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Issue 1

Newsletter

The Dark.Risk project isfinancially supported bythe 7th Frameworkprogramme of theEuropean Commission.

nside this issue

Main Objective p!

First reults

"# $ ROSA p%

"#!&% SERGAS p!

HMGU p%

ENEA p'

UAB p'

Meeting & Events p(

Website p(

Partners p(

Welcome

The Dark.Risk project has now been running for one year) with a European researcherteam. "e welcome you to this first issue of Dark.Risk newsletter. This will be bi*annual.The newsletter is launched to reach a wider audience seeking information about the

project. "e look forward to launching useful and informati+e issues. "e welcome anyfeedback and contributions.

Short introduction o the !roject

n the late $,(-s public health initiati+es in se+eral Europeancountries mandated scalp depilation as the standard of carefor the treatment for children infected with a fungal diseaseof the hair roots Tinea capitis/. The standard treatment

protocol used a mild dose of 0*irradiation to the head to killhair cells) and pro+ed highly effecti+e in combating thefungus. 1nly decades later it was noted) firstly in srael) then

in the 234 and other countries) that there was an increased fre5uency of cancer in thetreated indi+iduals.

The long term health effects following e6posure to low andmoderate ioni ing radiation remain uncertain. 4t doseslikely to be recei+ed from medical diagnostic procedures

e.g. Computer Tomography CT/ or #ositron EmissionTomography #ET// there is a lack of con+incing e+idencefor or against a risk to health. "ithin the Dark.Risk project we ha+e the uni5ue opportunity to study long*term healtheffects in a large cohort of indi+iduals 8 !(.---/ e6posedduring childhood to a highly uniform dose of radiation to the head and to create the3erbian Registry of Tinea capitis children 3RTCC/.

Facts and igures

Total cost999999999999!.% :;EC contribution9999999$.7:;Duration999999999%< :onths

From999999!-$!*$-*-$To99999999!-$(*-,*%- "ebsite9999 "ar#$%is#

Studies on a cohort of Serbian children exposed to x-irradiation to determine thecontribution of the non-coding genome to susceptibility at low doses

It is the mission o "ar#$%is# to evaluate the !otential o the non coding genometo deliver in ormation on disease outcome and association with radiation$


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! a g e ' (Main objective

Dark.Risk will prepare the way to use 3RTCC biomaterials to 5uantifythe contribution of indi+idual differences in sensiti+ity to risk from

low doses of ioni ing radiation. The focus will be on a no+el anduni5ue process to identify indi+idual differences. 2ntil recently ,(=of the human genome was considered to be non*translated >junk?D@4. Aowe+er) almost all of this >dark matterB is acti+ely transcribedinto a +ariety of non*coding R@4 species ncR@4s/ that regulateinteractions with the e6ternal en+ironment) such as repressingretrotransposons) regulating gene transcription responses to stress)

go+erning chromatic remodeling and coordinating the translation of mR@4s. The ncR@4s play an essential rolein coordinating critical cellular responses to radiation such as maintenance of genomic stability) senescence)apoptosis and sur+i+al.

First results

WP 1 "ithin the Dark.Risk project) "ork #ackage $ ismain responsibility of ZENSKA ASOCIJACIJA"ROSA" team. "#$ is designed to firstly establish the 3erbianRegistry of Tinea Capitis Children. This will befollowed by the recruitment of the cohort forepidemiological in+estigation on late healthconse5uences related to TC treatment) that is applied 0*ray irradiation used in inducing scalp hairremo+al. iological samples will then be collected from a selected number of indi+iduals in this cohort) andanalysed for targeted genetic traits by other Dark.Risk partners. 4t the end of year one) R134 s work can be briefly recapitulated as follows. n this period the primaryobjecti+e was to carry out identification of patients registered at the elgrade :ycosis Aospital from $,(-*$,(,. t was planned that )--- indi+iduals from the hospital records be identified by 1ctober $ st !-$%. Thisnumber has been reached) and we e6pect that it will continue to rise steadily) as scheduled by dynamics of

data collection. 4ll information concerning patients identity is stored into a digital database and is readyto be secured at the 3erbian nstitute of Aealth. n accord with 3erbian legislation >4ct on Conducting:edical ResearchB these records will be held there on behalf of R134. "e belie+e that this year was truly successful to us) and we hope that further stages of our work willcontinue to progress in an e5ually successful manner) meeting objecti+es and timetables of Dark.Risk.

WP 2/3SERGAS

#re+ious studies of the members of this consortium showed that reduced e6pression of Rb$ pre+entsradiation*induced cell cycle arrest and simultaneously creates shortening of the telomeres leading to anincrease in genomic instability Gonzalez-Vasconcellos et al., Cancer Research, 2013 /.These findings were linked to the changes in e6pression of a telomere specific long non coding R@4

lncR@4/ called TERR4. Together these two acti+ities can e6plain the increase in susceptibility toradiation*induced cancer in Rb$ insufficiency.

The goal of Dark.Risk is to establish a proof of concept by analysing the health status of Tinea capitis treated Serbianchildren with the aim of obtaining information on long-term health effects of low dose radiation exposure.


