Radiobiological experiments for carbon ion prostate cancer therapy [Elektronische Ressource] : interplay of normal and tumor cells in co-culture and measurement of the oxygen enhancement ratio / von Cläre von Neubeck
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Radiobiological experiments for carbon ion prostate cancer therapy [Elektronische Ressource] : interplay of normal and tumor cells in co-culture and measurement of the oxygen enhancement ratio / von Cläre von Neubeck

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GSI Helmholtzzentrum für Schwerionenforschung GmbHTechnische Universität DarmstadtRadiobiological experiments for carbon ionprostate cancer therapy: Interplay of normaland tumor cells in co-culture and measurementof the oxygen enhancement ratioVom Fachbereich Biologie der Technischen Universität DarmstadtzurErlangung des akademischen Gradeseines Doctor rerum naturaliumgenehmigte Dissertation vonDipl.-Ing. Biotechnologie Cläre von Neubeckaus Frankfurt (Main)1. Referent: Prof. Dr. Gerhard Thiel2. Prof. Dr. KraftTag der Einreichung: 31.07.2009Tag der mündlichen Prüfung: 28.09.2009Darmstadt 2009D17ZusammenfassungCo-Kultur Systeme sind häufig ein geeignetes Modell um die Reaktion von Gewebenin vitro zu untersuchen, die bei einer Strahlentherapie notwendigerweise gleichzeitig alsZiel- und Nachbarvolumen exponiert werden. In dieser Arbeit wurde ein Co-Kultur Mod-ell auf Basis von Rattenzellen eines Prostatakarzinoms (Dunning R-3327-AT-1) und desDünndarmepithels entwickelt um die Bestrahlungssituation eines Prostatakarzinompa-tienten zu simulieren. Beide Zelllinien wurden in Mono-Kulturen auf ihre Strahlensen-sitivität gegenüber 250 kVp Röntgen sowie 270 MeV/u, 100 MeV/u und 11.4 MeV/uKohlenstoffionen untersucht.

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Publié par
Publié le 01 janvier 2009
Nombre de lectures 31
Langue Deutsch
Poids de l'ouvrage 14 Mo

Exrait

GmbHSchwerionenforschungfürHelmholtzzentrumGSITechnischeUniversitätDarmstadt

Radiobiologicalexperimentsforcarbonion

prostatecancertherapy:Interplayofnormal

andtumorcellsinco-cultureandmeasurement

ratioenhancementoxygentheof

VomFachbereichBiologiederTechnischenUniversitätDarmstadt

zur

desErlangungGradesakademischen

naturaliumrerumDoctoreines

onvDissertationgenehmigte

Dipl.-Ing.BiotechnologieClärevonNeubeck
(Main)Frankfurtaus

1.Referent:Prof.Dr.GerhardThiel
2.Referent:Prof.Dr.GerhardKraft

31.07.2009Einreichung:deragT

TagdermündlichenPrüfung:28.09.2009

2009DarmstadtD17

Zusammenfassung

Co-KulturSystemesindhäufigeingeeignetesModellumdieReaktionvonGeweben
invitrozuuntersuchen,diebeieinerStrahlentherapienotwendigerweisegleichzeitigals
Ziel-undNachbarvolumenexponiertwerden.IndieserArbeitwurdeeinCo-KulturMod-
ellaufBasisvonRattenzelleneinesProstatakarzinoms(DunningR-3327-AT-1)unddes
DünndarmepithelsentwickeltumdieBestrahlungssituationeinesProstatakarzinompa-
tientenzusimulieren.BeideZelllinienwurdeninMono-KulturenaufihreStrahlensen-
sitivitätgegenüber250kVpRöntgensowie270MeV/u,100MeV/uund11.4MeV/u
Kohlenstoffionenuntersucht.AusdenÜberlebensdatenwurdendieParameterdesLinear-
QuadratischenModells,αundβ,errechnetunddierelativebiologischeWirksamkeit
(RBW)bestimmt,wobeidieRBWαderstrahlenresistenterenProstatakarzinomzellline
imGegensatzzurDünndarmzellliniegrößerwar.DerVergleichvonRBWWertener-
rechnetmitdemlocaleffectmodel(LEM)zeigtesehrguteÜbereinstimmungenmitden
gemessenenWerten.DieDatenausMono-KulturenwurdenverglichenmitCo-Kulturen
unbestrahlterundbestrahlterZellen(250kVpRöntgen,100MeV/uund11.4MeV/u
Kohlenstoffionen)beiderZelllinien.DiegemesseneSynergieindenCo-Kulturenwurde
aufbestrahlungsabhängigeund-unabhängigeFaktorenzurückgeführt.UmdieseEffekte
zuuntersuchenwurdedieSekretionderinammatorischenZytokineTGFβ,TNFαund
IL-2gemessen.Eszeigtesich,dassderenSekretionunabhängigvonderBestrahlungund
derCo-Kulturwar.
InderTumortherapiestellenhypoxischeTumorarealeaufgrunderhöhterStrahlenresistenz
einProblemdar.UmZellenunterdefiniertenSauerstoffbedingungenzukultivierenund
zubestrahlenwurdeeineKammerentwickelt,inderderSauerstoffgehaltgezieltreg-
uliertwerdenkann.IndieserKammerwurdendieProstatakarzinomzellenunteroxischen
undhypoxischenBedingungenmitKohlenstoffionenmiteinemLETvon100keV/µm
bestrahlt.DerSauerstoffverstärkungsfaktor(OER)wurdefürRöntgenbestrahlungenmit
OER=2,35undfürKohlenstoffionenmitOER=1,5bestimmt.DieErgebnissederCo-
KulturundderExperimentemitdefiniertemSauerstoffgehaltwurdenunterdemGesicht-
diskutiert.Prostatakarzinomtherapiederspunkt

iii

Summary

Co-culturemodelsarehelpfultoexaminecelltocellinteractionsinvitroandtoassess
thecross-communicationbetweentwoparticularcellpopulations.Co-culturesystems
partiallyreectthecomplexinvivosituation:inthisstudyaninvitroco-culturemodel
ofprostatecancercells(DunningR-3327-AT-1)andsmallintestinecells(intestinalep-
itheliumcellline6)oftheratwasestablishedtosimulatethecarboniontreatmentof
prostatecancerpatientsatGSI.Bothcelllineswerecharacterizedinmono-culturesfor
theirradio-biologicalresponseagainst250kVpx-raysandcarbonionsof270MeV/u,
100MeV/u,and11.4MeV/u,respectively.Theparametersofthelinearquadraticmodel,
αandβ,forcellsurvivalcurvesweredeterminedaswellastherelativebiologicalef-
fectiveness(RBE).ThemeasuredRBEvalueswerecomparedtocalculationsofthelocal
effectmodel(LEM)andwereinagreementtothecalculations.TheRBEαvalueincreased
strongerforthemoreradio-resistantprostatecancercelllinethanfortheepitheliumcell
line.Thesurvivalofunirradiatedandirradiatedcellsfromco-cultures(250kVpx-rays;
100MeV/uand11.4MeV/ucarbonions)wascomparedtomono-culturesunderthesame
conditions.Themeasuredeffectswereattributedtoirradiationindependentaswellasto
irradiationdependentfactors.Tostudytheseeffects,theinammatorycytokinesTGFβ,
TNFα,andIL-2wereanalyzed,buttheirsecretionwasindependentofirradiation.
Tostudytheproblemofhypoxiccellsintumortreatmentahypoxiachamberwasdevel-
opedinwhichcellsweregrownunderadefinedoxygenstatus.Prostatecancercellswere
irradiatedwith250kVpx-raysandcarbonionswithaLETof100keV/µmunderoxic
andhypoxicconditions.Theoxygenenhancementratiosfor10%survivalwerefoundto
beOER=2.35forx-raysandOER=1.5forcarbonions.Theresultsoftheco-culture
modelandtheexperimentsunderdefinedoxygenstatusarediscussedinrelationtoongo-
ingprostatecancertherapy.

v

Contents

Zusammenfassung

Summary

Contents

iii

v

1oductionIntr11.1Objectives..................................1
1.2Physicalpropertiesofionizingradiationandradio-biologicalfundamentals2
1.3Theprostateanditscancers.........................6
1.3.1Dunningprostatecancercellsystem................7
1.4Theintestineandradiationsideeffects...................8
1.4.1Intestinalepitheliumcellline6...................9
1.5Cytokines..................................9

15methodsandMaterials22.1Celllinesandcultureconditions......................15
2.1.1DunningR-3327-AT-1.......................15
2.1.2Intestinalepitheliumcellline6...................15
2.1.3Co-culture.............................16
2.1.4Cryopreservation..........................19
2.1.5Growthcurves...........................20
2.1.6Clonogenicsurvivalassay.....................20
2.1.7X-Galstaining...........................21
2.2Chromosomepreparation..........................22
2.2.1Multicoloruorescenceinsituhybridization...........23
2.3Detectionofpro-inammatorycytokines..................23
2.4Flowcytometry...............................24
2.4.1Cellcycleanalysis.........................24
2.4.2FluorescencemembranelabelingwithPKH67andanalysis....25
2.5Hypoxiachamber..............................26
2.5.1Construction............................27
2.5.2Cellsamplesandhandling.....................28
2.5.3Gassingmodalities.........................29
2.6Cellirradiationprocedure..........................29

vii

Contents

3

4

viii

2.7Statisticaldataanalysis...........................31
2.7.1Errorestimationforsurvivalexperiments.............31
2.7.2CalculationofRBE.........................33
2.7.3Analysisofchromosomesamples.................34

37Results3.1CharacterizationofratadenocarcinomacelllineR-3327-AT-1......37
3.2Characterizationofintestinalepithelialcellline6.............41
3.3Survivalexperiments............................45
3.3.1RAT-1cells:radio-resistanceagainst250kVpx-rays.......45
3.3.2RAT-1cells:radio-resistanceagainstcarbonions.........46
3.3.3IEC-6cells:radio-resistanceagainst250kVpx-rays.......47
3.3.4IEC-6cells:radio-resistanceagainstcarbonions.........49
3.3.5Relativebiologicaleffectiveness(RBE)..............52
3.4Identificationofsub-populationsandclonesinIEC-6cellculture.....53
3.4.1ChromosomeanalysiswithmFISH................53
3.4.2Existenceofradio-resistantIEC-6sub-populations........54
3.4.3AnalysisofIEC-6singlecellcolonies...............57
3.5Co-culture..................................60
3.5.1Survivalexperiments........................61
3.5.2Cytokinemeasurements......................64
3.6Hypoxiachamber..............................67

71outlookandDiscussion4.1Celllines..................................71
4.1.1RAT-1Dunningprostateadenocarcinomacellline........71
4.1.2Developmentofaco-culturesystem-asuitablecelllinetoRAT-1
cellsisneeded...........................72
4.1.3TheIEC-6cellline.........................73
4.1.4IEC-6cellsub-populationsandclones...............73
4.1.5AlternativecelllinestoIEC-6cells................76
4.1.6Changingthecellsystemtohumancells..............77
4.2Mono-culturesurvivalexperiments.....................78
4.3Co-cultureexperiments...........................81
4.3.1Comparisonofmethods:cellpre-seedingversustrypsintreatment81
4.3.2Inuenceofserumonsurvivalexperimentsandcytokinedetection82
4.3.3Survivalexperiments........................82
4.3.4Cytokines..............................84

Contents

4.4Hypoxiachamber-measurementoftheoxygeneffect...........87
4.4.1OERforx-rayirradiation......................88
4.4.2OERforcarbonionirradiation...................90
4.4.3Gassingmodalities:acuteorchronichypoxia...........92
4.4.4Hypoxiachamber:outlook.....................94

yBibliograph

wledgementAckno

ErklärungEidesstattliche

Annex

solutionsUsed

esFigurofList

ablesTofList

ListofAbbreviations

vitaeCurriculum

95

111

113

I

I

III

V

VII

IX

ix

oductionIntr1

esvObjecti1.1

ThecancertreatmentwithheavyionsstartedatGSIinDecember1997.Uptotheendof
July2009,440patientswithchordomas,chondrosarcomas,andadenocysticcarcinomas
weresuccessfullytreated.Thefiveyeartumorcontrolrateincreasedfrom30-40%after
conventionaltreatmenttovalueshigherthan80%inallthesecases[123,122,21].In2006
astudyonlocallyadvancedprostatecancerwasstarted[97].Butforfuturedevelopments
intherapythenumberofindicationshastobeincreasedandthetreatmentadvantageshave
tobeextendedtoothertumortypes.Therearestillsomeunsolvedproblemsinheavyion
radiationtherapy.Onthebasisofprostatecancer,asanexample,theseunsolvedproblems
canbeexplained.Prostatecancerpatientswereirradiatedwithacombinationofintensity
modulatedradiotherapy(IMRT)attheUniversitätsklinikumHeidelbergandwithsixfrac-
tionsofcarbonionboostirradiationatGSI(activebeamscanning).TheIMRTdelivers
thephotondoseinmanysmallirradiationfieldsofdifferentbeamdirectionstoachieve
athreedimensionaltumorconformaloverlappingdose.Thecarbonionsaredeliveredin
onlytwoopposingfieldswithoutreducingthedelivereddoseinthetumorvolume.Since
theprostateisadeep-seatedorgan,bothmethodsirradiatehealthytissueandincluding
partsoftheradio-sensitiveorgansliketherectumandbladder.Duetothegreaterpreci-
sioncarbonionirradiationexcludesmorehealthytissuethantheIMRTirradiationwhich
protectsthenormaltissuesufficiently.ForcarboniontherapyinJapan(passivebeam
control)sideeffectslikeinammatoryreactionsintheco-irradiatedorgansarepublished
[134,66,137]andreducethemaximaldeliverabledosetothetumor.Abettercompre-
hensionofthehealthytissuereactionaloneandincombinationwithprostatecancercells
couldhelptoreducesideeffects,tomedicatetheinammationortoenhancethedeliver-
abledosetothetumor.

Inafirstreactiontotheseproblemsitwasplannedtoestablishaninvitroco-culturesys-
temofprostatecancercellsandnormalcellsofthesurroundingtissuetosimulatethe
patienttreatmentsituationandtoanalyzetissuereactions.

1

oductionIntr1

raisedquestionsThewere:

➢Howisthesurvivalofirradiatedtumorcellsinuencedbynormalcells(irradiated
unirradiated)?/➢Howisthesurvivalofirradiatedorunirradiatednormalcellsinuencedbytumor
unirradiated)?/(irradiatedcells➢Whichbiochemicalsubstancesmediatetheinuenceonthesurvival?
➢Aretheredifferentreactionsbetweencellsirradiatedwithcarbonionsorx-rays?

Acommonfeatureofprostatetumorsisthattheyarepoorlyoxygenatedtissuewhichis
moreradio-resistant[89].Furthermore,hypoxiahasbeencorrelatedwithlocaltumorre-
currences[131]andconsequentlywithapoorprognosis[89].Duetotheoxygeneffect
thehypoxictissueismoreradio-resistantthanwelloxygenatedtissue.Byusinghigh
LETcarbonionstheoxygeneffectisreduced[52].Forcarbonionirradiationwithmixed
LETtheoxygeneffectisunclear.Ifitwerepossibletodeactivateallhypoxiccellsmore
efficiently,thepoorprognosisintermsoflocalrecurrenceswouldberevised.There-
fore,morebiologicaldataofhypoxiccellsareneededtoimprovethetreatmentplanning
system.

Thesecondintentionofthisthesiswastodevelopameasurementsystemforhypoxiccells
whichfulfillsthefollowingcriteria:

➢adjustableanddefinedoxygenstatusofthecells,
➢measurableacuteandchronichypoxia,and
➢suitableforphotonandcarbonionirradiation.

