontology and palliative care Belgrade. In 2007 ROSA became member of the Serbian NGO network "Humana's".

The research group - Summary of experience relevant to the research project

The research group headed by Goran Sevo currently comprises of 6 members: 2 senior researchers (epidemiologists by training), one post-doctoral researcher, and 3 technical assistants. Its main research focus is epidemiological evaluation of the TC campaign in Serbia during 1950s.

 The research team 

In 1950 Tinea Capitis (TC, ringworm) eradication campaign was initiated by former Yugoslavia authorities to tackle the major epidemic of this fungal infection of the scalp hair. It occurred following WW2 due to mass population migrations, poor sanitation and the hygienic standards. The campaign received financial assistance and sponsorship from the UN Children's Fund (UNICEF). The treatment of choice (standard of care at the time) was X-ray induced scalp hair removal, followed by topical application of available anti-fungal drugs. In 1970s, however, it was conclusively established that ionizing radiation used in this protocol could be associated with significantly increased risk to various health disorders, primarily head and neck tumors, occurring many years after the treatment (mean latency estimated to 35 years). The first evidence of this association came from the cohort of 11,000 children treated in Israel upon immigration in early 50s (Modan et al. 1974, Lancet.157:410-418).

Investigation of Serbian TC campaign started in 2005 by two current ROSA members Dr Sevo and Dr Tasic, in collaboration with Israeli scientists Prof. Shvarts and Prof. Sadetzky. Due to general lack of information, and to some extent possibly even deliberate “oblivion” created by the authorities, it took several years to comprehensively demonstrate historical aspects of the campaign.

It was established that almost 2 million of children in former Yugoslavia had undergone the field screening for the disease, while X-ray treatment was provided to some 100,000, at least 50,000 in Serbia alone. This information created considerable surprise in the scientific community, making the campaign one of the largest of its kind ever. In addition, several hundred former TC patients were interviewed by qualitative methodology, to obtain their personal impressions and experiences on the campaign. A number of them had already suffered delayed health consequences related to TC treatment. These findings were reported on several occasions at scientific meetings in Serbia and abroad (Shvarts et al. 2008, AAHM 81st Annual Meeting, Rochester NY, Sevo et al. EAHMH, September 2009, Heidelberg Germany, Tasic et al. 10th Serbian Congress of Gerontology, May 2010 V. Banja Serbia, Sevo and Tasic, Institute of Gerontology, Belgrade 2011). Crown of this work certainly represents joint publication which made cover page of the British medical journal Lancet, August 2010 issue (Shvarts et al. 2010, 10:571-576)

 

In addition to other findings, an intensive archive search yielded probably the greatest scientific benefit and potential from this work: discovery of hospital records for some 25000 patients treated in Belgrade TC children's hospital in the respective period. This information provided opportunity for individual identification of the treated patients, forming the basis for in-depth epidemiological evaluation of the campaign.

 

TC Children’s Hospital BelgradeRegistry Book (1950-1959)

In 2011 Serbian Ministry of health sponsored ROSA's one year pilot study aimed to transcribing hospital records into a digital database. Its desired outcome was opportunity to form the national registration of former TC patients, which could in turn facilitate meeting their specific health needs arising due to TC treatment.

Next logical step was to joint efforts with research partners investigating other aspects of exposure to low dose ionizing radiation (identification of genetic markers of exposure, susceptibility to delayed health outcomes etc.). This gave rise to the current Dark.Risk Project.

 

ROSA will act as work package leader for WP1, where they will coordinate fieldwork and manage data for the creation of Serbian Registry of Tinea Capitis Children, from which will be selected the Study cohort, and carried out the Feasibility Study for collection of biomaterials.

