Constitutive expression of tdTomato protein as a cytotoxicity and proliferation marker for space radiation biology

GermanAerospaceCenter(DLR),InstituteofAerospaceMedicine,RadiationBiology,LinderHöhe,D-51147Köln,Germany

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Articlehistory:

Received25September2014

Receivedinrevisedform21December2014 Accepted30December2014 Keywords: Mammaliancells Radiationresponse Fluorescentprotein Cytotoxicity Proliferation tdTomato

The radiation risk assessment for long-term space missions requires knowledge on the biological effectiveness ofdifferent spaceradiation components,e.g. heavy ions,on the interaction ofradiation and other spaceenvironmentalfactors such as microgravity, and onthe physical and biological dose distribution in the human body. Space experiments and ground-based experiments at heavy ion acceleratorsrequirefastandreliabletestsystemswithaneasyreadoutfordifferentendpoints.Inorderto determinetheeffectofdifferentradiationqualitiesoncellularproliferationandthebiologicaldepthdose distributionafter heavy ionexposure,astable humancell lineexpressinganovel fluorescent protein was established and characterized. tdTomato, a red fluorescent protein of the new generation with fast maturationand high fluorescenceintensity,was selected asreporter ofcell proliferation.Human embryonic kidney (HEK/293) cells werestably transfected with aplasmid encoding tdTomato under the control of the constitutively active cytomegalovirus (CMV) promoter (ptdTomato-N1). The stably transfectedcelllinewasnamedHEK-ptdTomato-N18.Thiscytotoxicitybiosensorwastestedbyionizing radiation(X-raysandacceleratedheavyions)exposure.Asbiologicalendpoints,theproliferationkinetics andthecelldensityreached100hafterirradiationreflectedbyconstitutiveexpressionofthetdTomato wereinvestigated. Bothwerereduced dose-dependentlyafter radiationexposure.Finally, thecell line was used for biological weighting of heavy ions of different linear energy transfer (LET) as space-relevant radiation quality. The relative biological effectiveness of accelerated heavy ions in reducing cellular proliferationpeaked atanLET of91 keV/μm.The resultsof thisstudydemonstratethat the HEK-ptdTomato-N1reportercelllinecanbeusedasafastandreliablebiosensorsystemfordetectionof cytotoxicdamagecausedbyionizingradiation.

©2015TheCommitteeonSpaceResearch(COSPAR).PublishedbyElsevierLtd.All rights reserved.

Abbreviations: A260, absorbanceat 260nm; A280, absorbanceat 280nm; BP, bandpass; CellRad, Cellular Responses to Radiation in Space; CMV, Cy-tomegalovirus;Cu,copper;DMSO,dimethylsulfoxide;DsRed,redfluorescent pro-teinfromDiscosomasp.;EGFP,EnhancedGreenFluorescentProtein;ESA,European SpaceAgency;F,fluence;FA,formaldehyde;FBS,fetalbovineserum;FT,“Farbteiler” –dichroicmirror;GANIL,GrandAccélérateurNationald’Ions Lourds;GCR, galac-ticcosmic rays;Gy,Gray;GSI,GSIHelmholtzzentrumfür Schwerionenforschung GmbH;HEK/293,Humanembryonickidney;IBER,InvestigationsintoBiological Ef-fectsofRadiationUsingtheGSIAcceleratorFacility;ISS,InternationalSpaceStation; LEO,lowEarthorbit;LET,linearenergytransfer;LP,longpass;MDR,multidrug resis-tance;MTT,3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazoliumbromide;NADH, reducednicotinamideadeninedinucleotide; PBS,phosphatebufferedsaline;RBE, relativebiologicaleffectiveness;RBE50%PR,RBEfor50%proliferationreduction;SCR,

solarcosmic rays; SpaceLife,Helmholtz SpaceLifeSciences ResearchSchool;Sv, Sievert;tdTomato,tandemdimerTomato;μ×g,microgravity.

*

Correspondingauthor.Tel.:+4922036013243;fax:+49220361970.

E-mailaddresses:arif.chishti@dlr.de(A.A. Chishti),christine.hellweg@dlr.de

(C.E. Hellweg),thomas.berger@dlr.de(T. Berger),christa.baumstark-khan@dlr.de

(C. Baumstark-Khan),sebastian.feles@dlr.de(S. Feles),tkaetzel@uni-bonn.de

(T. Kätzel),guenther.reitz@dlr.de(G. Reitz).

1 _{Presentaddress:KarachiInstituteofBiologyandGeneticEngineering(KIBGE),}

UniversityofKarachi,Karachi-75270,Pakistan.

1. Introduction

Longstaysinspace,whetherontheInternationalSpaceStation (ISS)oronreturnflights toMoonorMars,arenotonlyaphysical and mental challenge for the astronauts because ofmicrogravity (

μ

×

g) andlivingina confinedenvironment, butarealso associ-ated withincreasedionizing radiationexposure due tothe com-plexnaturalradiationenvironment(Reitz,2008).TheISSislocated in low Earthorbit (LEO)at an altitudeof approximately415 km fromtheEarth’ssurface.TheradiationfieldinLEOconsistsofsolar cosmicrays(SCR),galacticcosmicrays(GCR)andtrappedparticles of the Earth’sradiation belts, resulting in thepresence of highly energeticprotons,baryons,electrons,neutrinos,

γ

-rays,

α

-particles (helium nuclei) and nucleiof atomsheavier than helium.Highly

2 _{Presentaddress:InstitutfürHumangenetik,BiomedizinischesZentrum,}

Univer-sitätsklinikumBonn,Sigmund-Freud-Str.25,D-53127Bonn,Germany.

http://dx.doi.org/10.1016/j.lssr.2014.12.005

energeticparticles ofSCRandGCR, withhighvelocityand pene-trance,representahealthriskforastronauts(Hornecketal.,2010). TheradiationfieldinsidetheISSisalsohighlyvariableduetothe changesinISSattitude,altitudeandduetothelocalshielding en-vironmentforvarious sectionsinsidetheISS,leadingto absorbed dose valuesintherangeofa fewhundredμGray/day (μGy/d) in-side and up to the mGy/d range outside the ISS (Berger et al., 2013). Thebiological relevantdose (effectivedose)asa prerequi-siteforhumanradiationriskassessmentwasdeterminedapplying relevantphantomexperiments tobe intheorderof

∼

550 μSiev-ert/d(μSv/d)forexposuresinsidetheISS(Puchalskaetal.,2014).