Withthissystem,theoxygenenhancementratioforx-rayandcarbonionirradiation
measured.beshould

1.2Physicalpropertiesofionizingradiationandradio-biologicalfun-
damentals

Thedepthdoseprofilesofphotonsandcarbonionsdifferbecauseoftheirphysicalprop-
erties(seefigure1.1).Concerningtherapyconditionscarbonionshaveabetterdose
distributionthanphotonsi.e.abettertumorconformitycanbereached.Sparselyioniz-
ingradiationlikephotonsdepositsitsenergyviaphotoeffect,Compton-process,andpair
production.Theresultingsecondaryelectronsofhighenergyphotonsarepreferentially
scatteredforward.Thisshiftsthemaximumenergydepositionprofileofphotonstohigher

2

1.2Physicalpropertiesofionizingradiationandradio-biologicalfundamentals

Figure1.1:Therelativedosedistributionforx-rays,photonsandcarbonionswithdepthinwater.
CourtesyofU.Weber.

depths,butafterafewcentimetersthedepthdoseprofiledecreasesexponentially.Incon-
trast,particleirradiationlikeprotonsorcarbonionsexhibitsaninversedepthdoseprofile.
Theenergydepositionincreasesslowlywithdepthtoasharpmaximum,theBragg-peak.
Thedepositedenergyalongthecovereddistanceisdefinedasthelinearenergytransfer
(LET).

dE(1.1)=TLEdxEquation1.2showstherelationbetweenLET[keV/µm],doseD[Gy],uenceF[cm-2],
anddensityoftheirradiatedmaterialρ[cm3/g]

D=1.6∙10−9∙F∙LET∙ρ−1,(1.2)
wherethenumericfactoraccountsfortheconversionoftheunitsasgivenabove.The
biologicaleffectofsparselyanddenselyionizedradiationdiffers.Therelativebiological
effectiveness(RBE)allowsacomparisonbetweenthetwobeamqualities.TheRBEis
theratioofastandardradiationdosetoatestradiationdosewhichinducesthesame
biologicaleffect(seeEq.1.3).

DioneffectisoRBE=Dx−ray(1.3)
Figure1.2illustratestheRBEwithtwotypicalsurvivalcurvesofsparsely(x-rays)and
densely(carbonions)ionizingradiation.Thecellularsurvivalafterx-rayirradiationfol-
lowsashouldercurvewhichcanbedescribedwithEq.1.4.

3

oductionIntr1

S=S0∙e−αD−βD2(1.4)
HereisS0andSthesurvivalbeforeandaftertheirradiation,respectively,α(Gy-1)the
initialslope,andβ(Gy-2)thecurvatureofthelinearquadraticsurvivalcurve.Theshoul-
deredshaperesultsfromrepairabledamagesinthelowdosearea(αvalueisdominant).
Theaccumulationoflesionsleadstoirreparablecomplexdamagesforhigherdoses(β
valueisdominant).Theratioofαandβischaracteristicforacelllineandisaparameter
forradio-sensitivity.Apronouncedshouldercorrespondstoahighradio-resistanceanda
ratio.β/αsmall

Figure1.2:Relativebiologicaleffectivenessof11.4MeV/ucarbonionscomparedto250kVp
x-rayirradiation,CourtesyofW.K.-Weyrather.

Thecellsurvivalafterionirradiationwithhighenergydepositionvaluesfollowsanexpo-
nentialdeclinewithincreasingdoses(LET>100keV/µm).ThehighLETofionsinduces
complexandmostlylethaldamagesinthecell.Iftherepaircapacityofthecellisover
strained,theterm(βD2)convergestozero.Thecellinactivationorthemitoticdeathof
cellsafterirradiationcanbedeterminedinacolonyformingassay[104].
Figure1.2showstheRBEαwhichisgivenbytheratiooftheinitialsslopαinthelimitof
dose,anishingvαionRBEα=αx−ray.(1.5)

ForcancertherapywithcarbonionsatGSItheRBEiscalculatedwiththelocaleffect
model(LEM;[120,33]).Themodelincludesthreecentralingredientswhichrevealthe

4

1.2Physicalpropertiesofionizingradiationandradio-biologicalfundamentals

RBEcharacteristics:a)thetargetsize,e.g.sizeofthecellnucleus,b)trackstructureof
theionsused,sincetheRBEisparticledependent,andc)x-raysurvivalcurveofused
celltype,sincetheRBEiscelltypedependent.ItisnotpossibletodeterminetheRBE
valuesforpatienttreatment.SotheLEMcalculationisneededforeachvoxelofthetarget
volume.FortheRBEcalculationofpatienttreatmentthedataofphotonradiotherapyare
usedtocalculatethecorrespondingclinicaldatainsteadofcellRBEvalues.Thephysical
optimizationofthetreatmentplanisdonewiththetreatmentplanningsoftwareTRiP98
[78].Thecarbonionbeamiscontrolledwiththisoptimizedplanandscansoverthetumor
volume.FormoredetailsseeHabereretal.[50].

Thebeamabsorptioninthetissueleadstotheproductionofradicals.Hallpostulated
thattheradicalsreactwiththesurroundingmoleculese.g.withtheDNA.Inthepresence
ofoxygentheDNAradicalsreactwithoxygenmoleculestoperoxides,andthelesionin
theDNAisfixed.Intheabsenceofoxygen(hypoxia)nofixationoftheDNAdamages
withoxygentakesplace.ThefreeDNAendscouldberealignedwhich,inconsequence,
increasedthecellsurvival[52].Thistheoryisunderdiscussion,andtherealmechanism
mightbemorecomplex.ThedamagesintheDNAareofspecialinterestsinceMunro
identifiedthecellnucleusasthemostsensitivetargetinthecell[93].

Thecellularsurvivalunderhypoxicconditionsisincreased.Theratioofadoseunderhy-
poxictoadoseunderoxicconditionswhichinducesthesamebiologicaleffectisdefined
astheoxygenenhancementratio(OER).

DxicoeffectisoOER=Dhypoxic(1.6)
IncontrasttotheRBE,theoxygeneffectseemstoenhancethesurvivalbyaconstant
factorindependentfromsurvivallevel.Dependingonthecelllineandtheoxygenstatus
theenhancementfactorisintherangeofonetothree,whereasatissuesupplyof≤3%
oxygenissufficientfortheoccurenceofthefulloxygeneffect[52].Inradiotherapy
theoxygeneffectisofgreatimportancesincesometumorsshowlargehypoxicareas,
e.g.prostatetumors[89].DuetotheOERthesehypoxicareasaremoreradio-resistant
tosparselyionizingradiation.TheOERisLETdependent.ThehighertheLETthe
smalleristheenhancementfactorandtheOERconvergestoone.Bytheuseofdensely
ionizingradiationlikecarbonionstheOERcanbereduced.Therefore,particletherapy
iswellsuitedtoeffectivelyinactivatehypoxiccells.Furthermore,thecellularresponse
toradiationdependsontheexposurelengthtohypoxicconditions.Itisreportedthat
theradio-sensitivityofchronicallyhypoxiccellsisreducedto30%comparedtoacute
hypoxiccells[89].

5

oductionIntr1

1.3Theprostateanditscancers

Theprostatetogetherwiththetestes,theepididymis,andtheseminalvesiclesformthe
innergenitalsoftheman.Theprostateadjoinscranialtothebladderanddorsaltothe
rectum.Thechestnutshapedandsizedorganenclosesthebeginningoftheurethra.The
tubuloalveolarexocrineglandiscomposedofthirtytofiftysingleglandswithexcretory
ductsintotheurethra.Theglandsaresurroundedbysmoothmusclecellsandconnective
tissue.Theprostateisexternallyborderedbyaconnectivetissuecapsuleandcanbeclas-
sifiedinhistologicalzones:peripheral(70-75%ofglandmass),central(25-30%ofgland
mass),periurethral,transition,andanteriorfibro-muscular(noglands)zone.Prostatecan-
cerdevelopsinmostcasesintheperipheralzone[85,91].
TheprostatecarcinomaisthemostfrequentlyoccuringcancerformeninGermanyand
responsiblefor10%ofcancerrelateddeathperyear.Theriskofprostatecancerincreases
withageand90%ofthepatientsareolderthan60.Theimportantdiagnosticprocedures
aredigitalrectalpalpation,serumlevelofprostatespecificantigen(PSA),sonography,
andpunchbiopsy[113].
Prostatecarcinomascanpathologicallybedividedintofourcategories:latent(undetected
inlife,autopsy),incidental(notdetectablewithclinicaldiagnostic,incidentallydiscov-
ered,T1),occult(primarytumornotdetected,metastasesinducedfinding),andclini-
calprostatecancer(assureddiagnosis,T2-T4).95%ofthetumorsareadenocarcinomas
whereasurothelialorsquamouscellcarcinomasaswellassarcomasappearrarely.The
aggressivenessofprostatecancerisevaluatedwithGleasonscoring(factor2-10).With
thisthedifferentiationpatternofthecellstructuresarejudged[34].Inaddition,differ-
entmorphologicalgrowthpatterns(cribriform,anaplastic,wellandpoorlydifferentiated
glandular)canbefound[10].Thetumorclassificationisperformedaccordingtothe
TNMsystemoftheinternationalunionagainstcancerwhichdifferentiatesbetweenpri-
maryTumor,regionallymphNodeanddistantMetastasis(seetable1.1).Prostatecancer
metastasizesfirstintothelymphnodesofthepelvisminorandlater,metastasescanbe
foundinthebones,liver,andlung.Thetherapeuticalproceduredependsonthetumor
state.Await-and-seestrategywithcontrolsisusedinstateIwhileinstateII,acura-
tiveapproach,(radiotherapy,hormone-therapy)orsurgerycanbechosen.ForstateIIIno
therapeuticallyguidelineexists(alltherapyformsexceptchemotherapy),andinstateIV
asystemictherapyofhormone-,chemo-andpain-therapyissuggested[34].
However,themethodofcancertreatmenthastocomplywiththegeneralconditionandthe
expectedlifespanofthepatient,theseverityoftheillness,andtheexpectedsideeffects
ofthetreatment.Forradiotherapytwomethodsareused.Methodone,thebrachytherapy
appliesirradiantmaterialsdirectlyintotheprostatewhiletheIMRT,thesecondmethod

6

1.3Theprostateanditscancers

Table1.1:Prostatecancerclassificationaccordingtotheinternationalunionagainstcancer,ex-
cerptofTMNsystem[34]
DefinitionCategoryStateTXprimarytumornotrateable
T0noindicationforprimarytumor
I-IIT1a-cincidentallydetectedtumor
IIT2tumoronlyinprostate(onelopeT2a,bothlopesT2b)
IIIT3tumorwithcapsularpenetration(externalexpansionT3a,sem-
T3b)infiltrationesiclevinal.IVT4tumorisfixedand/orinfiltratingotherstructures
NXregionallymphnodenotrateable
metastasesnodelymphnoN0N1regionallymphnodemetastases
MXdistantmetastasesnotrateable
esmetastasdistantnoM0M1distantmetastases(otherlymphnodesM1a,bonesM1b,rest
M1c)bodytheof

irradiatesexternallyinthreedimensionalconformalmanner.Theprimarysideeffectsin
bothcasesareimpotence,incontinence,andintestinaldisorders[113].

1.3.1Dunningprostatecancercellsystem

TheoriginaladenocarcinomawasdiscoveredbyDunninginthedorsallopeoftheprostate
ofamaleCopenhagenrat(rattusnorvegicus)in1961andwasnamedR-3327.Partsof
thetumorweresuppliedtootherinstitutes,andovertheyearsseveralsub-lineswerede-
velopedwithdifferentcharacteristics.ThegenealogyoftheR-3327ratprostatecanceris
presentedinreference[64].Inthisthesisthesub-lineR-3327-AT-1(RAT-1)isusedwhich
occurredatJohnHopkinsOncologyCenterin1976.RAT-1cellsforminvivoafastgrow-
ing,androgen-andestrogen-insensitive,andanaplastictumorwithalowmetastaticability
[65].Anaplasiaischaracterizedbythelossofstructuralandfunctionaldifferentiationof
normalcells[34]andishistologicalgradedwithfive(Gleason)[20].Invitrothecell
doublingtimeis32.5±3.7handtheplatingefficiency(PE)is16.7±0.9%.Themean
chromosomenumberwasdeterminedwith60±7[65].Thenormalratkaryotypefor
rattusnorvegicusis42(2n)whichmeansthatRAT-1cellshaveananeuloidchromosome

7

oductionIntr1

number.Aneuploidy(conditionunderwhichacellhasmissingorextrachromosomes)
canoccurwithtwoprinciplemechanisms:chromosomelossandnon-disjunction.Acom-
pletechromosomecangetlostifthekinetochoreofthechromosomeisnotattachedtothe
spindlefiber.Duringanaphasethechromosomeisthereforenotmovedtothepolesof
thedividingcellnucleus.Themembraneformationintheendofthemeiosisexcludes
thechromosomefromthedaughtercells,andtheformationofamicronucleusispossible.
Non-disjunctiondescribesthefailureindivisionofthesisterchromatidsinthebeginning
oftheanaphase.Thereby,ahyperploid(1n+x)andahypoploid(1n-x)daughtercell
elops.vdeTheRAT-1cellsgrowinvivotolargertumorswhereasthesmallertumorsaresignificantly
lesshypoxic.Butevenlargetumorsdonotformacentralnecrosis.Thebetteroxygenated
tumorsshowanenhancedresponsetoradiationwhenthecellswereirradiatedwithasin-
gledoseof30Gyof4MVx-rays[12].Furthermore,RAT-1celltumorsareknowntobe
].101[radio-resistantmoderately

1.4Theintestineandradiationsideeffects

Theupperintestineiscomprisedbythemouth,pharynx,esophagus,andstomach.The
lowergastrointestinaltractconsistsofthesmallintestinewhichhasthreeparts(duode-
num,jejunum,ileum).Thelargeintestinehasthreepartsaswell:cecumwithappendix,
colon(ascending,transverse,descendingcolon,sigmoidexure),andrectum.Thegen-
eralstructureoftheintestinalwallisfromoutwardstoinwardscomposedofaserosa,
alongitudinalmusclelayer,acircularmusclelayer,asub-mucosa,andamucosa.The
mucosaofthesmallintestineisfoldedandcoveredwithfingershapedand1mmhigh
villi.Thevillisurfaceiscomprisedoutofabsorbingepitheliumcells[30].Theintestinal
epithelialcells(IEC)havethreeessentialfunctions:a)nutrientuptake,b)immunologi-
caldefense,andc)formingabarrier.Thebarrierarethecellsthemselvesthroughtight
junctionsealing(zonulaoccludens)andmucus(glycocalyx)secretion[19].TheIECs
desquamateatthevillustips[102]andarereplacedbyundifferentiatedstemcellsfrom
theproliferouszonewithinthecrypts[99].Acompletecellexchangecorrespondstothe
functionallifetimeofaepithelialcellwhichise.g.invivoformurinedifferentiatedep-
ithelialcellsfivetotendays[121].
Independentfromthetypeofradiotherapy,inprostatecancertreatmentthesurrounding
normaltissue(intestine,bladder,urethra)ispartlyco-irradiatedandshowsdosedepen-
dentsideeffects.Tissueswithahighproliferatingratesuchasintestinalmucosaaremost
sensitivetoionizingradiationandhavethegreatestriskofinjury.Theradiation-induced
gastrointestinalsyndromeisanacutereactiontoradiationandischaracterizedthrougha

8

Cytokines1.5

massivedepletionofintestinalepitheliumcells,disordersofuids,andsolutetransports.
Therateofapoptosisinmurineepithelialcryptcellsafterirradiationwasfoundtobe
higherthanthatinthevilliandshowedasaturateddoseresponseatapproximately1Gy
ofphotons[127,121].Theseresultsaresupportedbyareportofbeginningchangesin
thehumanintestinalepitheliumafterirradiationwith1-2Gyofphotons[34].Long-term
complicationsintheintestinecanbethemanifestationofprogressivevasculitis,enteritis,
andfibrosis[127].Prostatecancertreatmentwithcarbonionsattheheavyionmedical
acceleratorinChiba,Japan(passivebeamcontrol)[134],deliveredatotaldoseof66.0
Gy(RBE,Japan)in20fractionsoverfiveweeks.Thepatientsdevelopedsideeffectswith
gradeI(17.3%)andgradeII(2.7%)complicationsintherectum.Inasecondstudythe
dosewasreducedto57.6Gy(RBE,Japan)in16fractionsover4weeks.Thesideeffects
couldbelimitedwiththismethodtogradeI(10.3%)andgradeII(1.1%)complications
rectum.thein

1.4.1Intestinalepitheliumcellline6

Theintestinalepithelialcellline6(IEC-6)originatedfromtheintestinalcryptcellsof
arat,asjudgedbymorphologicalandimmunologicalcriteria.AsdescribedbyQuaroni
etal.thecellsarenon-tumorigenicandretaintheundifferentiatedcharacterofintestinal
epithelialstemcells[106].Forthecelllineastablekaryotypeof42chromosomes,a
constantpopulationdoublingtimeof20h(19-22h)whichtendstodecreasewithage,and
aPEof2.3%weredetermined.Thelifespanofthecellsislimitedto30-40passageswhile
themorphologyisstable[106].TheIEC-6cellsareusedforawiderangeofexperiments
andarewellcharacterizedinliterature.Wroblewskietal.analyzedtheradiationeffect
invivoinratsandinvitrowithIEC-6cells.Theyconcludedthattheobservedeffectsin
cryptcellsinvivowereidenticaltotheeffectsintheIEC-6cellline[147].Thisconclusion
makestheIEC-6celllinesuitableforsideeffectinvestigation.Inaddition,IEC-6cells
areacommonmodelfornormalhumanintestinalepithelialbiology[138].