  1. Serbian Registry of Tinea Capitis Children -The Belgrade TC Hospital was created to implement highly standardized depilatory X-irradiation treatment of children suffering TC between 1950 and 1959. Records of 23.951 Serbian patients from this hospital have been secured, and experienced epidemiologists from ROSA have transcribed this material into a digital database. Preliminary evaluation has confirmed that health history of these patients is traceable using public sources. Municipal and regional public records will be used to identify individuals' current residence, vital status, causes of death and location of their medical records. Wherever this methodology does not provide satisfactory results, additional information will be sought from the Interior Ministry Citizen Database and from the Serbian Institute of Statistics. From this evaluation we expect to be able to identify and trace 18.248 cases (i.e. 80% out of 22.810 unique identities from the registry book). This work will be conducted under ethics approval already granted by the Ethics Committee of the Serbian Medical Association. In accord with Serbian legislation “Act on Conducting Medical Research” the completed database will be held on behalf of ROSA by the Serbian Institute of Health.
  2. Recruitment of the epidemiological cohort and record collection - ROSA epidemiologists will initially select 500 living patients from the Register and contact their primary care physicians to identify the patients’ current health status, disease history and confounding factors relevant both to cancer and non-cancer end points. In addition, all individual records of the cohort will be cross-referenced to the Serbian Cancer Registry. Having established the validity and efficiency of the procedures for the first 500 cases, data collection will be expanded to include at least 5000 irradiated individuals and all of their living siblings. Due to large family size at the time, we expect to be able to use siblings as another non-irradiated control group (in addition to 1,000 controls selected from the general population, appropriately matched with cases by all relevant characteristics). A similar cohort in Israel has proven invaluable in providing insight into late health effects of this treatment. Dosimetry data is already available for the patients, courtesy of previous Serbian-Israeli research project (Prof. Sadetzki, SAB member).
  3. Feasibility study of the collection of samples (biological material) from the cohort for use in retrospective biological analysis (ROSA, HMGU, SERGAS) - In this task we will ascertain how feasible it is to collect biological samples. Using the initial sample of 500 cases, we will undertake to collect biological samples following participants' informed written consent. Our goal is to collect materials from at least 100 individuals and controls. In addition to detailed evaluation of their health status, including relevant confounders, all participants will be requested to provide biological samples (buffy coat, plasma, an oral epithelium swab and hair bulb samples). These materials will be stored in a repository and small sample subjected to quality control analysis to determine suitability for analysis of DNA, RNA, non-coding RNA, epigenetic markers. In other words it will be used to establish methodology, validate quality and to perform the initial molecular epidemiology studies for elucidating the contribution of individual variation to the health effects of low dose radiation.
Application of X-ray protocol in TC treatment

Photo: historical Archives 

ENEA

Participant 3

ENEA -Agenzia Nazionale per le Nuove Tecnologie, l'Energia e lo Sviluppo Economico Sostenibile, Italy

         

 

Brief description of the organisation and summary of experience relevant to the project

ENEA (Agenzia Nazionale per le Nuove Tecnologie, l’Energia e lo Sviluppo Economico Sostenibile, Italy) is a public research centre operating in the fields of energy, environment and new technologies to support competitiveness and sustainable development. ENEA has unique competences in Radiological Protection and Safety in Italy, and is a "repository" of scientific knowledge, technical competence and data in the country. ENEA has in house know-how, laboratories, equipment and unique infrastructures in support of radiobiology- and dosimetry-related activities. The infrastructures available include X-ray generators and high- and low-energy neutron facilities equipped with biological channels, as well as facilities for proton irradiation. These facilities comply with EU standards and are periodically checked by the INRIM (National Institute of Metrology of Ionizing Radiation).

The ENEA Unit of Radiation Biology and Human Health (http://utbiorad.casaccia.enea.it/), located at ENEA Casaccia Research Centre, has long and internationally recognized experience in radiobiology at molecular, cellular and whole-animal level. Medical/veterinarian staff for animal care and histopathology, and biological staff skilled in tissue processing and molecular techniques are available for evaluation of clinical, pathological and genetic alterations in experimental animals. The Laboratory of Radiation Biology and Biomedicine has participated in several EURATOM projects, the most recent of which are the FP7 EU projects DoReMi, ProCardio and CEREBRAD.

    

 

ENEA "key tasks" in Dark.Risk

Participating in WP2 (Task 2.2)

In collaboration with HMGU, ENEA will study the changes in the transcription profile of non-coding RNAs that occur during the development of radiation-induced medulloblastoma in a mouse model heterozygous for the Ptch1 gene. This gene activates a growth and differentiation programme that is a strong candidate for regulation through the non-coding genome. Consequently will be compared the expression of microRNA and long non-coding RNA in mouse brains irradiated with low (0.01 to 0.1Gy) and moderate (0.1 to 2Gy) X-ray doses. 

Leading WP3.