Thebiologicaleffectsofionizingradiationdependonitsability to ionize atoms or molecules. The most critical target of ioniz-ing radiation within a cell is the cell nucleus, particularly DNA. DNA damage induced by ionizing radiation may lead to either early or late effects, such ascarcinogenesis, if DNA repairis un-successful (Tubiana, 2009). Early after exposure or with a delay ofdays,ionizing radiationcan elicitcell death.Dependenton ra-diationquality, dose, dose rateandcell type andstatus, ionizing radiationcancausedifferenttypesofcelldeathandreducecellular survival(Takashi,2013; Panganibanetal.,2013; Yangetal.,2010; Pettersenetal.,2007).Acommonlyusedparametertodescribe ra-diationqualityisthelinearenergytransfer(LET3_{)inmatterwhich} depends on mass, charge and energy of the particles. For many biological endpoints including cell killing, it was shownthat the relative biological effectiveness (RBE) ofdifferent heavy ions de-pends on LET with a peak at about 100–200 keV/μm(Cucinotta andDurante,2006).

Theeffectofradiationoncellsurvivalcanbemonitoredby sen-sitive bioassays orreporter systems.Such bioassays will comple-mentthephysicaldetectorsystemsusedinspaceandonEarth,in view ofthefactthat theyyield intrinsicallybiologically weighted measures of cellularresponses tothe complex radiationfield for thespacehabitat.Previouslyconventionalvitaldyesand histolog-ical stainingmethods to examine membrane integrity were used asmarker toolsforgrowth determination.For example,the MTT test is used as a cost-effective standard assay to determine cell viability, cell growth and cytotoxicity. It requires the addition of 3-(4,5-dimethyethiazole-2-yl)-2,5-diphenyltetrazoliumbromide as substrate,whichdestroysthecellsbyformationofspicularcrystals afterreductionbycellularreducingagentssuchasreduced nicoti-namideadeninedinucleotide(NADH)(Berridgeetal.,2005).Ithas tobesolubilizedbyadditionofdimethylsulfoxide(DMSO), there-foretime-courseexperimentscanonlybe carriedout byrepeated addition of the reagent to separate culture vessels (Rampersad, 2012). While additionof a substrate andcell lysis mightstill be feasible forscreeningof a large setof radiationqualities and bi-ological depth-dose distribution tests (obviating the need for it wouldsavetimeandresources),forspaceexperiments,the devel-opmentofa screeningtest withaninherent reporter isrequired. Asliquidhandlinginspacerequiresspecialdevicesandastronaut timeisvery limited,additionofa substrateandcelllysishaveto beavoidedinaspace-suitablecytotoxicitytest.

Morerecently,fluorescentproteinshavebecomemorecommon astheir expression is instantaneous,quantitative, non-invasive in living tissues and it reduces the possibility of staining artifacts (Jiangetal.,2004).Fluorescencedoesnotrequireanysubstrateor cofactor, itcan easily be usedin livingcells tomonitor signaling pathways (Davidson and Campbell, 2009; Verkhusha, 2001). Ex-pressionoffluorescentproteinscanbemeaningfullyimplemented intumorcellresearch,includingstudiesonmetastasisand angio-genesis where real-time imagingcan be used during therapeutic

3 _{LETisdescribedastheaverageenergydeposition(E)onthebeampath(S)}

ofanioninmatter,E/S (keV/μm).

studiesandtounderstandthemetastaticprocessandefficiencyof potentialtherapeutics(Hoffman,2005; Lietal.,1997).Constitutive expressionofafluorescentproteinasabiomarkerinreportercell lines allows thequantification of toxin levels and aids in under-standingmechanismsoftoxicity(Baumstark-Khanetal.,2010).

Fluorescent protein spectrums range from UV-excitable green to far-red (Shaner et al., 2004, 2005). The discovery of the red fluorescent protein (DsRed) fromthe seaanemone Discosomasp.

has openeda newaspectofbiological imaging(Campbell,2002). Different variants of DsRed are widely used in reporter studies ofgene expressionandproteinlocalizationduetoimproved con-trastanddecreasedphototoxicity,autofluorescence,andscattering (Davidson and Campbell, 2009; Hendriks et al., 2011). A whole family of fruit fluorescence proteins was constructed by altering DsRed, including the tandem dimer Tomato(tdTomato), mCherry and mStrawberry (Davidson and Campbell, 2009; Shaner et al., 2004). Shaner et al. improved the maturation kinetics through fiveroundsofdirectedevolutionresultingintheextremelybright fluorescence of dTomato, a feature that makes the protein a de-sirable choice when visualizing a sparse target (Shaner et al., 2004). dTomato has the highest brightness (product of extinc-tioncoefficient, 138,000Mol−1_cm−1_{,andquantumyield,0.69)at}

the cost of doubling the molecular weight (Shaner et al., 2004; Stronginetal.,2007).TwocopiesofthedTomatogenewerefused togethertocreatethetandemdimertdTomatowithexcitationand emission maxima at554 and 581 nm, respectively (Hinterdorfer andvanOijen,2009). Thisnewly designedtdTomatoshowed bet-tersuitabilityandbrighterflorescenceincellimagingtasksandis exceptionally photostablebecauseofits twinfluorophores. There-fore,anewtestsystemusingtdTomatowasestablishedtoreplace anearliercytotoxicityassayusingapromoterreportersystemwith EnhancedGreenFluorescentProtein(EGFP)(Hellwegetal.,2007a), originallyisolatedfromthebioluminescentjellyfishAequorea victo-ria (Shaneretal.,2008).

Theaimofthisstudywastoquantifycytotoxiceffectsof ioniz-ingradiationbyfluorescencemeasurementoftdTomatoexpressing HEK-ptdTomato-N1 cells. The fluorescence spectrum and inten-sity, and the growth kinetics of this cell line were determined. The newly constructed reporter system has a constitutively ac-tive promoter sequence that allows continuous expression of td-Tomato and thereby growth determination after radiation expo-sure. As growth determination islinked withfluorescent protein synthesis the loss of tdTomato fluorescence following irradiation indicates celldeath. Quantificationoffluorescenceincells canbe accomplishedby differentmeans,includingtheuseofmicroplate readers, flow cytometry anddigital fluorescence microscopy. The relative biological effectiveness (RBE) for growth reduction was comparedforheavyionsofabroadLETrange.