Cytokines1.5

Inammatorycytokinescanbedividedintotwogroupsaccordingtotheirinvolvement
intheinammatoryresponse:cytokinespromotingacuteversuschronicinammation.
Importantcytokinesfortheacutereactionareinterleukin(IL)-1,IL-6,IL-8,IL-11,and
TNFα.Thechronicinammationcanbesubdividedintocytokinesmediatinghumoral
responses(IL-4,IL-5,IL-6,IL-7,IL-13)andcytokineswhichmediatecellularresponses

9

oductionIntr1

(IL-1,IL-2,IL-3,IL-4,IL-7,IL-9,IL-10,IL-12,interferons,TGFβ,TNFα,TNFβ).
Mostcytokinesaremultifunctionalandpleiotropicwhichmeansthattheyelicittheiref-
fectlocallyorsystemicallyinanautocrineorparacrinemanner[35].Inthisthesisthe
cytokinesIL-2,TNFα,andTGFβwereanalyzed.ThedecisiontoanalyzeTGFβ,TNFα,
andIL-2wasmotivatedbytheresultsoftheco-culturesurvivalexperimentsandthe
publishedeffectsofthecytokinesonprostateandintestinalepitheliumtissue.Undersub-
section4.3.4thedecisionisdiscussedindetail.Inthefollowingthecurrentknowledge
aboutthethreecytokineswillbepresentedingeneralandincontexttotheusedcelllines
(ratanaplasticprostatecancercells,ratintestinalepitheliumcryptcells)inparticular.

pathwaysignalingβTGFIndependentoftheinducedregulatoryeffecttheTGFβsignalingpathwayisconserved
fromiestohumans.ThethreeisoformsofTGFβ(1-3,25kDahomodimer)aresyn-
thesizedaspro-hormonesinthecellandareconvertedintobioactivemoleculesinthe
extracellularmatrix[98,25].TheeffectofTGFβ-1andTGFβ-3ismediatedthroughthe
receptorsRI(53kDa)andRII(70kDa).Botharetransmembraneserine/threoninekinase
receptorsatthecellmembranethatinduceaphosphorylationafterTGFβbinding[7,145].
TGFβ-2needstheinteractionwithbetaglycan,whichisformallycalledRIII,tobedeliv-
eredtoRII[25].TheprimaryintracellulareffectorsofTGFβsignalsareSMAD-2and
SMAD-3whichdisplacetheactivationviainteractionswithSMAD-4andnucleoporins
fromthemembraneintothenucleus.InthecytosolSMAD-proteinsactivatecaspase-1,
anapoptosisinitiationfactor,whileinthenucleustheactivationofseveraltranscription
factorsistriggered[112,25].

ationvactiβTGFTGFβisstoredinitslatentforminextracellularreservoirsinanabundantmannerand
needstobeactivated.Ionizingradiationinducestheformationofreactiveoxygenspecies
(ROS).ItcouldbeshownthatROSareresponsibleforthedosedependentactivationof
TGFβafterirradiation[2].TheinhibitionofactiveTGFβleadstoinhibitionofautophos-
phorylationofthenuclearproteinkinaseATM(ataxiatelangiectasiamutated).ATMis
oneofthefirstandcentralproteinstorespondtoDNAdamage.TheabsenceofTGFβ-
ATMinteractionsincreasedtheradio-sensitivityofe.g.epithelialcells[2].

TGFβsignalinginprostatetissue
Growthfactorswhichaffecttheprostatecanbedividedintothreecategories:positive

10

Cytokines1.5

growthfactorsstimulateproliferationandgrowth;negativegrowthfactorsinhibitgrowth
andpromoteapoptosiswhileangiogenicfactorsstimulatevascularformation.Itiscon-
troversialastowhichcategoryTGFβbelongs.ReynoldsandKyprianousuggestthecate-
gorynegativegrowthfactorsinceTGFβisreleasedfromthestromalcellsintheprostate
inaparacrinemannerandinduceintheprostaticepithelialcellsgrowthinhibitionand
apoptosis[112].Incontrast,Tomlinsonetal.reportedthattheinhibitoryeffectofTGFβ-
1dependsonthedifferentiationstatusofthetargetcells.Undifferentiatedcellsinthe
prostateperiphery(distaltourethra)arestimulatedintheirproliferationwhilethehighly
differentiatedcellsinthecenter(proximaltourethra)oftheorganareinhibited[133].Lee
etal.considerTGFβ-1tobeapleiotropicgrowthfactorthatinhibitsproliferationandin-
ducescelldeathinnormalprostaticepithelialandstromalcellsfromrodentsandhumans.
Furthermore,theyreportedthatalowconcentrationofTGFβ-1increasesproliferation
ofprostaticcells[83].AccordingtoWikströmetal.TGFβinhibitstheproliferationof
epithelialcellsandinducesapoptosisinanormalratprostate[145].
TheexpressionofTGFβinprostatetumorstendstobeincreased.Theinhibitorygrowth
effectofTGFβismediatedviatheTGFβRII.Withongoingtumorprogressiontheexpres-
sionofTGFβRIIisdecreasedwhichmightenhancetheaggressivepotentialofthetumor
[112].TheDunningR3327ratprostaticadenocarcinomacellsystemseemstoacquire
aresistancetoTGFβmediatedgrowthinhibitionwithtumorprogression[145].Butthe
RAT-1celllinewasfoundinvivoandinvitrotoexpressinbothcaseselevatedTGFβ-1,
TGFβRIandTGFβRIImRNAlevelscomparedtonormaldorsalprostatetissue[145].

TGFβsignalinginintestinetissue
IninvivointestinethehighestTGFβmRNAlevelscanbefoundinthecryptcells.Here
TGFβpromotesintestinalepithelialrestitutionandinhibitionofproliferationafterintesti-
nalwoundinge.g.enhancedcelldeathafterradiotherapy.Restitutionisinvivoindepen-
dentofproliferationandinvolvesthemigrationofviableepithelialcellfromtheedgesof
theinjuredareatocovertheexposedarea.Theseeffectscanbestudiedinawoundheal-
ingmodelbasedonIEC-6cellmonolayer.TheTGFβexpressionintheIEC-6cellline
isregulatedbyanautocrinemechanism,andTGFβpreventsthedownregulationofex-
tracellularmatrixtranscriptswhichareneededforrestitution[102].Furthermore,TGFβ
inhibitstheDNAsynthesisinIEC-6cells[149]andhasonlysmalleffectsonepithelial
differentiation[99].TheproductionofTGFβisenhancedbyIL-2whichalsopromotes
cellmigrationinvitro.Podolskyconcludedthatcytokinesexpressedintheintestinalmu-
cosapromoteepithelialrestitutionaftermucosainjurythroughincreasedproductionof
activatedTGFβ-1inepithelialcells[102].

11

oductionIntr1

pathwaysignalingαTNFThepro-inammatorycytokineTNFα(17kDa)ismostlyproducedbyT-lymphocytes
andmacrophagesinamembrane-boundorinasolubleform.TheeffectofTNFαis
mediatedviatworeceptorcomplexes(TNFRIandTNFRII)[68].Inthefollowingthe
receptorcomplexbindstotheproteinsTNFreceptorassociateddeathdomainwhichac-
tivatestheTNFreceptorassociatedfactor2.Fromherethreepathwaysarepossible.Two
ofthemactivatethenuclearfactorNF-κBwhichpromotessurvivalfactorssuchasbcl-2,
ananti-apoptoticprotein.Thethirdpathway,phosphorylatesactivatorprotein-1,stimu-
].115[apoptosislates

TNFαsignalinginprostatetissue
TheeffectofTNFαoncellsurvivalandproliferationdiffersfrominvivotoinvitrostud-
ies,whilethespeciesplaysanimportantroleaswell.Inaprostatecancerpatientstudy
itwasobservedthattheTNFαlevelincreasedwithseverityofillness[6].Theexpres-
sionofTNFαandbothreceptorsareincreasedinepithelialcellsofprostatecancer[115],
too.Butthecellproliferationinprostatecancerisenhancedsincethepro-apoptoticway
ofTNFαisinhibitedbyp21andthesignalregulatingkinaseASK1[115].Inhumans
prostatecancercelllineLNCaPincreasedthenumbersofapoptoticcellsaftertreatment
with40ng/mlTNFαwhichisincontrasttotheinvivoresults[73].Invivoexperimentson
aratDunningsub-line,intra-tumorinjectionsofTNFαresultedinaslowertumorgrowth
incomparisontountreatedcontrolanimals[125].However,invitroexperimentswiththe
samecelllineandTNFαtreatmenthadnosignificanteffectoncolonyformation[135],
whichmeansnoeffectoncellsurvivalorgrowth.TheDunningsub-lineRAT-1,which
isusedinthisthesis,wasnotinvolvedinthisstudy.ItisexpectedthatTNFαcanhavea
positiveornegativeeffectonRAT-1cellproliferation.

TNFαsignalinginintestinetissue
TheintestinalmucosaproducesandsecretsTNFα[102].Underinammatoryconditions
thedensityofmucosacellsincreasessignificantlyandproducesTNFα,IL-2andIL-6
[149].IEC-6cellsarepartoftheintestinalmucosa.TheIEC-6cellsareabletosecret
TNFαwhichwasdemonstratedwithlipopolysaccharidesstimulation(LPS)[87].The
effectofTNFαonIEC-6cellshasbeenstudiedbyZachrissonetal..TheDNAsynthe-
sisofIEC-6cellswasenhancedinadose-dependentmanneraftertreatmentwith0.57x
10-9-10-11MTNFα.Thiswasassociatedwithincreasedcellproliferation[149].Higher
proliferationactivitywasexhibitedbyIEC-6cellsafterTNFαorIL-1βstimulationwas
comparedtountreatedcontrols.BothcytokinestogethersuppressedIEC-6cellprolifera-

12

tionbyanunknownmechanism[90].

Cytokines1.5

pathwaysignalingIL-2Il-2(14-17kDa)isthemajorgrowthfactorforactivatedT-lymphocytes[79].Besideac-
tivatedT-lymphocytes,othernormalandtumorcellsarefoundtoexpressandsecretIL-2
aswell[110].Thereceptorcomplex(IL-2R)consistsofaβ-andaγ-chainandhasanin-
termediateaffinitytoIL-2.Therefore,thecellsreactonlyonhigherIL-2concentrations.
Throughbindingofanα-chaintotheβ-andγ-chains,areceptorwithhighaffinityto
IL-2isformed[68].TheIL-2effectismainlymediatedthroughtyrosinekinaseswhich
stimulatesgeneexpression[79].IL-2seemstomediatethecellcycleprogressionintu-
morcellsviatheCDKinhibitorp27[110].

IL-2signalinginprostatetissue
InaprostatecancerpatientstudyitwasreportedthatstageIIandIIItumorshaveslightly
increasedIL-2levels.InstageIVtumorstheIL-2levelsdecreasedramaticallydueto
thedegradationoftheimmunesystem[6].Royuelaetal.analyzedthepresenceofIL-
2anditsreceptorinhumanprostatecancerandpreliminarystages.Ahighlyincreased
expressionofIL-2anditsreceptorwasfoundincellsofprostatecancercomparedtonor-
malprostatetissue[114].Ingeneral,thereceptorIL-2Rα-chainhasbeenfoundtobe
overexpressedinallprostatecancerstageswhichcorrespondstoincreasedproliferation,
drugresistance,transformingactivities,andanti-apoptoticproteinexpression[79].Inhu-
manprostatecancercelllinesacorrelationbetweenandrogen-sensitivityandgrowthin
responsetoIL-2wasfound:androgen-sensitivecelllinesshowedenhancedproliferation
whileandrogen-insensitivecellswerenoteffected[57].Thereareseveralstudiesshow-
inginvivoaninhibitorygrowtheffectofIL-2totheDunningprostatecancersystemin
generalandtotheandrogen-insensitiveRAT-1inparticular[54,53,55,70,71].Fora
combinedtreatmentofIL-2andirradiation(6MVx-rays,totaldoseof18Gyin3frac-
tion),tumorgrowthwasmoreefficientlyreducedthanbyirradiationalone[71].These
resultsdemonstratethatconclusionsbasedonobservationsmadeinpatientsorhumancell
linesarenottransferabletotheDunningsystem.

tissueintestineinsignalingIL-2TheIEC-6celllineoftheratisamodelfornormalhumanintestinalepithelium[138].
ThecelllinehasafunctionalIL-2receptor[102]andisabletosecretIL-2whichwas
shownwithLPSstimulation[87].Inaddition,IL-2enhancestheproliferationofIEC-6
cells[126].IL-2treatmentcombinedwithradiation(4Gy,fastneutronor4,8,12Gyγ

13

oductionIntr1

rays)increasedtheproliferationofirradiatedIEC-6cellsandinducedenhancedexpres-

sionofIL2-Rβ-chain[37,38].