In collaboration with other partners (HMGU, UAB, SERGAS), ENEA will develop WP3 tasks using information on the radiation response of the non-coding genome gathered in WP2 to examine the feasibility of biomarker analysis in samples obtained from radiation-exposed individuals in the SRTCC cohort. Whether individual differences in the genotype of the non-coding RNAs can account for differences in cellular responses will also be determined.

ENEA staff involved in the project

Dr. Mariateresa Mancuso

Senior Researcher with expertise in mouse models and pathology
Phone: +39 06 30484993
Fax: +39 06 30483644

e-mail:

Dr. Barbara Tanno

 

Researcher with expertise in molecular oncology

Phone: +39 06 30483984
Fax: +39 06 30483644

e-mail:

Dr. Simona Leonardi

 

Researcher with expertise in molecular radiation biology

Phone: +39 06 30484850
Fax: +39 06 30483644

e-mail:

Dr. Simonetta Pazzaglia

 

Senior Researcher with expertise in experimental oncology

Phone: +39 06 30486535
Fax: +39 06 30483644

e-mail:

 

Dr. Anna Saran

 

Head of the Laboratory of Radiation Biology and Biomedicine

Phone: +39 06 30484304
Fax: +39 06 30483644

e-mail:

 

UAB

Participant 4

UAB-Universitat Autònoma de Barcelona, Spain     

             

 

 

Description of the organization Universitat Autònoma de Barcelona

The Universitat Autònoma de Barcelona (UAB) is a public university. The UAB Campus brings together more than 40,000 students and almost 3,000 researchers. It is a pioneering institution in terms of fostering research and has established firm links with research centres abroad, especially with those of the EU.

The Research group -Summary of the experience relevant to the research project

The research group headed by Anna Genescà currently comprises 10 members: 2 permanent staff, 1 post-doctoral researcher, 4 PhD students, 2 master students and 1 technical assistant. The research group is well balanced in the type of training of its members. The main research interests of the group headed by Anna Genescà are in the areas of Radiobiology, DNA Damage Responses and Genome Integrity.

The Research group

The vast majority of cancers in the adult human population are of epithelial origin (carcinomas). Tumor cells in carcinomas are characterized by their inability to maintain a stable genome at chromosome level. They display a wide range of chromosome aberrations, including non-reciprocal tranlocations, gene amplifications, gains and losses of whole chromosomes and abnormalities in the number of chromosome sets.

Chromosomal instability most likely occurs at initial steps in the development of cancer, and might fuel the multiple genetic changes required for the initiation of the disease.The results obtained by our research team have contributed to establishing a link between the genomic instability and telomere dysfunction. Loss of telomere function occurs frequently as a consequence of progressive telomere shortening that normally occurs in proliferative cells in most of somatic tissues. The studies carried out by our research group point that telomere dysfunction contributes to chromosome instability mainly through the formation of dicentric chromosomes originated by end-to-end fusion of chromosomes with eroded telomeres (Tusell et al. Biochem Soc Transac 2010, IF 3.989; Frías et al. Frontiers Bioscience 2012, IF 4.048).

When fused chromosomes are pulled to opposite poles during the anaphase, a nucleoplasmatic bridge is formed. This triggers fusion-bridge-breakage (BFB) cycles, which are able to reorganize the cell genome through the formation of unbalanced translocations (Soler et al, Genes Chrom Cancer 2005, IF 3.306).

When sister chromatid are fused instead of chromosomes, nucleoplasmatic bridge breakage gives rise to segmental chromosome amplification events (Tusell et al. Cytogenet Genome Res 2008, IF 1.533). However, chromosome bridges do not always resolve by breakage (Tusell et al. Multiple Path Cancer Dev, 2008). Chromatin bridges may also persist unbroken during cell division giving rise to unfaithful segregation of chromosome between daughter cells and consequent chromosome number abnormalities, the so called aneuploid daughter cells (Pampalona et al. Genes Chrom Cancer. 2010, IF 3.990; Pampalona et al. Mutat Res. 2010; IF 3,.204).And yet an additional possibility exists when chromosome bridges remain unbroken: they may ultimately originate polyploidy cells (4n) through cytokinesis failure by bridged chromatin in the cleavage furrow causing cytokinesis abortion (Pampalona et al. Plos Genet. 2012, IF 9.543).