2. Materialsandmethods 2.1. Plasmidcloningandpreparation

The plasmid ptdTomato-N1 (Fig. 2A) was obtainedfrom Clon-tech (Palo Alto, USA). In this vector, the tdTomato gene is un-der control of the immediate early promoter of human Cy-tomegalovirus (CMV). The vector contains the aminoglycoside phosphotransferasegenereferringkanamycinresistanceinbacteria andG418 resistanceinmammaliancells. CompetentE.coli DH5

α

Fig. 1. Heavy ionexposureofHEK-ptdTomato-N1cells.The4-well-stripswereadaptedintoaholder,closedwithsiliconlids(A)andexposedtoacceleratedheavyions throughthebottomofthewell(B).PicturesweretakenatGANILinroomD1,withthebiologicalsampletransporterinfrontofthebeamexit.

preparationswithanA260/A280ratioover1.8wereusedfor trans-fectionofmammaliancells.

2.2.Cellstrainsandcultureconditions

Humanembryonickidney(HEK/293,ATCCCRL-1859)cellswere culturedaccordingtostandardproceduresin80cm2 _{flasks(Nunc,}

Wiesbaden,Germany)in

α

-medium (modifiedMEM, Pan-Biotech, Germany) with 10% fetal bovine serum (FBS) at 37◦C in a sat-urated humidity and a 5% CO2/95% air atmosphere. For weekly

sub-cultivation, cell lines were washed with PBS before detach-ingwithtrypsin/EDTAsolution(PANBiotech,Aidenbach,Germany). Subsequently,cellswere seededatadensity3

×

104 cells

/

cm2 in newflasks.Mediumwas changed every 4 days.Toavoid detach-ment of HEK cells during experimental procedures, cell culture vessels (Petri dishes,microtiter plates) were coated with poly-D-lysine (10 mg

/

cm2, Sigma-Aldrich Chemie, Steinheim, Germany) for15 min at 37◦C and washed three times with sterile deion-izedwaterbeforeuse.

Enhancedgreen fluorescent protein expressing HEK-pEGFP-N1 cells (Hellweg et al., 2007a) were used for comparison and cul-turedin

α

-mediumwith10%FBSand0.6 mg

/

mlG418.

2.3.Generationofastablytransfectedreportercellline

HEK/293 cells were transfected with the ptdTomato-N1 vec-tor using liposome-mediated DNA transfer (Fugene 6, Promega, Madison, WI, USA). The cells were seeded at a density of 3

×

104 _cells

_/

_cm2 _{into 24-well-plates (Falcon, Becton Dickinson}

Lab-ware,Heidelberg,Germany)andincubatedfor3days.Semi conflu-entcellculturesweretransfectedfollowingtheinstructionsofthe supplier.Forstabletransfection,thetransfectedcellswere trypsi-nated48 haftertransfection,diluted1:10withmediumcontaining 1.5 mg

/

ml G418 (Calbiochem, La Jolla,CA, USA) and cultured in Petri dishes.After10days,G418resistantcellswereseededin mi-croplates at a concentration of 50 cells per plate andcultivated for10–20days.TheresultingcolonieswerescreenedfortdTomato proteinexpressionusingthefluorescencemicroscopeAxiovert135 (Carl Zeiss, Oberkochen, Germany) as described in Section 2.8. Clones expressing tdTomato protein were reseeded into 24-well-plates. In addition, those that gave highest fluorescence without anymorphologicalorgrowthchangewereselectedbyflow cytom-etry. From such stably transfected clones, the tdTomato express-ingcell lineHEK-ptdTomato-N1 8wasderived andmaintainedin mediumcontaining0.6 mg

/

mlG418.

2.4. Radiationexposureandproliferationdetermination

HEK-ptdTomato-N1 8 cells were seededtwo days before irra-diation to reach a density of 20–30% at the time of irradiation. CellswereseededintoCostar9102stripwellplates(Sigma-Aldrich Chemie) containing 8wells per strip witheach a growtharea of 0.3 cm2andavolume320 μl.Afterseeding,thecellsadheretothe bottomofthewell.Thewellswerecompletelyfilledwithcell cul-turemediumimmediatelybeforeirradiationandclosedbymeans of sterile silicon lids (Abgene, Thermo Scientific, Schwerte, Ger-many). The 8-well stripswere brokeninto pieces of 4wells and positioned intospeciallydesignedholders(Fig. 1A)andirradiated asdescribedbelow.

Afterirradiation,thestripswereremovedfromtheholdersand thestripswerereorganizedintothe96-well-plateframe.The sili-conlidswereremoved.Mediumwaschanged,cellswereincubated andgrowthofcellswasfollowedbyfluorescencemeasurementof tdTomato. Its fluorescence intensity was measured twice daily in themicroplatereaderforupto

∼

100 hasexplainedinSection2.9. Forthefinalmeasurementat

∼

100 hafterirradiation,thedose ef-fectcurvesforproliferationreductionwerecalculatedasdescribed inSection2.10.

2.4.1. X-rayexposure

Cells were exposed to low LET (0.3–3.0 keV

/

μm) X-rays us-ing theGulmay X-raysourceRS225 (X-Strahl, Surrey,UK)at DLR Cologne. The X-ray tube was adjusted to 200 kV and 15 mA. To eliminate softX-rays,a Copper (Cu) filterwitha thickness of 0.5 mmwasused.Doseanddoseratewere determinedusingthe dosimeter UNIDOSwebline with the ionization chamber TM30013 (PTW, Freiburg, Germany). The distance of the sample from the X-raysourcewassetto450 mmtoprovideaconstantdoserateof 1.0 Gy

/

min.The temperatureinside the X-raychamber was kept at37◦Candsamplesweretransferredafterexposuretothe incu-bator.AstheX-raysourcewas locatedabovethesamples,cells in stripswereexposedinhorizontalpositionbelowtheexitwindow.