Summary

Itisconfirmedbypublisheddatathatbothusedcelllines(RAT-1cellsandIEC-6cells)

havefunctionalTGFβreceptorsandareabletosecretTGFβ.Moreover,theIEC-6cells
expressIL-2andTNFαaswellasthecorrespondingreceptors.Thethreechosency-
tokinesshouldcoveracuteandchronicinammatoryresponsewhichareexpectedtobe

irradiation.afteredobserv

14

methodsandMaterials2

2.1Celllinesandcultureconditions

-1TR-3327-ADunning2.1.1

Inthisworkthesub-lineR-3327-AT-1(RAT-1)oftheDunningprostatecancercellsys-
temwasused.ThecelllinewasobtainedfromATCC(No.JHU-29)inpassage31(P31),
propagated,andstoredinliquidnitrogen(P35).ThecellswereculturedinRoswellPark
MemorialInstitutemedium(RPMI)supplementedwith10%FCSand100U/mlpeni-
cillin/100µg/mlstreptomycin(allBiochromAG,Berlin,Germany)in95%humidified
airat37°Cand5%CO2.Thepassagewasdoneweeklywith5x105cellsin75cm2cul-
tureaskswith15mlculturemedium.Therefore,thecelllayerwasushedwith1.5mlof
trypsin-EDTA-solution(0.05%trypsin,0.1%EDTA,Pan,Aidenbach,Germany)andin-
cubatedwith3.0mlfreshtrypsin-EDTA-solutionfor4minintheincubator.Thetrypsin
reactionwasstoppedbyadding10mlculturemedium.Thecellswereseparatedand
countedwithanelectronicparticlesizeanalyzer(Z2CoulterCounter,BeckmannCoulter,
Krefeld,Germany)withinthelimits9-26µmbeforethecellswerereseededindesired
numbers.ordilutions

2.1.2Intestinalepitheliumcellline6

Theintestinalepitheliumcellline6(IEC-6)wasisolatedfromamalerat(rattusnorvegi-
cus)andwasestablishedbyQuaronietal..TheysuggestedthatIEC-6cellsareundiffer-
entiatedsmallintestinecryptcells.ThecellswereobtainedfromATCC(No.CRL-1592)
inpassage(P)13,werepropagated,andlongtermstoredinseveralvesselsinliquidni-
trogen(P15).ForcultivationthecellsweregrowninDulbecco´smodifiedEaglemedium
(DMEM)supplementedwith10%FCS,50U/mlpenicillin/50µg/mlstreptomycin(all
BiochromAG,Berlin,Germany),and4.5µg/mlinsulinfrombovinepancreas(Sigma
Aldrich,Germany)in95%humidifiedairat37°Cand10%CO2.Thecellswerepassaged
weeklywith3-4x105cellsin75cm2cultureaskswith15mlculturemedium.There-
fore,thecelllayerwasushedwith1.5mloftrypsin-EDTA-solution(0.25%trypsin,
0.02%EDTA,Pan,Aidenbach,Germany)andincubatedwith3mlfreshtrypsin-EDTA-
solutionfor7minintheincubator.Thetrypsinreactionwasstoppedbyadding10ml

15

methodsandMaterials2

culturemedium.Thecellswereseparatedandcountedwithanelectronicparticlesize
analyzer(Z2CoulterCounter,BeckmannCoulter,Krefeld,Germany)withinthelimits
9-18µmbeforethecellswerereseededindesireddilutionsornumbers.
ToisolateclonesfromtheIEC-6population,thecellswereseededin96-wellplateswith
acelldensityofonecell/wellin200µlmedium.Thefreshculturemediumwasmixed
withone-thirdofoldculturemediumfromapassagetoenhancethegrowthconditions
ofthecells.Amediumfiltrationwasnotperformed,sincenoviableIEC-6cellsdetach
andoatinthemedium.Afteroneweekeverywellofa96-wellplatewascontrolledfor
colonies.Onlyfromwellswithdefinitelyonecolonywerethecellstrypsinizedandtrans-
ferredinto12.5cm2cultureasksforcontinuingcultivation.Thecloneswerepropagated,
thechromosomenumberwasdetermined,andthecloneswerestoredinliquidnitrogen.
Afterdefrosting,thecloneswereweeklypassaged.Thetotalcellnumberofthepassage
wasdetermined,addedtothetotalcellnumberoftheweekbefore,andplottedagainstthe
passagenumber.Theslopofthelinearfitcouldbeconsideredasadoublingtimeand
allowsacomparisonofgrowthrates.

eCo-cultur2.1.3

Forco-cultureexperimentsbothcelllineshadtogrowinthesamecellculturemedium.
Therefore,thegrowthofIEC-6cellsandRAT-1cellswastestedinbothculturemediums.
ThecomparativecellcycleanalysisofRAT-1cellsandIEC-6cellsinDMEMmediumas
wellasinRPMImediumdidnotshowaneffectonbothcelllinesthroughtheinverted
medium(seefigure2.1).ThePEofRAT-1cellsinnormalmedium(RPMI)wastestedto
be38.6%whilethePEininvertedmedium(DMEM)wasreducedto28.2%.Incontrast
tothedecreasedRAT-1cellPE,theIEC-6cellswerenotinuencedbythemedium.In
normalmedium(DMEM)aswellasininvertedmedium(RPMI)thePEwasconstant
with63.5%and62.4%,respectively.Forco-cultureexperimentsIEC-6cellsandRAT-1
cellswerecultivatedinRPMImediumsupplementedwith10%FCSand100U/mlpeni-
cillin/100µg/mlstreptomycinin95%humidifiedairat37°Cand5%CO2.Theco-culture
weretrypsinizedwithatrypsin-EDTA-solution(0.25%trypsin,0.02%EDTA)andwere
countedwithinthelimits9-26µmwithanelectronicparticlesizeanalyzer.
Theco-culturesurvivalexperimentswerecarriedoutwithtwodifferentset-ups.Inthe
firstexperimentalapproachthecellswereseeded24hpriortotheexperimentin25cm2
cultureasks(45asks/cellline).Therefore,thePE(seesubsection2.1.6)andthecell
doublingtimewasestimated,andanappropriatecellnumberwasseeded.Thedoubling
timehastobeintegratedintothecalculationsincethecellsdoubleevery24h(seefig-
ures3.1and3.5).Onthedayoftheexperimentthecellswereirradiatedandafterward,

16

2.1Celllinesandcultureconditions

Figure2.1:CellcycledistributionofRAT-1cellsandIEC-6cellsinnormalandinvertedmedium.
Solidlines:RPMImedium,normalforRAT-1cells,dashedlines:DMEMmedium,normalfor
cellsIEC-6

unirradiatedcellswereadded.Inthesecondapproach,thecellswereirradiatedormock-
irradiatedinmono-cultures,trypsinized,andseededtogether.Inbothexperimentalset-
upsthecellswereseededseventimesin25cm2cultureasksfortheclonogenicsurvival
assay(seesubsection2.1.6).Figure2.2showstheseedingscheme.Theunirradiated
cellsaredepictedindarkblueanddarkgreen,whiletheirradiatedcellsareshownin
lightblueandlightgreen.Mono-culturesofunirradiatedandirradiatedcellsservedas
controls.Thesesamplesareshowninthecolorcombinationblueorgreenwithwhite.
Thecombinationofirradiatedandunirradiatedcellsofonecelllinewasnottestedsince
theresultingcoloniescannotbedistinguishedinirradiatedorcontrolcells.Insteadof
fullsurvivalcurvesonlythreesurvivallevelsofRAT-1cellswerechosen(80%,50%,
and15%)correspondingtoadoseof1,3,and6Gyofx-rays.ThesurvivalofIEC-6
cells,whichresultsafterx-rayirradiationwiththesedoses,wasdeterminedtobe73%,
38%,and6%.Thex-raydoseswereconvertedwiththeaidofRBEtocarboniondoses.
Table2.1givesanoverviewofthechosensurvivallevelsaswellasthecorresponding
gies.eneranddosesabsorbedFordataanalysisthesurvivalofmono-cultureswasnormalizedonthePEformono-
cultures(PEmono).Theco-culturedatapointswerenormalizedontheco-culturePE(PEco)
ofunirradiatedIEC-6cellsandRAT-1cells.Whenseveralexperimentswereperformed
toonedatapointthemeanvalueswiththeirstandarddeviationweredetermined.Ifonly
oneexperimentwasperformed,anerrorestimationwascarriedoutasdescribedunder

17

methodsandMaterials2

Figure2.2:Seedingschemeforco-culture.RAT-1cellsaredisplayedinblueandIEC-6cells
ingreen.Darkcolorsrepresentunirradiatedcellswhilelightcolorsshowirradiatedcells.The
mono-cultures(controls)areindicatedwithwhitefields.

Table2.1:Thetableshowsdosesandbeamenergiesusedinco-cultureexperiments.

Survivallevels[%]Dose[Gy]
RAT-1IEC-6250kVpx-rays12C100MeV/u12C11.4MeV/u
0.170.61.073800.531.73.038501.464.16.0615

subsection2.7.1.Toanalyzethepossibleco-cultureeffectthenormalizedco-culturesur-
vivalwasdividedbythenormalizedmono-culturesurvival.
Forcytokineexperiments(seesection2.3)thecellswereirradiatedwith0Gyand6
Gyofx-raysor1.46Gy(11.4MeV/u)ofcarbonions.Afterirradiationthecellswere
trypsinized,seededwithvariablecellnumbers,andcultivatedina6-wellplate/insertsys-
tem(Insert:PETmembranewith3.0µmporediameter,GreinerBio-OneGmbH,Ger-
many).Theinsertmembraneisporousandallowsthecellcommunicationwithsoluble
factorsviathemedium.Inthissystemdifferentgrowthbehaviorbetweentheplateand
theinsertarepossible.Therefore,bothseedingpossibilitiesweretestedforasamplepair:
plat/RAT-1cells+insert/IEC-6cellsandinverse.Thex-rayirradiationwasperformed
twiceandthecellswereseededwithlowerorhighercellnumberstoclarifyifthecell
numbersinuencethecytokineproduction.TheratioofseededRAT-1cellstoIEC-6
cellswastentosevenliketheseededcellnumbersincolonyformingassays(seesub-
section2.1.6).Atcertainpointsduringtheexperimentthecellculturesupernatantwas
harvestedforcytokinemeasurementsandcentrifugedat4°Cfor3minat300g.The
supernatantwasstoredlong-termat-20°C.Inparallel,thetotalcellnumberswerede-
terminedinthewellandintheinsert.Foranalysis,themeasuredcytokineamountinthe

18

2.1Celllinesandcultureconditions

Figure2.3:Schematicdrawingof6-wellplate/insertsystemusedinco-cultureexperiments.Left
panel:insert,rightpanel:6-wellplatewithtwoinserts,Illustration:BDBiosciences,SanJose,
modifiedUSA;CA,

supernatantwerecorrelatedtothecellnumbers.
Inaclonogenicsurvivalassay(seesubsection2.1.6)thecellshavepartialdirectcellto
cellcontact.Withthe6-wellplate/insertsystemthedirectcelltocellcontactinco-culture
wasexcludedandthecytokinescouldonlybemediatedviacellculturemedium.Tomea-
sureifthedirectcelltocellcontactsareimportantforthecytokinesecretioninco-culture,
thecellswereseededtogetherin6-wellplatesandthesupernatantswerehandledasmen-
tionedabove.Inamixedsuspensionofbothcelllines,aseparatedcellnumberforeach
celllinecannotbedeterminedwhichisneededfordataanalysis.Inaparticlesizean-
alyzerthecellhistogramsarecongruentsothatonlythetotalcellnumberwasdetected.
Tocircumventthisproblemonecelllinewasstainedwiththevitalmembranelabeling
uorescentdyePKH67(seesubsection2.4.2).Intheowcytometerthepercentagedis-
tributionofthetwopopulations(stained/unstained)wereanalyzed.Incombinationwith
thetotalcellnumberofbothcelllinesthecellnumberofeachcelllinecouldbedeter-
minedandcorrelatedtothecytokineamount.

2.1.4Cryopreservation

Forordertopreservethecellslong-termcellswerestoredinliquidnitrogenat-196°C.
Therefore,thecellsweretrypsinizedandpelletedbycentrifugationfor10minat4°Cwith
107g.Themediumsupernatantwasdiscarded,andthecellswereresuspendedincooled
culturemediumcontaining20%FCSand10%dimethylsulfoxide(DMSO,Applichem,
Darmstadt,Germany)besidethenormalmediumcomponents.2mlsuspensionwith1-2
x106/mlcellswastransferredintocryotubes.Thesampleswerefrozento-80°Cinpre-
cooledisopropanolvessels(NalgeneCryo1°CFreezingContainer,VWR,Germany)in
acontrolledmanner.After24hthetubesweretransferredintoliquidnitrogen.
Forcultivation,thecellsinthetubeswererapidlydefrostedbyshakinginawaterbath

19

methodsandMaterials2

at37°C.Thecellsuspensionwastransferredintoatubewith10ml4°Cpre-cooled
cellculturemedium.ToremovetheDMSOthecellswerepelletedbycentrifugationfor
10minat4°Cat107g.Themediumsupernatantwascompletelyremoved,thepellet
separated,andthecellsreseededin15mlnormalculturemediumpre-warmedto37°C
in75cm2cultureasks.

2.1.5Growthcurves

ThecelldoublingtimetDwasdeterminedingrowthcurves.Agrowthcurveissubdivided
inthreephasesofgrowth.Intheinitiallag-phasethecellsattachafterseedingandenter
theexponentialphasewhenthecellsproliferatewithaconstantcelldoublingtime.Fi-
nally,thecellsreducetheirproliferationwhenthegrowthareabecomeslimitedandenter
thestationaryphase(overviewinreference[1]).Intheexperiments1-5x104cellswere
seededinacultureaskwith12.5cm2or25cm2growtharea.Overseveraldaysthetotal
cellnumberwasdeterminedinduplicates.Intheexponentialphaseofthegrowthcurve
thecelldoublingtimewasdeterminedwiththeGSIinhouseprogramgd(©M.Krämer,
2003).

2.1.6Clonogenicsurvivalassay

Todeterminethecellsurvivalrateafterirradiationaclonogenicsurvivalassaywasper-
formed.Aftertreatmentthecellsweretrypsinized,separated,andcountedbeforethecells
werereseededin25cm2cultureasks.Inaddition,beforecountingRAT-1cellsneeded
acentrifugationover8minat521gtoremovetrypsinfromthecellsuspension.The
inoculum(I)forcellseedingwascalculatedaccordingtothefollowingEq.

NCIml=(Nml∙S∙PE),(2.1)
whereasNCisthecolonynumber/cultureaskwhichshouldgrow,Nmlisthecellnum-
ber/mlinthecellsuspension,Sthereducedsurvivalafterirradiation,andPEtheexpected
platingefficiency.ThePEdescribesthepercentageofcellswhichareabletoproliferate
andformcolonieswithmorethan50daughtercellswithinacertaintimeinterval.Since
thePEdependsonthecellhandlingandthegeneralstatusofthecellsithastobede-
terminedineveryexperiment.ForRAT-1cellassaysNCwas100andthesampleswere
reseededintriplicates.InIEC-6cellexperimentsNCwas70sothatthecellshaveto

20

2.1Celllinesandcultureconditions
bereseededinquadruplicatestoachievecomparablecolonystatisticstoRAT-1cellex-
periments.Inco-cultureexperimentswithIEC-6cellsandRAT-1cellsbothNC-values
remainedconstantbuttheasknumberwasincreasedtoseptuplicatewiththeobjective
ofincreasedstatisticanddetectionofevensmallco-cultureeffects.RAT-1cellsandIEC-
6cellswereincubatedfor11days.Afterincubationthegrowncolonieswerefixedand
stained.Thestainingprocedurestartedbydiscardingthemedium.Thegrowncolonies
werefixedwith2mlof70%ethanolfor15minatroomtemperature.Theethanolwas
aspirated,andthecolonieswerestainedwith3mlthreefoldmethyleneblue(seeannex)
for8min(RAT-1cells)or15min(IEC-6cells).Thestainingsolutionwasdiscarded,the
cultureaskswerewashedwith3mlpurifiedwatertoremovesurplusstainingreagent,
andthesampleswereairdriedunderthefumehood.Thestainedcolonieswerecounted
underastereomicroscope.InthefollowingEq.theobtainedPEiscalculatedwhereasNR
meanstheaverageoftheresultingcolonynumber/sample.Inthiscasethesampleisan
unirradiatedcontrolandthesurvivalisassumedwithone.
NPE=(Iml∙RNml)(2.2)
Thesurvivaloftheirradiatedcellsampleshastobecalculatedaccordingtothefollowing
Eq.andhastobenormalizedonthePE.
NRS=(Iml∙Nml∙PE)(2.3)
stainingX-Gal2.1.7X–GalstainingwasusedwithIEC-6cellsforthedetectionoftheenzymeβ-galactosidase
(β-gal)whichisdiscussedtobeageneralindicatorforcellsenescence[28,67].Leeetal.
demonstratedthatsenescence-associatedβ-galactosidase(SA-β-gal)correspondstolyso-
somalβ-gal[82]whichisconsistentwiththeobservationthatsenescentcellsincreased
thenumberoflysosomes[80].Lysosomalβ-galhasitsmaximalenzymaticactivitybe-
tweenpH4and4.5andclearlyloweractivityatpH6[92].Theunusuallyhighactivityof
β-galinnon-optimalpHistypicalofsenescentcells[46].Theindolederivative5-bromo-
4-chloro-3-indolyl-β-D-galactoside(X-Gal)isanartificialsubstrateoftheenzymeβ-gal.
Whenβ-galcleavedtheX-Galasolubleandcolorlessindoxylmonomerisproduced.
Twoindoxylmonomersformadimerwhichisoxidizedonair.Theresultanthalogenated
indigoisaverystableandinsolublecyancompound[17].Forthestaining1x105cells
in2mlculturemediumwereseededinPetridisheswithadiameterof3cm48hbefore
theexperiment.Onthedayoftheexperimentthemediumwasdiscardedandthecell
21

methodsandMaterials2

layerwasrinsedthreetimeswith1mlPBS–.Thecelllayerwasfixedwith1mlofa3%
formaldehydesolutionfor5minatroomtemperature.Toremoveformaldehydesurplus
thecellswerewashedthreetimeswith1mlPBS–.Thestainingwasperformedwith3ml
ofX-Galstainingsolution(seeannex)for18hat37°C.Afterward,thestainingsolution
wasremoved,andthecelllayerwasrinsedthreetimeswith1mlPBS–.Theanalysisof
theairdriedsampleswasperformedunderastereomicroscopeatwhichX-Galpositive
(blue-greencoloredcells)andnegativecellswerecounted.