In summary, telomere dysfunction has long been held responsible for the initiation of BFB cycles and their accompanying rearrangements, including non-reciprocal translocations, regional amplifications and segmental deletions. The finding that aneuploidy and polyploidy is an additional corollary of unmitigated telomere attrition adds to this inventory of telomere-related genome instability and provides a framework for the genesis of human tumors carrying heavily rearranged subtetraploid genomes (Genescà et al. Adv Cancer Res. 2011, IF 6.080).

                     

Besides these wide range manifestations of telomere-dependent chromosome instability, the presence of short dysfunctional telomeres in cells can also cause cells to become highly sensitive to radiation exposure (Genescà et al. Bioessays. 2006, IF 4.479). Human individuals often exhibit important differences in their sensitivity to radiation, which have important consequences in the use of radiation in cancer therapy. Given that ionizing radiation is also an important and widespread diagnostic and therapeutic tool, it is important to investigate further factors and mechanisms that underlie individual radiosensitivity. Evidence is accumulating that telomere function may well be involved in cellular and organism responses to ionizing radiation, expanding still further the currently complex and challenging scenario (Soler et al. Aging Cell. 2009, IF 7.148). Telomere function now emerges as a factor potentially contributing to increasing the sensitivity of aged human cells to DNA-damaging agents. Since eroded telomeres are sensed and act as DNA double strand breaks, they can interact with radiation-induced DNA breaks, thus sharply increasing the possibility of mis-rejoining and revealing itself as an important factor that definitely contributes to genomic instability and radiation sensitivity. In the light of more recent work carried out in the Genescà laboratory, it is clear that age-dependent radiation sensitivity is not only linked to telomere dysfunction but also to a progressive deterioration of DNA damage cell response. This is even more relevant when the malfunction of DNA-repair proteins has been observed in the scenario of low-dose radiation exposure. Concerned about the risks of mammography screening, this research group has analyzed the ability of human mammary epithelial cells to cope with mammogram-induced DNA damage. In a recent study, the Genescà laboratory shows that the dose received by the breast surface per mammogram X-ray exploration induces increased frequencies of DNA double strand breaks to aged—but not to young—human mammary epithelial cells (Hernández et al, Plos One, 2013, IF 4.092). This is consistent with recent IRCP published data that classifies breast tissues amongst those that are most sensitive to radiation and also with epidemiological studies that reveal increased carcinogenic risks of radiation exposures at older ages. When faced with low-dose radiation induced DSBs, aged mammary epithelial cells trigger a slow response, thus inducing increased amount of genetic damage (Hernández et al, Plos One, 2013, IF 4.092). In this new scenario where telomere dysfunction and DNA repair impairment is produced by the sole act of proliferative cell aging, radiation sensitivity might acquire a temporal aspect: shortened telomeres and decreased repair efficiency in aged cells may potentially increase radiation sensitivity in elderly organisms

                         

Organisms are continuously exposed to DNA damaging agents; consequently, cells have developed a complex defense system in order to detect DNA lesions, signal their presence and promote their repair. This system, known as the DNA damage response (DDR), is a hierarchical cascade of proteins composed of sensors, mediators and effectors. Altogether, they are responsible for recognizing the induced lesions and delay the cell cycle in order to provide the cells with time enough for protein effectors to repair the DNA lesions or alternatively induce apoptosis or senescence. To restore genome integrity, the cell must overcome important hurdles in space and time to rapidly sense and initiate the correct signaling and repair programs.

Basic cytogenetic studies and double strand break (DSB) rejoining kinetics studies carried out by our research group have demonstrated that DSB repair must be fast and complete to avoid open ends from accumulating, which would increase the probability of eventual illegitimate rejoining. Two proteins have been shown to carry out complementary activities during DBS repair. Our research group has shown that the interaction between ATM and DNA-PKcs at the DSB is essential to achieve fast and complete DSB repair (Martín et al. Mutat Res. 2012, IF 8.741).

The presence of DNA-PKcs guarantees that most breaks will join in a timely manner (Martín et al. Cancer Res. 2005, IF 7.856), while the presence of ATM protein ensures the complete repair of DSBs (Martín et al. Genes Chrom & Cancer. 2009, IF 3,990). Both changes in the DSB rejoining kinetics and the incapacity to rejoin a subset of DSBs cause increased genome instability and radiosensitivity (Martín et al. Mutat Res. 2012, IF 8.741). In the case of ATM deficient cells, one of the causes of this characteristic defect has been identified: deficient signaling of DNA damage is responsible for the incapacity of certain breaks to be repaired (Martín et al. Genes Chrom & Cancer. 2009, IF 3,990). When we shift from the time to the space dimension, we realize that only the intact nucleus provides the adequate local concentrations of DDR proteins capable of fixing the DNA lesions.