2.4.2. Acceleratedheavyionsexposure

Exposure to 13_{C (LET 33 keV}

_/

_μm), 22_{Ne (LET 91 keV}

_/

_μm), 56_{Fe (LET 151 keV}

_/

_μm), 58_{Ni (LET 175 keV}

_/

_{μm) and} 58_{Ni (LET}

905 keV

/

μm) ions was performedat the GrandAccélérateur Na-tionald’IonsLourds(GANIL,Caen,France)andtheGSI Helmholtz-zentrum für Schwerionenforschung GmbH (GSI, Darmstadt, Ger-many)aslistedinTable 1.

sam-Table 1

Acceleratedheavyionsandreferenceradiationusedinthisstudy. Ion Location Energy

(MeV/n) Energyontargeta (MeV/n) LET (keV/μm) X-rays DLR 0.5–3 13_{C GANIL 75 71 33} 22_{Ne GANIL 80 75 91} 56_{Fe GSI 1000 997 151} 64_{Ni GSI 1000 985 175} 58_{Ni GANIL 75 56 905}

a _{Effectiveirradiationenergyatthecellmonolayeraftertheenergylossesintwo}

detectors,theexitwindow,air(GANIL:1 cm,GSI:100 cm)andthebottomofthe culturevessel(1200 μmpolystyrene).

ples insample holderswere positioned on rectangular blockson a conveyor belt and were transported in front of the beam. The correctpositioningofthesampleswasverifiedbyavideocamera system. AtGANIL,thesampleholderswereinsertedinthe biolog-ical sample racks in upright position (6 samples per rack). Four sample racks were inserted in the biological sample transporter andmoved infrontofthebeam(Fig. 1B).Astemperaturecontrol was not available at GSI and at GANIL, samples were irradiated atroom temperature. The irradiation time per sample was up to 8 min, depending on dose, and the total time in the irradiation room was 20–30 min at both facilities. Controls were treated as the irradiatedsamples exceptthat they were not exposed tothe beam.Theywerestoredintheirradiationroominuprightposition forthesametimeastheirradiatedsamples.

Dosimetrywas performedby thestaffattheaccelerator facili-tiesanddose rateswere adjustedto

∼

1 Gy

/

min.The fluence (F) ofheavyions(particles/cm2_,P

_/

_cm2_{)wasconvertedtoenergydose}

inGy by thefollowing formulaunderconsideration ofthe linear energytransfer(LET,inkiloelectron-volt,keV,perμm) ofthe re-spectiveheavyion:

Dose

[

Gy

] =

1

.

6

×

10−9

×

LET

[

keV

/

μm

] ×

F



P

/

cm2



(1)

2.5. Growthdetermination

For growth determination, HEK-ptdTomato-N1 8 cells were seeded into Petri dishes (

∅

3 cm, Nunc, Wiesbaden, Germany) at a density of 1

×

104 cells

/

cm2 and harvested by trypsina-tion at regular time points. Cell numbers were determined by counting aliquots of the cell suspension in a counting chamber (Fuchs-Rosenthal). Incomparison, cell growthwas determined in the microplate reader as described in Section 2.9 after seeding HEK/293 and HEK-ptdTomato-N18 cells (1

×

104 _cells

_/

_cm2_{) into}

96-well-plates(Costar3603,CorningCostar,Cambridge,MA,USA). Fluorescence measurements were performed at the same time points, while medium changes for both experiments were per-formedinanintervalofeveryfourthday.

2.6. Flowcytometry

Forflow cytometry,cells were detached fromthe growth sur-faceusingtrypsinandfixedwith3 mlice-cold3.5%formaldehyde (FA) in phosphate buffered saline (PBS) for 30 min. The FA was dilutedwithPBS(1:3)andcells werestoredat4◦C.Priortoflow cytometricanalysis,cellswerecentrifugedandresuspendedinPBS. ForwardandsidescatterandtdTomatofluorescence(FL-2channel, 565–606 nm)ofthesamplesweremeasuredinafluorescence ac-tivatedcellscanner(FACScan,BectonDickinson,SanJose,CA,USA) withan argon laser (488 nm) asexcitation source andanalyzed bytheCellQuestsoftware(version1.2,BectonDickinson,SanJose, CA, USA).2

×

104 cells were analyzedat arate of200–600 cells per second. Forwardand side scatter ofthe samples were setin a dotplot asa measure of cell size andgranularity and a region

of intact cells was defined. In the FL-2 histogram, the markers for tdTomato(−) _{cells and tdTomato}(+) _{cells were set by means} of non-fluorescent and fluorescent cells within the gated intact cell population. Thepercentage oftdTomato(+) _{cells was usedas} ameasureofthefluorescentcellpopulation.

2.7. Fluorescencespectroscopy

Todetermine thefluorescence excitationandemission spectra oftdTomatoprotein, aspectrofluorimeter (F-2700,Hitachi, Tokyo, Japan)was used.Formeasurement, 4

×

106cells were fixedwith 3.5%FAinPBS,incubatedfor30min,centrifugedandresuspended in4 mlPBSforanalysis.

2.8. Fluorescencemicroscopy

The tdTomato expression in HEK-ptdTomato-N1 8 cells, culti-vated on 16-well slides (Nunc, Wiesbaden, Germany), was visu-alized using an inverted fluorescence microscope (Axiovert 135, CarlZeiss, Oberkochen,Germany), equippedwitha filterset suit-ableforredfluorescencedetection(Zeissfilterset20,excitationBP 546/12 nm,dichroic mirrorFT560,emissionBP575–640 nm).As excitationsource,amercuryvaporshortarclamp(HBO50Wtype 448006,CarlZeiss)wasused.Photographsweretakenbymeans of the high-resolution microscopy camera Mrc 5 andAxioVision Rel.4.4Software(CarlZeiss).

2.9. Fluorescencemeasurementinamicroplatereader

Fluorescenceintensitiesofgrowingcellsin96-well-plateswere determined using a microplate reader (Lambda Fluoro 320 plus, MWG Biotech, Ebersberg, Germany). The plates were measured withoutlid.tdTomatowasdetectedwiththeopticspositioned un-der thebottom of the plateand the filters540/25 forexcitation and590/35foremission.