Chr2.2eparationpromosome

ChromosomepreparationswereperformedwithIEC-6cellsandRAT-1cellsaccording
tostandardtechniques[61].Therefore,1x106cellswereseeded48hbeforetheexper-
imentin75cm2cultureaskswith10mlculturemedium.Ontheexperimentday10µl
colcemid/mlmedium(RocheDeutschlandHoldingGmbH,Germany)wasaddedtothe
cells.Colcemidarreststhecellsinmitosisbyblockingthechromosomedivisionthrough
thespindleapparatus.After4hofincubationthecellsweretrysinizedandharvested.The
cellsuspensionwaspelletedbycentrifugationfor6minat204g.Thesupernatantwas
discarded,andthepelletresuspendedbyknocking.5mlpre-warmed0.075Mpotassium
chloride(KCl,MerckKGaA,Darmstadt,Germany)solutionwasaddedtothetubefora
durationof8minwhichinducedchromosomeswelling.Thecellswereagainpelletedby
centrifugingfor8minat204g.Thesupernatantwasdiscarded.Thepelletwasresus-
pendedbyknocking,and10mloffixativewasaddedtothecellsforadurationof8min.
Thefixativeiscomposedof3partsmethanoland1partpureaceticacid.Thecellswere
incubated30mininfixativebeforethecellswerepelletedbycentrifugingover10minat
293g.Thefixativewasdiscarded,thepelletresuspendedbyknocking,andthecellswere
washedwith10mlfixative.Afteranadditionalcentrifugationstepat293gfor10min
thecellswereresuspendedin1mlfixative.Thepreparedsuspensionwasdroppedonwet
slides(purifiedwater).After24hofairdryingtheslideswerestainedfor10minina10%
Giemsasolution(MerckKGaA,Darmstadt,Germany)dilutedwithSoerensenbuffer(pH
6.8,seeannex)orwerestainedwiththemulticoloruorescenceinsituhybridizationtech-
nique(seesubsection2.2.1).ToremovesurplusGiemsasolutiontheslideswerewashed
inpurifiedwateranddriedwithcompressedair.Forpermanentconservationtheslides
weresealedwithEukitt(KindlerGmbH&Co,Freiburg,Germany)andcoveredwitha
thincoverslip.Foreachsamplethemitoticindexwasanalyzedin6000nuclei,andthe
numberofchromosomespermetaphasewasdeterminedin300metaphases.

22

2.3Detectionofpro-inammatorycytokines

2.2.1Multicoloruorescenceinsituhybridization

Themulticoloruorescenceinsituhybridization(mFISH)presentsallchromosomes
ofametaphasesimultaneouslyinasinglehybridization.Thereby,fivedifferentuo-
rochromesoracombinationoftheuorochromesbindtotheDNAandallowtheanalysis
andidentificationofinter-chromosomalexchangeswhichisnotpossiblewithGiemsa
staining(seesubsection2.2).HerethemethodwasusedtoanalyzetheaneuploidIEC-
6cellmetaphases.ThemFISHkitsareavailableforhumanandmousechromosomes
butnotforratchromosomes.Anattemptwasmadetoanalyzetheratschromosomes
witha21xMousemFISHProbeKitformouse(MetaSystems,Altlussheim,Germany).
Thechromosomesampleswerepreparedasdescribedundersubsection2.2.ThemFISH
procedurewasdoneaccordingtotheXCyteLabManual(manufacturersinstructions)
includingtheRNAsepretreatmentpriortothepepsinpretreatmentbutwithoutpostfixa-
tionafterpepsintreatment.Thecellswasincubatedwithprobecocktailthreedaysat37
°Candpreparedtillthefirstdetectionstep.Afterairdryingthesampleswerecounter-
stainedwithDAPI/Antifade(4,6-diamidino-2-phenylindole,B-tectGmbH,Hannover,
Germany),coveredwithaslide,andstoredprotectedfromlightat4°C.Allconcen-
trationsofusedsolutionswereconsistentwiththeprotocolofthemanufacturer.The
stainedmetaphaseswereanalyzedwithamotorizedandcomputer-controlleduorescent
microscopeequippedwithamonochromeCCD-camera.Themetaphaseswereexcited
withUV-light(DAPI-channel)andwereselectedmanuallyundera100xobjective.With
thesoftwareIkaros&Isisversion5.0(MetaSystems,Altlussheim,Germany)theauto-
maticfilterchangeandsampleexpositionaswellasimagerecordingandprocessingwere
controlled.Sincemicehave21andrats22differentchromosomestheallocationofthe
chromosomeshastobedonemanuallyforanalysis.

2.3Detectionofpro-inammatorycytokines

Todetectthepro-inammatorycytokinesTGFβ,TNFα,andIL-2,differentSandwich
Enzyme-LinkedImmunoSorbentAssay(ELISA)detectionkitsfromR&DSystemsGmbH
(Heidelberg,Germany)wereused.InaSandwichELISAanantigenspecificcapturean-
tibodyisimmobilizedtothesurfaceofa96-wellplateovernightatroomtemperature.
Thesamplewithanunknownamountofantigenwasaddedforbindingtothefirstanti-
body.Asecondantibodywasaddedandformedacomplexwiththeantigen.Thesecond
antibodywascoupledwithastreptavidin-horse-radish-peroxidase-complex.Theenzyme-
complexproducesthroughbindingtoanenzymaticsubstrateavisiblesignalwhichindi-
catesthequantityofantigeninthesample.ThedetectionofTGFβ,TNFα,andIL-2

23

2methodsandMaterials

wascarriedoutinthecellculturesupernatantsofirradiatedandunirradiatedco-cultureor
mono-culturesamplesofIEC-6cellsandRAT-1cells.Theprocedurewascarriedoutac-
cordingtothemanufacturersinstructions.Allantibodyandsolutionconcentrationswere
consistentwithguidelines.Theopticaldensitywasdeterminedinamicroplatereader
(BioTekEL808,BioTekInstrumentsGmbH,BadFriedrichshall,Germany)at450nm.
Thewavelengthcorrectionwasdonewith570nm.Thedataanalysiswascarriedoutwith
EXCEL®.

cytometrywFlo2.4

analysiscycleCell2.4.1

Forcellcycleanalysisthecellsweretrypsinized,separated,andcounted.Aminimumof
1x105toamaximumof1x106cellswerecentrifuged8minwith521gatroomtem-
perature.Thesupernatantwasremoved,andthepelletwasresuspendedinthereux.The
cellswerewashedtwicewith1mlPBS–(centrifugation521g,8min)andresuspended
in1mlicecold70%ethanolundervortexingforcellfixation.Thepreparedsamples
couldbestoredforfourweeksat-20°C.Alternatively,theethanolcouldberemoved
after1hoffixationbycentrifugation(521g,8min).Thecellswerestainedprotected
fromlightatroomtemperatureforaminimumof30minwith1µg/mlDAPIinPBS–be-
foremeasuring.ThesamplesweremeasuredandanalyzedwiththeowcytometerPAS
III(Partec,Münster,Germany)andtheWindowsTMbasedsoftwaresFloMax®(Partec,
Münster,Germany)andMultiCycle(PhoenixFlowSystems,SanDiego,CA,USA).
Thecellcycleofeukaryoticcellsissubdividedinfourphases(seefigure2.4).Duringthe
M-phase,thechromosomesarecondensedandthereforevisibleunderamicroscope.The
M-phasecontainsthemitosis(divisionofthesisterchromatids)andthecytokinesis(cell
division).Themitosisitselfissubdividedintotheprophase,prometaphase,metaphase
(earlymitosis),anaphase,andtelophase(latemitosis).TheM-phaseisfollowedbythe
interphase.TheinterphasecontainstheG1-,S-,andG2-phase.InG1-phasethecellsgrow
andduplicatetheirorganelles.InthefollowingS-phasetheDNAisduplicated.Thefinal
dimensionreachthecellsinG2-phasebeforethecellsenterthemitosisagain.Somecells
leftthecellcycleandstopdividing.ThisrestingphaseiscalledG0-phase(overviewin
reference[1]).BecauseofthesimilarDNAcontentadifferentiationofthesub-phases
oftheM-phase,adiscriminationofM-phaseandG2-phaseaswellasadifferentiation
ofG0-phaseandG1-phaseisnotpossiblebyusingaowcytometerforthecellcycle
analysis.

24

cytometryFlow2.4

Figure2.4:Cellcyclephasesofaneukaryoticcell[1],modified

2.4.2FluorescencemembranelabelingwithPKH67andanalysis

PKH67(SigmaAldrich,St.Louis,WO,USA)isagreenuorescentlabelingdyewhich
integrateswithitslongaliphaticcarbontailintolipidregionsofthecellmembraneof
intactcells.Thedyehasanextinctionmaximumat490nmandanemissionmaximum
at502nm.Thecellstainingwasperformedwithsmallchanges,whicharementioned
below,accordingtothemanufacturersinstructions.Theirradiatedorunirradiatedcells
weretrypsinized,separated,andcounted.Theneededcellnumberwastransferredinto
apolypropylenetube,andthecellswerepelletedat400gfor5min.Thesupernatant
wasdiscarded,thecellswereresuspendedin5mlPBS–,andcentrifugedat400gfor5
min.ThePBS–wasdiscarded,thereuxreducedtounder25µlbypipetting,andthe
cellswereresuspendedin1mlDiluentC.Inaseparatetube2µlor8µlPKH67staining
reagentper1x107cellsweremixedwith0.5mlDiluentC.Allfollowingstepswere
doneunderconditionsprotectedfromlight.Thestainingsolution(PKH67+DiluentC)
waspipettedintothecellsuspension.Thesuspensionwasmixedwellbypipettingand
incubatedfor5minatroomtemperature.DuringthistimethePKH67integratesintothe
cellmembrane.Thereactionwasstoppedbytheadditionof2mlFCSfor1minfollowed
byacentrifugationat400gfor5min.Thesupernatantwasdiscarded,andthecellswere
resuspendedin10mlculturemedium.Themediumwashstepwasrepeatedthreetimes
beforethecellswerecountedandreseededindesireddilutions.Thecellsweregrown
protectedfromlightforseveraldays.Atvariouspointsintimethecellsweretrypsinized,
counted,andpreparedforowcytometeranalysis.Therefore,thecellswerepelletedat
521gfor8min.Thesupernatant(cellculturemedium)wasdiscarded.Thecellswere
fixed15minin2%paraformaldehyde(PFA)or15minin70%ethanol.Thefixativewas
removedbycentrifugingat521gfor8min.Thecellswerewashedoncewith5mlPBS–,
centrifugedat521gfor8min,andresuspendedin2mlPBS–.Theauto-uorescenceof

25

methodsandMaterials2

thenonstainedcellswashighenoughandacounterstainingagainste.g.theDNAwas
notneeded.Thestainedsamplesweremeasuredwiththeargonlaser(488nm)oftheow
cytometerPASIII(Partec,Münster,Germany).Thedataanalysisweredonewiththe
WindowsTMbasedsoftwareFloMax®(Partec,Münster,Germany)andandMultiCycle
(PhoenixFlowSystems,SanDiego,CA,USA)andtheGSIinhouseprogramgd(©M.
2003).,KrämerThePKH67stainingwasperformedwithdifferentdyeconcentrations(2µlor8µlPKH67/
1x107cells),andthestainedcellsweremeasuredaliveintheowcytometer.Figure2.5
demonstratesthat2µlPKH67wasadequatetostainthecellsandallowadifferentiation
betweenstainedandunstainedcells(blackcurve,crosssymbol).Theamountofdyein
thecellmembraneisreducedwitheverycelldivision.Therefore,thenumberoflessor
unstainedcellsincreasedinaPKH67stainedsampleswithtime.Foranobservationpe-
riodoffourdaysthedyeconcentrationof2µlPKH67/1x107cellswasnothighenough
todistinguishbetweenthestainedandunstainedcells(cyancurves,crosssymbol).The
enhancedPKH67concentration(8µlPKH67/1x107cells)wasmoresufficientinthat
waythatafterfourdaysthetwopopulationswerestilldividablebytheiruorescence
(cyanandblackcurves,opencircle).
Forsomeapplications,e.g.long-termstorage,itisadvantageoustofixthesamplesand
prolongthetimeintervalinwhichthesamplecouldbemeasuredwithreproduciblere-
sults.Twosolutionsaretestedforthefixation:ethanolandPFA.Infigure2.5isdisplayed
thatthroughtheethanolfixation(greencurves,openorclosedcircle)thehomogeneous
peakofstainedsamples(blackcurve,opencircle)isdestroyedwhilethesignaloffixed
andunstainedsamples(blackcurve,closedcircle)wasshiftedtolowersignalintensi-
ties.ThePFAfixation(redcurves,openorclosedcircle)hadverysmallinuenceson
thestaining(blackcurves,opencircle,minimalpeakshift)andshouldbethepreferred
agent.ThegreatdifferencesbetweenthelivingIEC-6cells(blackcurve,closedcircle)
andthePFAfixedcells(redcurve,closedcircle)resultsfromaniniquitousseparationof
thelivingIEC-6cells.ThePFAfixedsamplescouldbestoredovereightweeksat4°C
withoutsignalloss.Overall,thestainingwithPKH67needstobeoptimizedforeachcell
linebutthemethodopenesinterestinganalysisoptions.

chamberHypoxia2.5

Becauseoxygenationwithintumorsplaysanimportantroleintheoutcomeaftertreat-
mentthesimulationofdifferentoxygenationsituationswithcellculturescouldhelpto
understandtheunderlingmechanismandtocollectdatafortreatmentplanningsystems.
Forthispurposeahypoxiachamberwasestablishedtoirradiatecellcultureswithx-rays

26

hambercHypoxia2.5

(a)IEC-6cells(b)RAT-1cells
Figure2.5:PKH67stainedorunstainedIEC-6cellsandRAT-1cells.Inblack:livingcells,in
red:PFAfixedcells,ingreen:ethanolfixedcells,incyan:growthfor96haftertreatment,PFA
fixed,closedcircle:unstained,cross:2µlPKH67/1x107cells,opencircle:8µlPKH67/1x107
cells

andheavyions,especiallycarbonions,underdefinedoxygenconditions.Thehypoxia
chamberandthesampleringweredevelopedinthediplomathesisofC.Schickerwhich
wassupervisedbyW.K.-WeyratherandC.vonNeubeck[117].Theresultsofthethesis
werepatentedinreferences[118,119].Furtherdetailsoftheconstructionarereportedin
]117[erSchick

Construction2.5.1

Thehypoxiachamberwasmilledoutofonepieceofpolyetheretherketoneandhasthe
externaldimensionsof90x74x57mm(lengthxwidthxheight).Thefrontwallis
reducedto1mmthicknesswhichisthesocalledirradiationwindow.Onbothside
wallsafemalehosecouplingwasscrewedwhichcouldbemadepenetrablebyplugginga
malecoupling(bothneoLab,Heidelberg,Germany).Thecouplingnexttotheirradiation
windowwasusedforthegasinletandtheotherforthegasoutlet.Thetopcoverwasmade
oftransparentpolymethylmethacrylatewhichallowspositioncontrolofthesample.In
themiddleofthetopcoverandonthebottomofthechamberachamferwasmilledwhich
fixesthecellsamplesinthecorrectpositionduringtheexperiment(seesubsection2.5.2
andfigure2.6).WithfourscrewsandanadditionalO-ring(DichtungstechnikBensheim
GmbH,Bensheim,Germany)aroundthetopcoverthechambercouldbeclosedairtight.