The Genescà research group has recently provided evidence indicating that DDR is impaired when DNA lesions are located inside micronuclei. DNA lesions sequestered in micronuclei generate a less efficient response (Terradas et al, In J Mol Sci, 2012 IF 2.598). This laboratory has investigated the presence of DSB repair proteins in the micronuclear DNA in primary human fibroblasts exposed to γ-rays. In contrast to nuclear radiation-induced DSBs, only a small fraction of micronuclear DSBs were able to recruit the DDR proteins 53BP1 and MRE11 at the site of damage (Terradas et al. DNA Repair. 2009; IF 4,293).

Similarly, UV-induced helix-distorting lesions did not recruit NER factors when present in micronuclear DNA (Terradas et al. Mutat Res 2012, IF 3,204). Micronuclear envelope defects cause deficient recruiting of proteins involved in cell cycle checkpoints and DNA damage repair (Terradas et al. Mutat Res Fund Mol Mech Mutagenesis 2012, IF 3,204). The data collected in this laboratory suggest that the DDR machinery is not ready for action anywhere. Only the cell nucleus with intact envelope structure provides the adequate concentration of proteins for interacting with damaged DNA and triggering an efficient DDR. In the case of micronuclear DNA lesions, the chromatin encapsulated in micronuclei does not benefit from the intricate and efficient web of DDR players of the cell, and chromosome instability and radiation sensitivity would be favored under these circumstances when micronuclei is eventually incorporated into daughter nuclei. Thus micronuclei, which have mainly been considered as indicators of ongoing genomic instability, now emerge as a source of instability at the same time (Terradas et al. Mutat Res. 2010, IF 8,741). Altogether, this reveals a new dimension in the significance of micronucleation within the carcinogenesis process.

                             

The technical expertise of this research group encompasses cell and tissue culture, classical and molecular cytogenetics, molecular biology techniques, immunofluorescence for detection of proteins in cells, flow cytometry, infection with retroviruses and lentiviruses for permanent expression or silencing of genes in cells, transfection with genes of fluorescent proteins to carry out time lapse microscopy studies in living cells and standard techniques for cell biology studies, in general.

This research group have been previously involved, as partners, in the EU projects on "Genetic Risks Associated to Ionizing Radiation" (FI3P-CT920055), in the contract TELORAD "Telomere Instability and the Formation and Transmission of Radiation Induced DNA Damage" (FIGH-CT1999-00009), in the contract TELOSENS "Telomeres and Radiosensitivity of Individuals" (FIGH-CT-2002-00217), in the contract RISC-RAD "DNA damage responses, genomic instability and cancer: the problem of risk at low and protracted doses" (FP6-CT-2003-508842) and have coordinated the EU contract "Role of Telomere Addition in the Stabilisation of Radiation Induced DNA Breaks" (FI4P-CT960045).

 

Main tasks attributed in Dark.Risk project

Dr. Genescà will lead WP2, where UAB will contribute to the identification of long non-coding RNAs (lncRNAs) involved in the response to low dose radiation. In order to assess it, we will isolate RNA from Breast Primary Epithelial Cells (BPECs) irradiated at different low doses of X-ray. Then, we will perform gene expression microarrays that include probes to the lncRNAs cataloged by the Broad Institute. Those lncRNAs significantly affected by low dose radiation, are going to be further analyzed by RT-qPCR. We will also compare DNA damage response and functional importance of normal cells with cells with silenced candidate-lncRNAs.

In WP3 UAB will participate in the analysis of non-coding RNA identified in WP2 as markers of low dose exposures. To achieve it, RNA Taqman assays will be applied in the biological material from the Serbian cohort to quantify expression changes.

 

 

 

SERGAS

Participant 5

SERGAS-Servicio Galego de Saúde, Galicia, Spain

                       

Partner 05 (SERGAS) is leaded by Dr. José Luis Fernández García, chief of the Unit of Clinical and Molecular Genetics from the “Complexo Hospitalario Universitario A Coruña”, in Galicia, Northwest of Spain. This is a group of four academic hospitals with around 3750 employees, belonging to the Public Galician Health Service (Servicio Galego de Saúde-SERGAS). The administration activities are performed by a foundation - Fundación do Complexo Hospitalario Universitario a Coruña. The research activities of the “Complexo Hospitalario Universitario A Coruña” focus mainly in transplantation, rheumatology, regenerative medicine, microbiology, and genetics.