2.10. Relativebiologicaleffectiveness

TheRelativeBiologicEffectiveness(RBE)forproliferation reduc-tion by heavy ions in comparison to X-rays was calculated from the regression lines of the dose-effect curves for growth reduc-tion 100 hafterradiationexposure.Thedoses-effect curvesshow therelativefluorescencedependentonradiationdose.Relative flu-orescence of living cells was calculated by subtraction of back-groundfluorescenceofHEKcells, microplatebottom andcell cul-turemedium100hafterradiationexposure.Forthispurpose,two wellsperdosewereseededwithnontransfectedHEKcells,andthe mean ofthesetwowells wassubtracted fromeach well contain-ing HEK-ptdTomato-N18cells thatwereirradiatedwiththisdose to correctforautofluorescenceofcells. The backgroundcorrected fluorescenceoftheirradiatedsampleswasnormalizedtothe unir-radiatedcontrol.Foreachdoseeffectcurve,aregressioncurvewas calculatedusingSigmaplot12.0andthedosereducingtherelative fluorescenceto50%was derived(50%proliferationreduction).The RBEfor50%proliferationreduction(RBE50%PR)wasthencalculated

by dividingthe required absorbeddose ofX-rays (200 kV, refer-enceradiation)bytheabsorbeddoseofheavyions(testradiation) usingthefollowing equation:

RBE50%PR

=

Energy dose of X-rays

[

Gy

]

Energy dose of heavy ion

[

Gy

]

(2)

2.11. Statistics

Fig. 2. Schematic diagramofptdTomato-N1vectorandtdTomatoexpressionofHEK/293cellsaftertransfectionofthisvector.TheplasmidptdTomato-N1(A)wasstably transfectedintohumanembryonickidneycells(HEK-ptdTomato-N1).Singlecolonieswerepickedafterstabletransfectionandcellswerepropagatedintissuecultureflasks. PhotographsweretakenwiththemicroscopycameraMrc5attachedtoafluorescencemicroscope(B:phasecontrast,C:redfluorescence,filtercombination540/590).

timeisveryrestrictedanditwasnotpossibletorepeatthe exper-imentsinindependentbeamtimesforeveryionanalyzedinthis work.MeansandstandarderrorswerecalculatedwithMicrosoft®

OfficeExcel2010andSigmaPlot12.0.

3. Results

3.1.Creationofstablytransfected,tdTomatoexpressingcelllines

HEK/293 cells were stablytransfected withthe ptdTomato-N1 vector containing the tdTomato gene under control of the CMV promoter (Fig. 2A). Stably transfected clones were screened for tdTomato proteinfluorescencebyfluorescencemicroscopy(Fig. 2B, C).AllcellsinacolonyofstablytransfectedHEK/293cellsshowed red fluorescence after cultivation in

α

-medium containing G418. tdTomatowas localized in thecytoplasm andthe nucleusof the cell(Fig. 2C). Clones that expressedthe tdTomatogene were ex-pandedandanalyzedby flowcytometry.Thisanalysisof26 HEK-ptdTomato-N1clonesindicatedthat clone #8exhibitedtdTomato expression in all cells and showed highest mean fluorescence (Fig. 3).Therefore HEK-ptdTomato-N1 clone# 8 was selected for furtherexperiments.

3.2.Fluorescencespectraandintensity

Theexcitation andemission spectraoftdTomato, expressedin HEK/293cells,weredeterminedusingaspectrofluorimeter(Fig. 4). Inthe HEK-ptdTomato-N1cells, the excitation spectrum peaksat 555 nm (fluorescence intensity, FI: 91.4). The emission spectrum showsitsmaximumat577 nm(FI:88.2).Excitationandemission maximaareratherneartoeachother withaStokes shift (excita-tion/emissionwavelengthdifference)of22 nm.

ThetdTomatofluorescenceofHEKandHEK-ptdTomato-N1cells was analyzed by flow cytometry. Wild-type HEK cells showed low autofluorescence andwere quantified using the tdTomato(−) marker. Cells with a strong red fluorescence were designated as tdTomato(+) _{(Fig. 5A). HEK-ptdTomato-N1 cells had superior} flu-orescence compared to formerly generated HEK-pEGFP-N1 cells (Fig. 5B).

3.3. GrowthoftdTomatoexpressingcellsusingfluorescence measurement

In order to exclude possible effects of stable transfection on growth characteristics, the growthkinetics of HEK-ptdTomato-N1 cells wascompared tothoseofHEK/293 cells.The growthcurves showed a lag phase, an exponential phase (log phase) and a steady-statephase (Fig. 6A).The lagphasesofbothcell lineslast for around 24 h after seeding, and then cells start growing ex-ponentially with a comparable slope up to 120 h. The doubling timeofbothcelllinesduringthelogphasewas

∼

20 h.Thesteady stateisreachedafter120 h.Thecellsreachamaximumdensityof 4

×

105 _cells

_/

_cm2_.

Cellularproliferationwasmonitoredbymeasuringfluorescence intensityofconstitutively expressedtdTomato. Tocalculate solely the fluorescence derived by fluorescence proteins, nontransfected HEK/293 cells were treated and measured the same way asthe HEK-ptdTomato-N1 8cells. The backgroundfluorescence (average autofluorescenceofHEKcellsandthefluorescenceofwellbottom andthecellculturemedium)wassubtractedfromthefluorescence valuesmeasuredforHEK-tdTomato-N18cells. Growthkineticsof HEK-tdTomato-N18cellswasdeterminedbyseeding1

×

103_cells

Fig. 3. tdTomato expressionofstablytransfectedHEKcellclones.Cloneswere se-lectedaccordingtotheirpercentageofthetotalpopulationexpressingthe fluores-centproteintdTomato(A)andtheirmeanfluorescence(B).Clone#1represents theuntransfectedwild-typecells(HEK/293).Clone#8showedhighestmean fluo-rescenceandwasselectedforfurtherexperiments.

Fig. 4. Fluorescence spectraoftdTomatoinHEKcells.ThetdTomatoproteinwas stablyandconstitutivelyexpressedinHEK/293cells.Formeasurementofthe emis-sionspectrumoftdTomato(A),theexcitationwavelengthwassetto510 nm.The excitationspectrumoftdTomatowasmeasuredatanemissionof610 nm.

aplateauafter12days(Fig. 6B).Measurementoffluorescence in-tensitiesofknowncellnumbersinthemicroplatereaderrevealed thattheredfluorescenceaugmentedasthecellcountperwell in-creased(Fig. 7).Thereby,thecellnumbercanbedeterminedfrom therespectivefluorescenceintensityvaluesusingthisfluorescence intensity–cellnumbercurve(Fig. 7).