27

methodsandMaterials2

Figure2.6:Hypoxiachamberwithsamplering.

handlingandsamplesCell2.5.2

Forexperimentsinthehypoxiachamberthecellshadtogrowonaspecialfoil.The
bioFolie25(InVitroSystems&Services,Göttingen,Germany)ispermeableforgases
suchasCO2(2.2µmol/cm2h)andO2(6.3µmol/cm2h).Celladhesionispossibleonthe
hydrophilicsideofthefoil.ForexperimentsthebioFolie25wascutintocircles(∅44
mm),sterilizedin70%ethanolfor15min,andairdried.Withhighviscoussiliconpaste
(Baysilone,BayerAG,Leverkusen)thefoilwasbondonasampleringtoformaPetri
dish.Thesampleringwasmadeofpolyvinylchloride,hasathicknessof3mm,anda
radialthroughboringwhichisclosedwithsiliconepasteoralittlescrew.48hpriorto
theexperiment3x104RAT-1cellsin1mlculturemediumwereseededonthefoil-Petri
dish.Onthedayoftheexperiment,asecondfoilwasstuckwithsiliconpasteonthe
ringtogenerateasterilevolumein-betweenthetwofoils.Thesampleringwasfixed
inaretainingbracketwiththeradialthroughboringontop(seefigur2.7).Thevolume

Figure2.7:Retainingbracketwithfixedsamplering.

createdbythetwofoilswasfilledwith1.4mloffreshculturemedium,andthethrough

28

2.6Cellirradiationprocedure

boringwasclosedwithsiliconepaste.Thecellsampleringwasinsertedintothehypoxia
chamberwiththecelllayerindirectionofirradiationwindow(seesubsection2.5.1).For
moredetailsseereference[117].Thecellsamplesinthehypoxiachamberwereaerated
withpureN2fortestmeasurementsor95%N2/5%CO2(LindeAG,Pullach,Germany)
forexperiments.Afterirradiationthecellsamplewasremovedfromthechamber,andthe
cellswereanalyzedinaclonogenicsurvivalassay.Therefore,thefoilwithoutcellswas
removed,themediumdiscarded,andthecelltreatedasdescribedundersubsection2.1.6.

modalitiesGassing2.5.3

Thecellsamplesinthechamberswereaeratedwithdifferentgasmixturestoreducethe
oxygeninthegasphaseandintheculturemediumundercontrolledconditions.The
partialO2pressurewasmeasuredwithanopticalO2microsensor(Needle-TypeHousing)
andameasuringinstrumentMicroxTX3(bothPreSens,Regensburg,Germany).Thegas
owwasmeasuredwithathermalmassowmetercalibratedfornitrogen(1000ml/min
N2,red-ycompactmeterGCM,VögtlininstrumentsAG,Aesch,Switzerland).Through
theusageofnitrogenbasedgasmixturesameasurementerrorof3%shouldbetakeninto
accountbesidetheinstrumentalerrorof1%(totalerror3.2%,errorestimationVögtlin
instrumentsAG).Thetimeintervaltoachievehypoxicconditions(thyp)isdependenton
thegasuxandwasdeterminedwithfivein-lineconnectedhypoxiachambers.Apure
nitrogenowof100ml/minreplacestheairinthechamberswithin5.54min.Tosimulate
experimentalconditionsthechamberswereloadedwithsampleringswhichwerefilled
withpurifiedwater.Aowof100ml/min,150ml/min,and200ml/minpurenitrogen
correspondstothypvaluesof164.4min,150.2min,and86.2min,respectively.The
experimentalprotocolforthecellexperimentsweredefinedtobea2hgassingwith200
ml/minwhichtakeintoaccountthedeviceerrorof3.2%,thechamberushingtimeof
5.54min,andthetimetoachievehypoxiaconditionsinthechambers.

2.6Cellirradiationprocedure

Allirradiationexperimentswereperformedwithasynchronousproliferatingcellcultures
incultureask,PetridishesoronbioFolie25forexperimentsinhypoxiachambers.In
everyexperimentunirradiatedcontrolcells(0Gy)fordeterminingthePEwereseeded,
incubated,andhandledunderthesameconditionsliketheirradiatedsamples.

irradiationX-rayThex-rayirradiationwasperformedwiththex-raymachineIsovoltDS1(Seifert,Ahrens-

29

andMaterials2methods

berg,Germany).Theacceleratingvoltagewassettobe250kVpwithacathodecurrent
of16mA.Atthebeamexitwindowafiltersystemof7mmberyllium,1mmaluminum,
and1mmcopperabsorbedthelongwavepartoftheradiation.Thedoseandthedose
rateweredetectedbyadosimeter(SN4,PTWFreiburg,Germany).Theirradiationwas
performedwithadoserateof1.3to4.0Gy/minatroomtemperaturewhiletheabsorbed
doseswerebetween0to27.0Gy.

irradiationCarbonTheGSIsUniversalIonLinearAccelerator(UNILAC)acceleratesthecarbonionupto
20%lightvelocity,andthefollowingheavyionsynchrotron(SIS)enhancestheacceler-
ationto90%lightvelocity.ForexperimentsatUNILACthecellswereculturedin3cm
PetridishesandwereirradiatedinPetridishcompatiblemagazines.Thestoppingcarbon
ionshadaprimaryenergyof11.4MeV/uand9.8MeV/uontargetwiththecorrespond-
ingLETof170keV/µm.Theapplieddoseswereintherangeof0to3.5Gy.Adetailed
descriptionoftheirradiationprocedureanddosimetryisgivenelsewhere[76].Afterir-
radiationthePetridisheswereremovedfromthemagazinesundersterileconditions.In
everyPetridishtheinnerborderwascleanedwithasterilecottonbud.Thishastobe
donesincethePetridishisirradiatedinaverticalposition,andamediumdropisformed
atthebottomoftheinnerborder.Inthemediumdroptheionsstoppedanddonothitthe
cells.Withthewipingstepallunirradiatedcellsareremoved.
ForirradiationatSISfacilitythecellswereculturedin25cm2cultureaskswhichwere
completelyfilledwithculturemediumandsealedwithanadditionalparafilmstrip(Amer-
icanNationalCanGroup,SanDiego,CA,USA).Thecellswereirradiatedwithinten-
sitymodulatedrasterscanningtechniqueasdescribedindetailelsewhere[50].Mono-
energeticfieldswithaprimaryenergyof100MeV/uor270MeV/uweredelivered(LET:
28keV/µmand13keV/µm,respectively).Theseenergieswerereducedtoenergylevels
of93.2MeV/uand267MeV/uontarget(LET:27.9keV/µmand13.7keV/µm,respec-
tively)duetotheiontraversalthroughthedetectorsystemandthecultureaskbottomin
thebeamline(1.3mmwaterequivalentthickness).Theapplieddoseswereintherange
of0to8.0Gy.
SamplesinthehypoxiachamberwereirradiatedwithaspreadoutBraggpeak(SOBP)of
1cmthicknesswithadose-averagedLET(LET)of100keV/µm.Theapplieddosesfor
oxicsampleswere0to3.0Gyandforhypoxicsamples0to5.4Gy.

30

analysisdataStatistical2.7analysisdataStatistical2.72.7.1Errorestimationforsurvivalexperiments
ThePE(seeEq.2.2)ofunirradiatedcontrolsampleswasdeterminedineverysurvival
experimentanditdefinesthe100%survivallevelforaspecificexperiment.Allirradi-
atedsamplesofthisspecificexperimenthavebeennormalizedtothisvalueaccordingto
Eq.2.3.Theratioofsamplenumbersofunirradiatedcontrolstoirradiatedsampleswas
inallexperimentsdescribedsmallerorequalto1:4.
Inthefollowingparagraphtheerrorestimationforsurvivalexperimentsispresented
withintwodifferentcasesandexplained.Inthefirstpart,theerrorestimationforcomplete
survivalcurvesispresentedwhileinthesecondpart,theerrorcalculationforisolatedsur-
vivalvaluesorexperiments,whicharedoneonceisshown.Theerrorcalculationforthe
survivalexperimentswithmultiplesampleshasbeencarriedoutasfollowed.Theaverage
survivalSofthesurvivalvaluesS1,S2...,Sncorrespondingtoonedosewascalculatedas
wellastheirstandarddeviationσSandtheirstandarderrorofthemeanSEMSinwhich
thenumberofsamplesisgivenbyn.
nS=1Si(2.4)
n=1i1n
σS=n−1i=1(Si−S)2(2.5)
SEMS=√σS(2.6)
nFromthelinear-quadraticfittothesurvivaldatatheparametersαandβweredetermined
withtheiraveragesαandβaswellastheirstandarddeviationsσαandσβ.
nσα=n−11(αi−α)2(2.7)
=1inσβ=n−11(βi−β)2(2.8)
=1iFortheradio-resistanceparameterα/βtherelativeerrorσα/βwasperformedaccordingto
.2.9Eq.31

methodsandMaterials2

Δα/β=σα/β=(σα)2+(σβ)2(2.9)
α/βαβTheerrorestimationforsurvivalexperimentswhichweredoneoncewasdeveloped[136].
Thesurvivalwithinthelinear-quadraticmodelisgivenbyEq.2.10,andEistheassociated
effectasdefinedinEq.2.11.

S=e−(αD+βD2)
E=αD+βD2

(2.10)(2.11)

Theerrorisontheonehandinuencedbytheuctuationofthedatapointsaroundthe
individualsurvivalcurve(ES)andontheotherhandchangedbythevariationoftheen-
sembleofthecurves(Eα,β).TheerrorcontributionsofEsumuptoΔEinEq.2.12.

ΔE=ΔES2+ΔE2α,β(2.12)
Thefirstfactorcontributingtotheerroriscausedbytheuctuationofsurvivallevels
aroundtheircorrespondinglinear-quadraticcurve.TheassociatederrorΔESiscalculated
asthestandarddeviationofthemeasuredeffectlevelsEiofunirradiatedsamples.
n1=1iΔES=n−1(Ei−E)2(2.13)
InsertingEq.2.11inthedefinitionofthestandarddeviationresultinginEq.2.14.
ΔEα,β=σα2D2−2σ2α,βD3+σβ2D4(2.14)
ThefactorsσαandσβweredeterminedaccordingtoEqs.2.7and2.8atwhichbeamand
energydependentmeanαandβvaluesweredeterminedtoαandβ.Forthecalculation
ofthecurveuctuationEα,βitwasassumedthatαandβareinverselycorrelated.The
valueswerejoinedinEq.2.15tocalculatethethirdparameterσα,β.

32

n1nσα,β=−1[(αi−α)(βi−β)]
−=1i

(2.15)

analysisdataStatistical2.7

Tpropagable2.2:ationThefortablex-raysshoandwstwtheoerrorcarbonionpercentageenerofgies.survivalforselecteddosescalculatedwitherror
CelllineDose[Gy]250kVpx-ray12C100MeV/u12C11.4MeV/u
RAT-109.39.39.3
9.410.210.6110.117.417.8314.664.141.3612.012.012.00IEC-612.014.813.3112.527.419.3315.362.632.86

TheupperandthelowererrorlimitsofthesurvivalwerecalculatedaccordingtoEqs.2.16
and2.17.Theerrorlimitshavealmostequaldistancestothemeansurvival.Henceforth,
theerrorintervalwasregardedtobesymmetricwithrespecttothemeansurvivalvalues.

S=e−(E±ΔE)

(2.16)

ΔS=Supper−Slower=e−(E−ΔE)−e−(E+ΔE)(2.17)
22Intable2.2theerrorpercentageofsurvivalforselecteddosesisshown.Bothcelllines
showinprinciplesimilarerrorvalues.Thevaluesincreasedwithincreasingdosesdueto
higheruncertaintiesandvariancesinthesurvivallevelstohigherdoses.Thisbiological
effectisexpressedbythetermσβ2D4inEq.2.14.Overall,theerrorsof11.4MeV/uirra-
diationaresmallerthantheotherirradiationerrorsincetheβcontainingtermsconverge
zero.to

RBEofCalculation2.7.2

Theeffectofdifferentbeamqualitiesoncellscanbecomparedwiththerelativebiological
effectiveness(RBE).TheRBEvaluecalculationforRAT-1cellsandIEC-6cellsdiffers
sincetheradio-resistanceofRAT-1cellsstaysstableandtheradio-resistanceofIEC-6
cellsagainstx-raysincreasesclearlywithcultivationtimeasdiscussedinsection4.2.
Thiscauseschangingαvaluesforx-rayirradiationandhigherdosesfor10%survival

33

methodsandMaterials2

levels.Itwasnotpossibletodetectthisbehavioralsoforcarbonionsirradiationdueto
thelimitedamountofdata.Inthefollowing,thecalculationoferrorsforbothcelllinesis
described.

cells-1TRAForeverysurvivalcurvetheparametersαandβaswellastheiraverageαandβwere
determinedfordifferentenergiesandbeams.Likewise,thestandarddeviationσαandσβ
werecalculated.TheRBEαwasdeterminedaccordingtoEq.1.5andtherelativeerrorof
RBEαwasdoneaccordingtoEq.2.18.

ΔRBEα=σRBEα=σα2+σα2(2.18)
RBEααionαx−ray
TheRBEfor10%survival(RBE10)wascalculatedaccordingtothesameformalismas
usedforRBEαwiththedosesfor10%survival.
σRBE10σD102σD102
ΔRBED10=RBE10=D10ion+D10x−ray(2.19)

cellsIEC-6Forcarbonionsurvivalcurvestheparameterαwasdeterminedwithitsmeansandstan-
darddeviationsasdescribedforRAT-1cells.Forx-raysurvivalcurvestheαvalueswere
plottedversuspassagenumberandfittedbyaline,seefigure2.8.Theerrorofαwassetto
bethe68%confidenceinterval.Atthemeanpassagenumberoftheperformedcarbonion
experiments(17.67)theαvaluewithitsdeviationwasevaluated(0.354±0.021).The
RBEfor10%survivalwascalculatedalongthelinesofRBEαwiththedosesfor10%
survival.ThedeterminedD10atpassagenumber17.7was5.0±0.13Gy.