Dr. Fernández has been working in radiobiology since 1985, developing research in the field of chromosomal structure and mutagenesis, especially in biological dosimetry and the genetic effects of ionizing radiation. He has published more than 140 articles. His laboratory has developed the technology DBD-FISH (DNA Breakage Detection-Fluorescence In situ Hybridization) to study the damage induced in specific DNA sequences, and the test SCD (Sperm Chromatin Dispersion) to determine the frequency of sperm cells with fragmented DNA. The latter has been patented, being commercially available worldwide. The research group has been involved in numerous collaborative research projects in radiation biology and radiation protection, in particular three supported by the European Community mainly related to telomere biology and radiosensitivity and several projects supported by the Nuclear Security Council of Spain. It currently leads the programme of DNA and chromosome damage (toxicogenetics) within SERGAS.

Dr. Iria Gonzalez-Vasconcellos is a molecular biologist, MSc in Radiobiology and Oncology (UCL, 2007), which has been recently incorporated to the research group as a postdoc after performing her PhD in the Helmholtz Zentrum München (HMGU) at the Institute of Radiation Biology. Her work was dedicated to elucidate the mechanisms of how haploinsufficient mutations of the tumour suppressor gene Retinoblastoma (RB1) influence susceptibility to radiation-induced osteosarcoma. She demonstrated that decreased levels of Rb1 cause telomere shortening and promotes radiation-induced genomic instability in osteoblast primary cells (Gonzalez-Vasconcellos et al 2013). The team has previously shown that naturally occurring variations of the same RB1 gene render mice much more likely to develop cancers after radiation exposure (Gonzalez-Vasconcellos et al 2011).

                                       

Increased telomere attrition and instability  in Rb1 haploinsufficient primary mouse osteoblasts. Representative images from cell lines that were established from 2 animals. Left panels: wild-type cells at passage 8; middle panels: haploinsufficient cells at passage 8. Right panels: Radiation induced genomic instability in Rb1+/- osteoblasts. Compromised divisions, chromatin (CB)  and anaphase bridges (AB) and telomere fragments (TF) lost during the divisions detected in DAPI-labeled nuclei of  Rb1+/- osteoblast.

 

 

Within Dark.Risk, SERGAS will contribute with activities in WP2 and WP3.

In WP2 under the objective: The contribution of non-coding RNAs to differences in individual sensitivity due to changes in telomere length will be examined (Task 2.4) SERGAS will collaborate with HMGU to determine how the radiation regulated non-coding RNAs are involved in regulating chromatin structure, telomere length and genomic stability. SERGAS shall establish a functional assay to investigate specific long non coding RNAs (lncRNAs) expression.

In WP3 under the task 3.3 SERGAS will study the contribution of non-coding RNA transcriptome to individual differences in the telomere length and genomic stability. Proving its success, the basic knowledge will be translated using biomaterials from SRTCC to determine the use of long non-coding RNAs as potential biomarkers.

The aims of SERGAS are reflected in the following tasks, developed in collaboration with HMGU:

-Developing basic research, the group will extend studying the mechanism of increase in susceptibility to radiation-induced cancer in Rb1 insufficiency. Reduced expression of Rb1 prevents radiation-induced cell cycle arrest and promotes telomere shortening leading to an increase in genomic instability. In this task we will investigate the regulation of lncRNAs expression following radiation exposure, to determine if this is the target that triggers the instability.

-Following a translational approach, it will be performed in collaboration with ROSA, HMGU, ENEA, UAB, an evaluation of the suitability of the collection of archived biological samples from Serbian subjects irradiated for tinea capitis and control individuals for studies of telomere length, lncRNA and epigenetic markers. This is an important step for future studies on biological biomarkers of radiation exposure and adverse effects.

-Finally, we want to go deeper in the study of telomere structure and function as a marker of radiation-susceptibility, focusing in the potential contribution of lncRNA transcriptome to individual differences in the telomere length and genomic stability. We will use molecular assays to determine possible telomere length variation between individuals after radiation exposure.