3.4. GrowthinhibitionafterX-rayexposure

Cells were exposed to X-rays (0, 2, 6 and 12 Gy) 48 h af-terseeding.Thefluorescenceintensitywasmeasuredsubsequently twiceadayover

∼

100 h inthemicroplatereader.Itwasobserved that the increase ofrelative fluorescenceintensity overtime was reduced withincreasing dose (Fig. 8A). The relative cellular pro-liferation decreased significantly after2 Gy andproliferationwas stronglysuppressedafterexposureto16 Gy.Thegrowthcurves af-terneonionexposurealsoshowareducedgrowthafterexposure to2 Gy(Fig. 8B).Thegrowthinhibitingeffectof6 Gyneonionsis strongercomparedto6 GyX-irradiation.Theresultsshowthatthe appliedrecombinantcelllineissuitableforscreeningthecytotoxic effectofdifferentradiationqualitiesincludingheavyions.

3.5. Cellularsurvivalafterheavyionsexposure

Relative fluorescence was calculated to compare the effect of differentradiationqualitiesoncellularproliferation(Fig. 9A). Sur-vivaldecreasedwithincreasingdose.HighLETradiationincluding

13_{C (33 keV}

_/

_μm), 22_{Ne (91 keV}

_/

_μm), 56_{Fe (151 keV}

_/

_μm), 64_Ni

(175 keV

/

μm)and58_{Ni(905 keV}

_/

_{μm)ionsshowedahigh}

damag-ingcapacityasseenbythesteepdecreaseofrelativefluorescence. Theslopeofthecurvesrepresentsthekillingeffectwhichdepends on theradiationqualityasdefinedby LET.Heavy ionsintheLET range of

∼

30–175 keV

/

μm showed a maximal killing effect. In caseof58_{NiparticleswithveryhighLET(905 keV}

_/

_{μm),thekilling}

effectwasonlyslightlystrongercomparedtoX-rays.

TheRBEforgrowthreductionwascalculatedbasedonthedose neededtoreducecellgrowthto50%with200kVX-raysas refer-enceradiation (Fig. 9B).The RBE50%PR reachesa maximumof4.3

at91 keV

/

μmfor22_{Neions.TheRBE}

50%PR iswell above2for13C, 56_{Fe and} 64_{Ni ions. It drops to 1.4 for}58_{Ni ions with an LET of}

905 keV

/

μm.

4. Discussion

Inthelast fewdecadesbacterial,yeast,insectandmammalian cell-based systems have been developed (Baumstark-Khan et al., 2010; Laietal., 2006; Robertoetal., 2002; Lagendijketal., 2010) as alow costand efficientalternative assaysto liveanimals. For the purposeof radiobiologicalstudies, a humancell linesuitable to be transfected with a fluorescent reporter gene was selected. HEKcellsareconsideredasausefultooltostudydifferentcellular processes,asthey canbeeasilytransientlyandstablytransfected. HEK cells havealready beenusedin pharmacological, endocrino-logical, and toxicological studies and also in other reporter gene studies (Sung et al., 2009; Lodeiro et al., 2009; Lau et al., 2009; Boraetal.,2008; Maoetal.,2007).HEKcellshavealsobeenused inradiobiologicalstudiesasabiologicalmodelfordose-depth dis-tribution ofa proton beamusingthe resazurin assay (Kim etal., 2007) and for analysis of cell cycle checkpoints after irradiation (Yuet al., 2001). In ourprevious studies withHEK cells, theLET dependence of cellular killing by heavy ions was comparable to thatobservedinothercelltypes(Hellwegetal.,2011).

Fig. 5. Fluorescence intensityoftdTomatoinHEKcells.RedfluorescenceintensityofastablytransfectedHEK-ptdTomato-N1clonewasmeasuredbyflowcytometryinchannel FL-2(tdTomato(+)_{)andcomparedtountransfectedHEK/293cells(tdTomato}(−)_{)(A).Forcomparisontoafluorescenceproteinofthefirstgeneration,thegreenfluorescence}

intensityofHEK-pEGFP-N1cells(B)isshown,whichexpressEnhancedGreenFluorescentProtein(EGFP)constitutivelyundercontrolofastrongviralpromoter(CMV).The greenfluorescenceintensitywasmeasuredinchannelFL-1oftheflowcytometer.UntransfectedHEK/293cellsareEGFP(−)_.

Fig. 6. Growth kineticsofHEK-ptdTomato-N1incomparisontonon-transfectedHEK/293.Forgrowthkineticscellswereseededin∅3cmPetri dishesatadensityof 1x104_cells/cm2_{.Cellswerecountedtwiceadayafterdetachingcellsbytrypsination.Thegraphshowsmeanandstandarderrorofthreeindependentexperiments.Ifthe}

barsarenotvisible,thestandarderrorsaresmallerthanthesymbol(A).Fordeterminationoffluorescenceincreaseduringgrowth,1×103_{cells perwellwereseeded}

intoamicroplateandfluorescencewasdeterminedinthemicroplatereadereveryday.Barsshowthestandarderrorforthreeindependentexperimentswitheacheight replicates (B).

Fig. 7. Fluorescence intensityofHEK-ptdTomato-N1celllayersdependsoncell num-ber.Thecellswereseededinincreasingcellnumbersinto microtiterplatesand fluorescenceintensityoflivingcellswasmeasuredinthemicroplatereader24 h af-terseeding.Barsshowthestandarderrorforthreeindependentexperimentswith each4replicates.

4.1. Preparationofstablytransfected,tdTomatoexpressingcelllines

Inordertosavetimeandtominimizetheexperiment variabil-ity,HEK/293 cellswere stablytransfectedwiththeptdTomato-N1 vector. Adrawbackofstabletransfectionisthatexpression ofthe fluorescentproteinisaffectedbythegeneintegrationsiteandthe numberof integratedgene copies.In some ofthe clones expres-sion of fluorescent protein was low which might be due to the integration intoan inactive partofa chromosome. The tdTomato expressingcloneswereexpandedinpresenceofG418after identi-ficationwiththefluorescencemicroscope.Someoftheclonesdid not survive in presence of G418 as cellular enzymes might de-stroythepromoterortheaminoglycosidephosphotransferasegene. Therefore,preparationofasuitablestablecelllinerequired screen-ingofmultipleclonesthatsurvivedinpresenceofG418.Clone# 8stablyintegratedtheforeignDNAintoitsgenome,survived the selectionbytheantibioticG418andshowedthehighestmean flu-orescence;thereforeitwasselectedforfurtherexperiments.