2.7.3Analysisofchromosomesamples

Foreachchromosomesample300metaphaseswereanalyzedaccordingtotheirchromo-
somenumbers.Furthermore,themitoticindexin6000cellspersamplewascounted.
Fromthereceiveddataaweightedmeanmandaweightedstandarddeviationσm,for
chromosomenumbersormitoticindex,werecalculatedaccordingtothefollowingEqs.,
wherexisthedataandwtheweightswithiwi=1.
wim=xiwi(2.20)

34

(2.20)

analysisdataStatistical2.7

(a)αvalues(b)D10values
Figure2.8:αvaluesandD10ofIEC-6cellx-raysurvivalcurves.Theverticallineindicatesthe
meancircles:passageserumbatchnumberIIofperformedcarbonionexperiments.Closedcircles:serumbatchI,open

σm=2wxiwi−m2
i

(2.21)

35

Results3

3.1CharacterizationofratadenocarcinomacelllineR-3327-AT-1

TheadenocarcinomacelllineR-3327-AT-1(RAT-1)isafastgrowing,androgen-and
estrogen-insensitive,andanaplastictumoroftheDunningsystem([65],seesubsection1.3.1).
ForexperimentsRAT-1cellsfromthestockinP35weredefrostedandpassagedoncea
weekinRPMImediumwithphenolredaspHindicatorandthepassagewasnumbered
consecutively.ThepHoftheRPMImediumdecreasedbetweentwopassageswhichwas
opticallydetectedbyacoloralterationfromred-violettolightorangeofthepHindicator.
Figure3.1showsthecellsthreedaysandsixdaysafterseeding.Aconuentcelllayer
couldnotbeachievedsincethecellsstarttogrowinmultilayerandpileupincellclusters
at70%conuence.Aseparationofthecellswasnotpossibleafterthreedaysgrowthof
clusters.ThecelldoublingtimetDwasdeterminedingrowthcurvesandwascalculated
tobe21±2hintheexponentialphaseofthegrowthcurve.ThetDremainedstableover
thewholepassageperiod(seefigure3.1).
ThecellcycledistributionofRAT-1cellsinP38ispresentedinfigure3.2.Theex-

Figure3.1:Leftpanel:RAT-1cellsthreedaysandsixdaysafterseedingincultureask.Magni-
fication≈20xRightpanel:GrowthcurvesofRAT-1cellsincultureasks.Thepassagenumber
PandthedoublingtimetDisgivenforeachcurve.

perimentwasperformedinduplicatesoverfivedaysaftermockirradiationand250kVp
x-rayor11.4MeV/ucarbonionirradiationwhichreducedthecellsurvivalto10%.Inthe
firsttwelvehoursafterirradiationaG2-blockwasinducedintheirradiatedsamples.The
maximumoftheG2-blockisbetweenfiveandeighthours.Thex-rayandthecarbonion
irradiatedcellsaccumulateto41%inG2-phasewhileinthecontrolsampleonly29%of

37

3Results

Figure3.2:CellcycledistributionofRAT-1controlcellsandafter250kVpx-rayor11.4MeV/u
carbonionirradiationto10%survivallevelinP38.

thecellswereenrichedinG2-phase.Atlaterpointsintimetheirradiatedsampleswere
notdistinguishablefromthecontrolcells.Attheendoftheobservationperiod76%of
thecontrolcellsaccumulatedinG1-phasewhichindicatesalimitationofgrowthareaor
nutrientincultureaskandanaccompaningproliferationstop.57%ofthecarbonion
irradiatedcellsand61%ofthex-rayirradiatedcellswereinG1-phaseafter120hincul-
ture.Thisindicatesthattheirradiatedculturesdidnotreducetheirproliferationsincethe
cellnumbersofgrowingcellswerelowerandnospaceornutrientlimitationtookplace.
Tumorcellsoftenshowvariationinchromosomenumbers(aneuploidy)andgrowthprop-
erties.Toexcludepossiblelargeshiftsofthechromosomedistributionand/orachange
inthemitoticindex(MI)achromosomeanalysiswasperformedwithtwoindividualcell
batchesfromstockinP35overseveralpassages.Figure3.4showsthemetaphaseanalysis
ofbothcellbatcheswhereeachsmallpanelrepresents300metaphasesofonepassage.
Forillustrationfigure3.3showstwometaphasesofRAT-1cells.Therewasnodetectable
variationinthechromosomedistribution,andsmalldifferencesareduetostatisticalrea-

38

3.1CharacterizationofratadenocarcinomacelllineR-3327-AT-1

Figure3.3:MetaphasesofRAT-1cellswith62(left)and64(right)chromosomes,
450xmagnification≈

(a)RAT-1cellbatch1(b)RAT-1cellbatch2
Figure3.4:HistogramsofkaryotypedistributionpermetaphaseinRAT-1cellswithtimeincul-
ture.Everysmallpanelistheanalysisof300metaphases.

sons.Foreverypassageaweightedaveragechromosomenumberwascalculated.Out
oftheseaveragechromosomenumbersameanchromosomenumberforRAT-1cellswas
calculatedtobe58.9±9.5.Table3.1showstheaveragechromosomenumbersbesidethe
correspondingMI.TheMIforapassagewasdeterminedin6000cells,andaweighted
meanwascalculated.Asitcanbeseentheindexesdonotvaryessentiallyandthecalcu-
latedmeanMIwas11.7±1.1%.Moredetailsoncalculationscanbefoundinsection2.7.

39

Results3

58.859.957.956.857.4

WCNΔ

8.97.17.88.68.4

MI[%]

9.113.212.913.110.3

MIΔ

0.90.20.70.81.0

Table3.1:AnalysisoftwoindividualRAT-1cellbatchesindifferentpassages.Theweighted
chromosomenumber(WCN)withitsweightedstandarddeviation(ΔWCN)wascalculatedin
300cellsandthemitoticindex(MI)withitsweightedstandarddeviation(ΔMI)wasdetermined
cells.6000inPassageWCNΔWCNMI[%]ΔMI
1batchCell0.99.18.958.8P380.213.27.159.9P430.712.97.857.9P480.813.18.656.8P531.010.38.457.4P572batchCell3.215.511.462.3P391.36.010.656.4P400.711.15.260.0P420.513.314.560.4P441.312.012.258.8P46

40

62.356.460.060.458.8

11.410.65.214.512.2

15.56.011.113.312.0

3.21.30.70.51.3

3.2Characterizationofintestinalepithelialcellline6

3.2Characterizationofintestinalepithelialcellline6

Theintestinalepithelialcellline6(IEC-6)oftheratisacommonmodelfornormal
humanintestinalepithelialbiology[138](seesubsection1.4.1).Afterdefrostingthesub-
cultivationofICE-6cellswasdoneonceaweek.Figure3.5showsthecellsthreedays
andsevendaysafterseedinginculture.Thecellsunderliecontactinhibitionandstartto
dieanddetachafteronedaygrowthinaconuentcelllayer.
ForcelllinecharacterizationthedoublingtimetDwasderivedfromgrowthcurves.For
anexplanationofthemethodseesubsection2.1.5.Theresultinggraphsareplottedin
figure3.5andtDisgivenforeverypassage.Withtimeincultureandwithincreasing
passagenumbertDdecreasedfrom36hto17h.

Figure3.5:Leftpanel:IEC-6cellsthreedaysandsevendaysafterseedingincultureasks.
Magnification≈20x,Rightpanel:GrowthcurvesofIEC-6cellsincultureasks.Thepassage
numberPandthedoublingtimetDisgivenforeachcurve.

Thecelllinewasfurtherinvestigatedwithacellcycleanalysis.Ineverysecondpassage
(P16,P18,P20,P22,P24)cellsweremeasuredaccordingtosubsection2.4.1.Infig-
ure3.6thecellcycleanalysisoftwodifferentsamplesisshown.Theuppertwopanels
werepreparedwithcellsinP16whilethelowertwopanelswerecellsinP20.Itbecame
obviousthattheIEC-6cellculturedevelopedseveralsub-populationswithage.Thesub-
populationsseemnottohaveintegermultiplesofthenormalDNAcontent.Thepeak
analysiswasnotpossiblesincetoomanysub-populationswerepresentandtheirpeaks
erlapped.voSimultaneouslythecellcycledistributionofIEC-6cellsinP17wasanalyzedindupli-
catesoverfivedaysaftermockirradiationand250kVpx-rayirradiationwhichreduced
thesurvivalrateto10%(seefigure3.7).InthefirsttwelvehoursafterirradiationaG2-
blockisinducedintheIEC-6cells.ThemaximumoftheG2-blockoccurredafternine
hoursatwhich38%oftheirradiatedcellsaccumulatedcomparedto22%inthecontrol

41

Results3

Figure3.6:CellcycledistributionofIEC-6cellsinP16andP20

cells.Atlaterpojntsintimethex-rayirradiatedsampleswerenotdistinguishablefrom
thecontrolcells.Attheendoftheobservationperiod70%ofthecontrolcellscompared
to60%ofthex-rayirradiatedcellsaccumulatedinG1-phaseduetolimitationofgrowth
areaornutrientincultureaskandanaccompaningproliferationstop.Furtherinvestiga-
tionstoanalyzethecellcyclewerenotmadebecauseofthedevelopingsub-populations
problems.analysisconcomitanttheandThecellcycleanalysisoverseveralpassagesleadstothequestionsofchromosomesta-
bilityandsenescenceoftheIEC-6cellline.ForidentificationofsenescentcellsaX-Gal
stainingwasperformedasdescribedinsubsection2.1.7.Insub-conuentIEC-6cellsam-
plesnopositiveX-GalstainingwasfoundfromP16toP24.Inareasofconuentgrowth
alightgreenstainingwaspartiallydetected.Theweakpositivesignalwasprobablyin-
ducedbycontactinhibitionofthecellsandwasnotscored.
Achromosomeanalysiswasperformedtoidentifypossiblesub-populationsandtoen-
surethattherewerenocrosscontaminationswithotherspeciesinthecellline.Figure3.8
showstwoexamplesofIEC-6cellmetaphases.Infigure3.9thechromosomeanalysis
oftwoindividualIEC-6cellbatchesoverseveralpassagesisshown.Eachsmallpanel
representstheanalysisof300metaphases.Thenormalkaryotypeofratcells(rattus
norvegicus)is2n=42.Ameanchromosomenumberof42couldonlybecountedinvery
earlypassages(P15toP18).Atlaterpointsintimetheweightedchromosomenumber
(WCN)increaseddramaticallybutdifferentlyinthetwocellbatches.Table3.2shows
indetailthechangingprogressionofcellswith42chromosomes.Inbothcellbatches
thepercentageofcellswith42chromosomesdecreasedrapidlyandtheamountofcells
withmorethan42chromosomesincreaseddramaticallyuntilnearlyallcellsshowedan
abnormalkaryotype.AfterP20asub-populationwith53chromosomesappearedinboth

42

3.2Characterizationofintestinalepithelialcellline6

Figure3.7:CellcycledistributionofIEC-6controlcellsandafter250kVpx-rayirradiationto
10%survivallevelinP17,duplicates

Figure3.8:MetaphasesofIEC-6cellswith42(left)and54(right)chromosomes,
700xmagnification≈

batcheswhichmightindicatethatthiskaryotypehasagrowthadvantage.Tetraploidcells
developedinbothbatchesbutwithalternatingfrequency.Duetotheweeklypassage,a
cellselectionprocesstakesplacewhichcouldinducethesestatisticaluctuations.The
celllineseemstohavethetendencytodeveloptetraploidcells.Table3.2showsthecor-
respondingmitoticindexes(MI)fortheanalyzedchromosomesampleswhichincreased
slowlywithculturetimeinparalleltotheabnormalkaryotypes.Theseresultsshowage-
neticalterationoftheIEC-6celllinewhichmighthaveinuencesontheradio-resistance.

43

Results3

WCN

Δ%NWCN=42

%N<42

%N>42

Tableweighted3.2:Analysischromosomeoftwonumbersindividual(WCN)IEC-6withcellregardbatchestoaccordingmetaphasestowiththeir42mitoticchromosomindex(MI)es.Fandor
everypassagetheMIwithitsstandarddeviation(ΔMI)wasdeterminedin6000cellsandthe
threeWCNwithcolumnsitsnameweightedthestandardpercentagedeofviation(metaphasesΔWCN)withwas42,lesscalculatedormorein300than42IEC-6cells.chromosomesThelastat
300.isNwhichPassageMI[%]ΔMIWCNΔWCN%N%N%N
>42<42=421batchCellP156.50.842.26.937.732.330.0
P1911.40.547.411.415.023.761.3
P268.70.846.97.23.312.384.3
P3014.70.547.43.12.34.793.0
P3418.30.749.63.71.02.097.0
2batchCellP166.51.141.83.329.342.328.3
P187.30.442.15.426.044.030.0
P2010.40.245.76.717.723.758.7
P2215.70.649.46.94.36.389.3
P2410.90.463.620.00.72.097.3
P2614.80.468.320.42.71.795.7

44

0.80.50.80.50.7

1.10.40.20.60.40.4

42.247.446.947.449.6

41.842.145.749.463.668.3

6.911.47.23.13.7

3.35.46.76.920.020.4

37.715.03.32.31.0

29.326.017.74.30.72.7

32.323.712.34.72.0

42.344.023.76.32.01.7

30.061.384.393.097.0

28.330.058.789.397.395.7

xperimentseSurvival3.3

(a)IEC-6cellbatch1(b)IEC-6cellbatch2
Figure3.9:HistogramsofkaryotypedistributioninmetaphasesofIEC-6cellswithtimeinculture
indicatedbypassagenumber.Eachsmallpanelistheresultof300analyzedmetaphases.

3.3Survivalexperiments

3.3.1RAT-1cells:radio-resistanceagainst250kVpx-rays

Todeterminetheradio-resistanceofRAT-1cellsagainst250kVpx-rayirradiationthe
cellswereseededincultureasksthreedayspriortotheexperiment,irradiatedwith
dosesbetween0to10Gy,andreseededincultureaskswithexpected100surviving
cellsforacolonyformingassay.Figure3.10showsethanolfixedandmethyleneblue
stainedcoloniesafterelevendaysofgrowth.ThemorphologyofRAT-1coloniesdiffers
evenincontrolsamplesbutinallcasesthecellsshowedanintensebluecolor.Thecell
survivalratewascalculatedasdescribedundersubsection2.1.6andsemi-logarithmically
plottedagainstthedose.Figure3.11showsthecumulativex-raycurve(closedcircles)of
sevenindividualexperiments.Themeanαvaluewascalculatedtobe0.174±0.052Gy-1
whilethmeanβvaluewas0.026±0.010Gy-2ofthecumulativecurve.Theresulting
α/βratiowas6.8±0.5Gywhichcharacterizesthecelllineasmoderatelyradio-resistant.
Themeandosefor10%survivalwas7.0±1.4Gy.
ThecolonyformingassayforRAT-1cellscontainedacentrifugationstepbeforethecells
werereseeded(seesubsection2.1.6).Infigure3.11oneexperimentwhichwasdone
withoutcentrifugationisshown(curvewithopencircle).Thecomparisonofthetwo
curvesshowsthatthecellsurvivalratedecreasedstepplyforhighdoseswhenthecells
werenotcentrifuged.Thiscanbeexplainedbytheinuenceofthetrypsininthecell

45

Results3

Figure3.10:RAT-1cellcolonies:ethanolfixatedandmethylenebluestained,magnification≈
10-20x

Figurprotocole3.11:withSurvicentrifugvalcurvation,esofn=7,250openkVpcircle:x-rayexperimentirradiatedRATprotocol-1cell.withoutClosedcentrifugcircle:eation,xperimentn=1

suspension.Forhighirradiatedcellsagreaterinoculumvolumeisneededtoseed100
expectedsurvivingcellscomparedtolowerirradiatedcellsamples.Inconsequence,a
higherconcentrationoftrypsinisretainedinthemediumforthecolonyformingassay.
Thehighertrypsincontentleadtoadditionalcelldeath.Therefore,acentrifugationstep
wasneededtoremovethetrypsinfromthecellsuspensionandtodetectonlythereduced
survivalcausedbyirradiation.