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! a g e ' )

During this first >Dark.RiskB year) the group of Dr. Fern ndepart of "#! and "#%/ is studying how Rb$ regulates

transcription of the TERR4 lncR@4) its action at the le+el of theTERR4 promoter and the possible telomere epigenetic alterations

arising from mutations in the Rb$ tumour suppressor gene.They are setting up an assay see figure/ to in+estigate theregulation of TERR4 e6pression in cells lacking one copy of theRb$ gene. Chromosomes $() $- and 0&G in human/ and $ ) $- and 0&G in mouse/ are being analysed at their subtelomeric regions

ensuring that the studies on TERR4 transcriptional le+els are both telomere length and location*independent. Furthermore) a standardisation of H*#CR methods to measure telomeric length in a largenumber of samples is being performed.Finally) a new techni5ue to discriminate between different epigenetic states of telomeres assayed at thechromatin condensation le+el is being de+eloped and e+aluated.

HMGU 4lthough) the data of the radiation effects on non*coding R@4 transcripts lncR@4s) miR@4s/ and deeperepigenetical control are in their infancy) we could confirmed the importance of miR@4s modulation incells after irradiation. A:I2 reports here the e6pression profiling of the radiation response of 8$()---lncR@4s that was undertaken with human microarrays 4rray3tar/ using R@4 from E4.hy,!< humanendothelial cell line/) 2!13 human osteosarcoma tumour cell line/ and T'7D human breast epithelialtumour cell line/) 'hr after !.(Iy gamma irradiation e6posure.

Differentially e6pressed lncR@4s were identified

following 5uantile normalisation) fold change filterprocessing Iene3pring/ with sample and control

sham irradiated/ comparison. 1f the se+en regulatedlncR@4s that were subse5uently selected for +alidation) all e6hibited the same up* or down*regulation following irradiation in each of the threecell lines. Conse5uently an lncR@4 that shows strongup*regulation in E4.hy,!< cells was also found up*regulated in T'7D and 2!13 cells) and +ice +ersa.This is the first indication that a gi+en radiation*regulated lncR@4 has a defined role across differentcell lines during the radiation response. 3uch a resultis a strong contrast to the situation obser+ed formiR@4s) which e6hibit highly cell*type specificchanges in e6pression.

:ean regulations of more than !*fold up*regulation were found for the lncR@4s #4@D4 and linc*p!$. 3ignificantly altered lncR@4s associated with genesin+ol+ed in cancer were selected for Ta5man real*time RT*#CR +erification in T'7D and :D4*: *%


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! a g e ' *E@E4

3tudies by E@E4 resulted in followingJ :edulloblastoma : / is the most common malignant braintumour of children) arising from cerebellar granule precursors CI#s/. Karge fraction of human : sre+eals mutations in 3AA pathway) one of the major regulators of cerebellar de+elopment. #atched$knockout mice #tch$L&*/) one of the most studied models of medulloblastoma due to 3AA pathway

deregulation) represent an ideal in +i+o model to in+estigate the role of ioni ing radiation in :de+elopment. Recent data demonstrate the rele+ance of miroR@4s in setting D@4 damage response.Therefore the #tch$L&*mice is a useful model for studies of changes in the transcription profile of non*coding R@4s after irradiation.

E@E4 carried out e6perimental settings to in+estigate the response to damage induced by radiation atpostnatal age ! #!/. CI#s) isolated from #tch$L&* and wt mouse cerebella) were irradiated with -.$) -.() $Iy. 4fter ' hours) cells were stained with propidium iodine # / and subjected to flow cytometry todetermine their D@4 content. Results show a statistically significant decrease of hypodiploid D@4 inmutant irradiated CI#s compared to their wt counterparts) for all e6perimental conditions) suggestingthat a lower rate of apoptosis could be caused by a more efficient D@4 repair after damage. These datacomply with the increased proliferation inde6 determined by = 3 phase plus = of I!&: phases/ managedby acti+ation of 3AA pathway in the mutant CI#s compared to wt.

E@E4 collected mutant and wt CI#s at #! after irradiation with $Iy of 0*rays and performed the >miR@4#CR 4rraysB. This arrays profile the e6pression of ' miR@4s known to alter their e6pression duringner+ous system*related carcinogenesis. #reliminary data show +arious microR@4s modulated in mutantIC#s +ersus wt. 3ome of the identified microR@4s are found deregulated in human medulloblastomas)supporting the hypothesis that could be in+ol+ed in this pathogenesis.

24 During this first year) at 24 we established a list of %!long intergenic non*coding R@4s lincR@4s/ induced in

reast #rimary Epithelial Cells #ECs/ after a low ormoderate dose of 0*ray e6posure. The biological material was obtained from normal breast tissue from disease*freereduction mammoplasties. Cells were irradiated using amammogram 0*ray de+ice and a Therapa6) which is an 0*ray source that is used to irradiate at higher doses butemulated the dose rate and the energy gi+en by themammogram. 4fter irradiation) R@4 was e6tracted and

hybridi ed in 3ure#rint I% Auman Iene E6pression +!:icroarray) which includes probes to lincR@4s cataloguedby scientists at the road nstitute.

:oreo+er) we in+estigated whether the humanhomologous region of the murine lincR@4*p!$ wasacti+ated after irradiation. The lincR@4*p!$ is acti+atedafter do6orubicin treatment in mouse. t acts as a repressor of genes normally repressed by p(% Huarte etal 2010/. Different primers co+ering all the human lincR@4*p!$ se5uence known so far were designed tocheck its e6pression by 5#CR after e6posure to !Iy of gamma*rays. @o significant results were obtainedand thus we can conclude that radiation*induced damage does not acti+ate its e6pression in #ECs and inhuman dermal fibroblasts.


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