4.2. FluorescencespectraoftdTomatoexpressingcells

spec-Fig. 8. Dose-dependent reductioninproliferationofHEK-ptdTomato-N18cellsafterexposuretoX-raysandenergeticneonions.Cellswereseeded,grownfor48hand exposedtoX-rays(LET3 keV/μm)(A)or22_{Neions(LET91 keV/μm)(B).Thefluorescenceintensitywasmeasuredinthemicroplatereaderwiththefilterset540/25and}

590/35twiceadayover115h.

Fig. 9. Proliferation reductioninHEK-ptdTomato-N1cellsafterexposuretoheavyions.Dose-effectcurvesforgrowthreductionafterexposuretolowLETradiation(X-rays, LET0.3–3 keV/μm)werecomparedwithhighLETradiation(33–905 keV/μm)(A)andtherelativebiologicaleffectivenessfor50%proliferationreductionwascalculatedwith thedosesdeductedfromtheregressionlines(B).HEK-ptdTomato-N18cellswereirradiated72 hoursafterseedinginmicrotiterplates.Fluorescenceintensitywasmeasured inthemicroplatereaderwiththefilterset540/25&590/35.Therelativefluorescencewascalculatedbydividingfluorescenceintensitiesafter100 hincubationvaluewith the0 Gy100 hvalueofrespectiveheavyionaftersubtractionofbackgroundfluorescence.HeavyionshaveamaximumkillingeffectupintheLETrangeof30–175 keV/μm. VeryhighLETheavyions≥900keV/μmshowedakillingeffectcomparabletoX-rays.Barsindicatethestandarderrorresultingfrom1–2independentheavyionexperiments witheightreplicateseach.Ifthebarsarenotvisible,theyaresmallerthanthesymbol.

trofluorimetrywhichdescribestherelationshipbetweenabsorbed and emitted photons at specified wavelengths (Lakowicz, 1999). tdTomato hasdetectableemission intensityfora broadexcitation range(

∼

500–580 nm).Maximum emission was achieved forthe excitation wavelength of 555 nm. When excited at 555 nm, the maximum emission occurs at 577 nm. In literature, the excita-tion and emission maxima of tdTomato were reported to be at 554/581 nm and a filter set of 535/20 and 615/100 for excita-tionandemission was recommendedrespectively forminimizing crosstalkwithother fluorescent proteinsand better spectral sep-aration (Shaner et al., 2005). The very minor differences in the maximabetweenourfindingandliteraturemightbeexplainedby thewidthofthe tipof thepeak stretchingover several nanome-ters.

4.3. FluorescenceintensityoftdTomatoexpressingcells

FlowcytometricanalysisofHEK-ptdTomato-N18cells revealed a strong tdTomato expression of the whole cell population. The mean fluorescence output of HEK-ptdTomato-N1 8 cells is about 1000times higher compared to non-transfected cells. The single peak inthered fluorescencehistogramconfirms

HEK-ptdTomato-N1 8 to be a stable cell linewith all the cells constitutively ex-pressing the red fluorescent protein tdTomato. The fluorescence intensityofnon-transfectedHEKcellsremainedbetween0and10 which was defined as the autofluorescence range (tdTomato(−)_). The fluorescent proteins EGFPand tdTomatohavedifferent phys-ical and optical properties (Shaner et al., 2008). The extinction coefficient andquantumyield oftdTomatoare muchhigher than forEGFP(138,000Mol−1cm−1versus56,000Mol−1cm−1,and0.69 versus0.60,respectively)(Shaneretal.,2004).Inthisstudyitwas noticed that,compared toHEK-pEGFP-N1cells, themean fluores-cenceintensityofHEK-ptdTomato-N18cellswas

∼

6timeshigher.

4.4. GrowthoftdTomatoexpressingcells

tensity was determined in the microplate reader. The minimum numberof cells that gives detectable fluorescence isabove 2400 cellsperwell,belowthiscellcountthemicroplatereaderwas un-able to distinguish HEK-ptdTomato-N1 8 cells fluorescence from background fluorescence of microplate and cell culture medium. The starting cell number of 3000cells per well for proliferation assayswaswellabovethedetectionthreshold,allowinggrowth de-terminationmeasurements starting immediatelyafter attachment ofcellstothewellbottom.

4.6.GrowthinhibitionafterX-rayexposure

Ionizingradiation (X-rays and heavy ions) is cytotoxic to the cells andinhibits cellular growth.The growth after radiation ex-posurewas monitored byfluorescence measurementoftdTomato content of the cell layer in a microplate. The tdTomato fluores-cence represents the cellular fluorescent protein content which wasevaluated asendpointforcytotoxicity.Thecytotoxiceffectof X-rayswasinvestigatedby measuringthe fluorescenceofthecell layer twice a day in a microplate reader. After X-irradiation,the tdTomato fluorescence increase was delayed because X-rays in-hibit proliferation due to its cytotoxic and genotoxic nature and effectsoncellcycleprogression.ThegrowthpatternofX-irradiated cellsshowedadose-responserelationship.Atlowerdoses(

≤

4 Gy), cells stayed in lag phase up to 36 hours but grew exponen-tiallyafterwards. Higher doses (

≥

4 Gy) inhibited cellulargrowth due to higher cytotoxic effects. This cytotoxicity bioassay is re-latedto proliferationassayswhichmeasure growthofacell pop-ulation based on cellular protein content (Hellweg etal., 2007a; Skehanetal.,1990).In aprevious study,lossoffluorescence fol-lowing low temperature exposure was explored as an indicator of cell death (Elliott et al., 2000). Fluorescence signalsrepresent differentprocesses whichcontribute to increasedcellular protein content: (i) cell division, which results in new protein synthesis inthedaughtercells and(ii) continuedproteinsynthesis ofcells in the interphase or during a cell cycle arrest. Direct effects of ionizing radiation on tdTomato are expected to be of negligible importance for two reasons. First, the test is predicated on the tdTomato thatis newly synthesized after radiationexposure and notonthetdTomatothatispresentinthecellduring irradiation. SoevenifthereisradiationdamagetotdTomato,itwillnotaffect thefluorescencemeasured100hafterradiationexposure.Second, the protein damage including that inflicted to tdTomato by ion-izing radiation exposure is expected to be extremely low in the doserangeunderinvestigation,asproteindamagestudiesare usu-allycarriedoutinthekGydoserange(Vuckovicetal.,2005).The sensitivityoffluorescentproteinbasedcytotoxicitybioassayswere proventobehighercomparedtootherassayssuchastheMTTtest (Hellwegetal.,2007a).

killingeffectcomparedto C, Ne, Feand NiionsintheLET range of 33 to 175 keV

/

μm, probably dueto compacted ioniza-tions within a very narrow track structure with extremely high dose deposition ina smallvolume withan increasingnumber of unhit cells, resulting in an overkill effect and lower cell killing potential forthe irradiatedcell population. Basedon fluence,the cell killingeffectatvery highLET (

≥

900 keV

/

μm) isless promi-nent andleast efficient in reducing cellular survival (Hellweg et al., 2011). Itwas concludedthatcellkillingcausedby heavyions dependson theion type,energy andtheLET value. TdTomato is expressedconstitutivelyandaccumulatesonlyinlivingcells.After irradiation a dying tdTomato expressing cell does not contribute to the fluorescence increase anymore. The reduced fluorescence mightbeduetothefactthattdTomatoproteinissoluble(Shaner etal.,2005),leaksoutofthedeadcellsandisremovedbyregular medium changes.ThegrowthtestusingtdTomatoallowsabetter estimation ofthe fractionofresidual replicativecells after radia-tionexposurecomparedtoothershort-termcytotoxicitytestssuch astheMTTtest.Becausetetrazoliumsaltbasedassaysdestroythe cells, time-courseexperiments cannotbe carriedout (Rampersad, 2012).Furthermore,thereducingpotentialresultinginconversion ofthe MTTreagentto thecolored formazan productdependson celltype,resultingincell-typespecificcalibrationcurvesofoptical densityandnumberofcells (Mosmann, 1983; Alleyetal., 1988). In other than radiobiological applications, the MTT test reagents might also interact with tested compounds or multidrug resis-tance(MDR)protein,thatisoftenexpressedintumorcells,leading to false positive or false negative results (Vellonen et al., 2004; Worle-Knirschetal.,2006).

Comparedtothe standardradiobiologicaltest forsurvival,the colony formingability test,the sensitivityof thefluorescent pro-teinsbasedgrowthtestislower, butthisassayrequiresmuchless time andmaterial (Hellweg etal., 2007a) andassuchthe test is morecost-effective.

thefindingsofStoll etal.(1995,1996)whofoundan RBEforcell survivalbyhighLETnickelions(

>

1000 keV

/

μm)around 1.

5. Conclusions

In order to measure cytotoxic effects, the fluorescent protein tdTomato isaveryusefulreporterproteinasitismorestablethan other fluorescentproteins.The fluorescenceintensityoftdTomato in stably transfectedcells is around 6 times higher than that of EGFP.tdTomatofluorescencecaneasilybemeasuredinmicroplates usingafluorescencemicroplatereaderequippedwithappropriate filtersets ormonochromators. HEK-ptdTomato-N1 8cells can be usedforestimationofthefractionofresidual replicativecells af-terradiation exposure. Compared to the standard radiobiological testforsurvival,thecolonyformingabilityassay,theeaseof per-formanceof thetdTomatoproliferationtest allows screeningofa largevarietyofcytotoxicconditionsinalabor-,time- and money-savingmanner,anditrequiresirradiationofonlyaverysmall sam-plearea.TheHEK-ptdTomato-N1reportersystemgivesalsostrong signals when measured by flow cytometry, which allows single cellanalysis.Automatedimageanalysisoffluorescencemicroscopic images could give more details about radiation-induced cytotox-icity in mammalian cells. Therefore, the constitutively expressed tdTomatoisasuitablereporterofradiationinducedgrowth reduc-tion ofhuman cells. HEK-ptdTomato-N1 8cells canbe alsoused formonitoringofothercytotoxicagentsthanradiationand subse-quentriskestimation.Afterthesegroundbasedpreparatorytests, thebiosensor systemwillbe usedto reduce uncertainties inrisk assessmentofspaceenvironmentalfactorssuchasspaceradiation andmicrogravity. ThespaceexperimentCellular Responsesto Ra-diationinSpace(CellRad),selectedbyNASA/ESAtobeperformed ontheISS, willsupplybasicinformationonthecellularresponse to radiation, including proliferation reduction, applied in micro-gravity. Preparatorytestsforthisspaceexperimentincludingtest ofuploadandstorageconditionsarecurrentlyperformed. Further-more,itcan beapplied torapidlymonitorthe effectofshielding measuresoncellularsurvivalbehindtheshieldingmaterial,andto determinea biological weighted depthdose distribution ina hu-manphantom.

Conflictofintereststatement

Theauthorsdeclarethattheyhavenoconflictofinterest.

Acknowledgements

Arif A. Chishti received a scholarship of the Helmholtz Space LifeSciences ResearchSchool (SpaceLife) which isfunded by the Helmholtz Association (Helmholtz-Gemeinschaft VO-KH-300) and the German Aerospace Center (Deutsches Zentrum für Luft- und Raumfahrt e.V., DLR). Part of this work was funded by the Eu-ropean Commission in the frame of the FP7 HAMLET project (Project # 218817). Beamtimes at GSI were supported by the European Space Agency (ESA) program “Investigations into Bi-ological Effects of Radiation Using the GSI Accelerator Facility” (Ground basedradiation field simulationof the MATROSHKA ex-periment: Physical and Biological Experiments for RadiationRisk Assessment–AO-08-IBER-12andAO-10-IBER). Theauthorswould like to thank the staff at the heavy ions accelerator facilities at the Centre Interdisciplinaire de Recherche Ions Lasers (CIRIL) at GANIL,Caen,France,andGSIHelmholtzzentrumfür Schwerionen-forschungGmbH,Darmstadt,Germany,fordosimetryandsupport ofthe heavy ionexperiments,especially Dr.Isabelle Testard, Flo-rentDurantel(GANIL),ChiaraLaTessa,DieterSchardtandMichael Scholz (GSI), andall themembers ofthe Biodiagnostics group at DLRfortheirhelpduringbeamtimes.

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