3.3.2RAT-1cells:radio-resistanceagainstcarbonions

ForirradiationatSISfacilitythecellswereseededincultureaskswhileforirradiationat
UNILACfacilityPetridisheswereusedforcellseeding.Thecellpreparationafterirradi-
ationwascarriedoutsimilarlytox-rayexperiments.Figure3.12showsthecellsurvival
curvesoffourindividualexperimentsafterirradiationwithcarbonionsof270MeV/u,
100MeV/u,and11.4MeV/u,respectively.Table3.3showsthecorrespondingfitpa-

46

Survival3.3xperimentse

Table3.3:RAT-1cells:αandβvaluesaswellasratioα/βandD10ofcarbonionsurvivalcurves.
Thevaluesareaveragesfromfourindividualexperimentswithstandarddeviation.
Energyα-1Δα-1β-2Δβ-2α/βΔα/βD10ΔD10
[Gy][Gy][Gy][Gy][Gy][Gy][Gy][Gy]
270MeV/u0.1970.0870.0310.0116.40.66.020.46
100MeV/u0.2920.0690.0340.0068.60.34.990.30
11.4MeV/u1.2370.0771.870.12

rametersofthelinearquadraticmodel.ThemeanPEofRAT-1cellsweredeterminedto
be0.446withitsstandarddeviationof0.113anditsSEMof0.026outofallirradiation
(n=19).xperimentse

Figure3.12:SurvivalcurvesofRAT-1cellsaftercarbonionirradiationwithdifferentenergies:
270MeV/u(black),100MeV/u(red),and11.4MeV/u(green).Themeansurvivalvalueswith
theirstandarddeviationaregiven,n=4.

3.3.3IEC-6cells:radio-resistanceagainst250kVpx-rays

Todeterminetheradio-resistanceofIEC-6cellsagainst250kVpx-rayirradiationthe
cellswereseededthreedaysbeforetheexperimentin25cm2cultureasks,wereirra-
diatedwithdosesbetween0to10Gy,andreseededincultureaskswithexpected70
survivingcellsinacolonyformingassay.Afterelevendaysofgrowththecellswere
fixedwithethanol,methylenebluestained,andthecolonieswerecountedmanually.The
IEC-6cellcoloniesshowedauniformmorphologyandwereweaklybluecolored(see
figure3.13).Figure3.14showsthreeandfourindependentx-raycurves,respectively,of
twodifferentIEC-6cellbatches(A1,A2).IntheleftpanelIEC-6cellbatchA1wasirra-
diatedinP19,P25,andP28andintherightpaneltheIEC-6cellbatchA2wasirradiated

47

Results3

Figure3.13:ColonyofIEC-6cells:ethanolfixatedandmethylenebluestained,
20xmagnification≈

(a)IEC-6cellbatchA1(b)IEC-6cellbatchA2
Figure3.14:SurvivalcurveofIEC-6cellsirradiatedwith250kVpx-rays

inP16,P20,P22,andP24.CellbatchA2increasedtheradio-resistancewithongoingpas-
sagenumber,whilecellbatchA1underwentnochangesinradio-resistance.Becauseof
thesystematicshift,anaverageαandβvaluesoverallperformedirradiationexperiments
wasnotcalculated.Table3.4showstheαandβvaluesoftheindividualexperiments.The
αandβvaluesofcellbatchA2donotreecttheincreasingradio-resistance.Duringthe
experimentalphaseofthisworkthecellcultureserumwasconsumedsothatnewserum
batcheshadtobetestedforsuitability.Inthefollowingtheserumbatcheswillbenamed
asI,II,andIIIinwhichserumIistheconsumedserumandserumIIthenewstandard
serum.TotesttheseruminuencetheIEC-6cellbatchA6wassplitinthreeparts,and
everypartwascultivatedeightweeksinculturemediumwithaspecificserum.Tocontrol
theserumeffectonthePE,survivalandthecolonygrowth,anx-raysurvivalcurvewas
performedeverysecondweek.Figure3.15showstheresultingcurves.InserumIthe
IEC-6cellbatchA6didnotdevelopanincreasingradio-resistancewithtime.Inserum
IItheIEC-6cellsdevelopedapronouncedradio-resistancewhileinserumIIItheIEC-6
cellsincreasedtheirradio-resistanceslightly.Interestingly,thecellsinserumIIIdidnot

48

ISerum(a)

3.3xperimentseSurvival

IISerum(b)

IIISerum(c)Figure3.15:Serumtest:250kVpx-raysurvivalcurvesofIEC-6cellbatchA6.Thepassageand
theexperimentswereperformedinculturemediumwiththreedifferentserums.

showshouldercurves.Table3.4presentsthefitparametersforthex-raysurvivalcurves.
Inconclusion,IEC-6cellscan,butdonotnecessarily,developanincreasingradio-resistance
withage.Apossiblereasonforthisvarianceisthecellselectionintheweeklypassage
ypothesis).h(test

3.3.4IEC-6cells:radio-resistanceagainstcarbonions

ForcarbonionirradiationatSISfacilitytheIEC-6cellswereseededincultureasks,
whileforirradiationatUNILAfacilityPetridisheswereusedforcellculturing.After
irradiationthecellsweretrypsinizedandreseededinacolonyformingassaywith70
expectedsurvivingcells.Theethanol-fixedandmethyleneblue-stainedcolonieswere
countedandanalyzedaccordingtosubsection2.1.6.Figure3.16showstheaveragesur-
vivalcurvesoverfourindividualexperimentsforthreedifferentcarbonionenergies11.4
MeV/u,100MeV/u,and270MeV/u,each.Thecurveswereperformedintwobeam

49

Results3

Table3.4:Thetablepresentstheαandβvaluesaswellasratioα/βandD10ofIEC-6cellx-ray
survivalcurvesindependencyofcellbatchandserumbatch.
CellbatchSerumPPEα-1Δα-1β-2Δβ-2α/βΔα/βD10
batch[Gy][Gy][Gy][Gy][Gy][Gy][Gy]
A1I190.3600.2600.0470.0290.0109.00.45.5
I250.4810.2780.0480.0310.0059.00.25.2
I280.5840.3250.0330.0210.00315.50.25.3
A2I160.2450.3380.0420.0250.00513.50.25.0
I180.3350.4980.0460.0010.00549.829.15.1
I200.4510.2460.0260.0300.0038.20.15.6
I220.5210.3710.0460.0130.00528.50.45.2
I230.6160.3410.0290.0140.00323.70.25.5
I240.6270.2600.0330.0190.00313.70.26.1
A6I160.2480.3450.0510.0190.00518.20.35.2
I180.3910.3240.0740.0210.00815.40.45.3
I200.4140.3120.0330.0260.00312.00.25.2
I220.2730.3880.0520.0130.00529.80.45.1
A6II160.2070.4190.0940.0300.01114.00.44.2
II180.3480.3150.1020.0300.01210.50.55.0
II200.4330.3500.0320.0230.00415.20.25.0
II220.2310.3440.1270.0080.01543.01.95.9
A6III160.2770.6250.1040.0020.012312.56.03.6
III180.3570.3610.0510.0190.00619.00.35.0
III200.4630.5300.0900.0020.011265.05.54.3
III220.2560.5100.0630.0070.00772.91.04.3

A6A6A6

50

xperimentseSurvival3.3

Figure3.16:SurvivalofIEC-6cellsaftertracksegmentirradiationwith11.4MeV/u(green),
100MeV/u(red),and270MeV/u(black)carbonions.Thecurvesaretheaverageoverfour
individualexperimentswithstandarddeviation,each.

Table3.5:IEC-6cells:αandβvaluesaswellasratioα/βandD10ofcarbonionsurvivalcurves.
Thevaluesareaveragesfromfourindependentexperimentswithstandarddeviation.
Energyα-1Δα-1β-2Δβ-2α/βΔα/βD10ΔD10
[Gy][Gy][Gy][Gy][Gy][Gy][Gy][Gy]
270MeV/u0.4390.0380.0080.00854.91.04.950.74
100MeV/u0.5510.1440.0070.04078.75.74.191.14
11.4MeV/u1.8040.0641.250.09

times.Thevariancesbetweencurvesofonebeamtimewereverysmallbutthediffer-
encesbetweenthebeamtimeswereonlyslightlymorepronounced.Thedifferencesare
significantathigherdoseswherethespreadofcolonynumberspercultureasksvaries
essentially.IndependentoftheirradiationmodalityineverycolonyformingassayaPE
hastobedetermined.ThecalculatedPEoffittedIEC-6cellsurvivalcurvesispresented
infigure3.17.ThebluepointsdisplaythePEwhichwasdeterminedinculturemedium
withserumI,theredpointsinmediumwithserumII,andthegreenpointsinmedium
withserumIII.Fromthescatteredplot,astronginuenceoftheserumonthePEcan
notbededuced.InalltestedserumstheIEC-6cellsincreasedtheirPEwiththeweekly
passage.ThiseffectseemstobelimitedsincethePEpassesintoaplateau.Acurvewas
notplottedduetoinsufficientdatawithhigherpassagenumbers.

51

Results3

Figure3.17:ThePEofIEC-6cellsdeterminedinculturemediumwithserumbatchI(bluepoints),
serumbatchII(redpoints),andserumbatchIII(greenpoints)isplottedagainstthepassagenum-
ber,whichweredoneweakly.

3.3.5Relativebiologicaleffectiveness(RBE)

TheRBEwascalculatedaccordingtosection1.2andsection2.7.Infigure3.18theRBEα
andtheRBE10forIEC-6cellsandRAT-1cellsareplottedagainsttheLEToftheparticle
beamoragainsttheresidualrangeinwaterofthecarbonions.Thephysicalparameters
ofthecarbonbeamaregivenintable3.6.TheRBEincreaseswithincreasingLETand
decreasingresidualrangeofthecarbonions.Forhighenergy,representingtheentrance
channel,theRBEisclosetoonewhichmeansthatthenormal,tumorsurroundingtissue
isnotfurtherdamagedthroughtheuseofcarbonionsincomparisontophotonirradiation.
TheRBEαofRAT-1cellsincreasedmorepronouncedthanthatofIEC-6cellswhichis
duetothesteepsurvivalcurveafter11.4MeV/ucarbonionirradiation.Thisindicates
thatcarbonionsarewellsuitedfortheinactivationoftheprostatecancercellline.

theFigurLETe3.18:andinRBEtheαandrightRBEpanel10agofainstIEC-6thecellsresidualandRATrange-1incellswareaterofplottedtheincarbontheleftions.panelTheagRBEainst
v(RAaluesT-1arecells)andcalculatedsixteenoutof(IEC-6theavcells)eragesovindependenterfourx-rayeindependentxperiments,carbonerespectivxperimentsely.andseven

52

3.4Identificationofsub-populationsandclonesinIEC-6cellculture

Table3.6:Thetabledisplaysthephysicalbeamparametersoftheusedcarbonionbeams.
Energy[MeV/u]LET[keV/µm]ResidualrangeinCorrespondenceto
applicationclinical[mm]waterchannelentrance137132701002819.8closetotumor
11.41700.41tumorvolume

3.4Identificationofsub-populationsandclonesinIEC-6cellculture

3.4.1ChromosomeanalysiswithmFISH

Thechromosomeanalysisinsection3.2showsthattheIEC-6cellsdevelopananeuploid
karyotypewithinafewweeksinculture.Toidentifytheadditionalchromosomesachro-
mosomeanalysiswithmFISHwasdone(seesubsection2.2.1).AcompletemFISHkitfor
allchromosomesisonlycommerciallyavailableforhumanandmousesamples.Sincethe
geneticrelationshipiscloseandthechromosomeprofileofmice(2n=40)ismoresimilar
torats,amousemFISHwasusedonratchromosomes.Forthechromosomepreparation
IEC-6cellsinP22wereusedtoachieveawidespectrumofkaryotypes.Itwasexpected
tofindsectionsofIEC-6cellchromosomesstainedlikebandsinwhichthechromosomes
oftheratandthemousematch.ThestainingwithTR,SpO,andDEACwentwell,the
hybridizationwithFITC-linkedprobeswasdiminishedandnearlynolabelingwithCy5
wasfound.Inthefirstanalysisstepthechromosomesweremanuallyarrangedalong
theirshapeandsizeintheDAPIimage.Basedonthefalsecolors,thearrangementwas
correctedinasecondstep.Figure3.19showsamFISHstainedmetaphasewith52chro-
mosomeswhichispresentedintheleftpanelinDAPIcounterstainingandintheright
panelinfalsecolors.Theredcircledemonstratesthatchromosomeswhichareclearlyof
thesamesizeandshapeinDAPIstainingwerenotnecessarilydisplayedinthesamefalse
colors.Incontrast,thegreencircleshowsthatchromosomesillustratedwiththesame
falsecolorsdonotmatchinsizeandshape.ItbecomesobviousthatamousemFISHis
notsuitablefortheanalysisofratchromosomes.DuetomissingmFISHalternativesor
otherhighthroughputmethodstheidentificationoftheadditionalchromosomeswasnot
up.wedfollo

53

Results3

(b)Metaphasedisplayedinfalsecolors

(a)MetaphasedisplayedinDAPIcounterstain-(b)Metaphasedisplayedinfalsecolors
ingFigure3.19:ApplicationofmousemFISHonIEC-6cellsinP22.Shownisametaphasewith52
700xMagnificationchromosomes.≈

3.4.2Existenceofradio-resistantIEC-6sub-populations

Thecombinationofincreasingradio-resistanceandPE,karyotypechangesbutdecreasing
doublingtimesuggeststhatadominantsub-populationofIEC-6cellsisresponsiblefor
thesedevelopments.Therefore,theIEC-6cellbatchA3wassub-dividedatP18inthree
parts.Onepartwasmockirradiated,onepartwasirradiatedwith5Gyofx-rays,andthe
lastpartwasirradiatedwith1.25Gyof11.4MeV/uofstoppingcarbonions.Theirradia-
tiondidnotonlyreducethecellsurvivalrateto10%butalsoshoulddeactivateespecially
thepossibleradio-sensitivesub-population.Tworadiationqualitiesweretestedbecause
ofthedifferentdamagecharacterswhichmightgeneratedotherclones.Afterirradiation,
theIEC-6cellsweretrypsiniziedandreseededincultureaskswithagrowthareaof75
cm2.Intheweeklypassagethetotalcellnumberwasdetermined,addedonthetotalcell
numberoftheweekbefore,andfittedagainstthepassagenumberafterirradiation(see
figure3.20).Theplotallowsacomparisonofthegrowthratesandreplacesthetimecon-
suminggrowthcurves.Throughtheirradiationnochangesinthegrowthcharacteristics
oftheIEC-6cellcultureswereobserved.Thelowercellnumberinthecontrolsamples
at21daysand28daysafterirradiationmightbeamistakeinthecalculationoftotalcell
numbersincelaterpointsintimeshowagoodagreementwiththex-rayandthecarbon
ionirradiatedcellsamples.Thediminishedslopofthecurvesafter40daysisnotdue
toalimitedgrowthareasincethecellsampleswerereseededweekly.Itmightbethat
theIEC-6cellbatchA3underwentdifferentiationorsenescenceandsloweddownits
time.doubling

Everysecondweekachromosomepreparationofallcultureswasdonetoidentifypossible

54

3.4Identificationofsub-populationsandclonesinIEC-6cellculture

Figure3.20:Thetotalcellnumberofmock,250kVpx-ray,and11.4MeV/ucarbonionirradiated
IEC-6cellsplottedagainstthetimeindaysafterirradiation.

developingsub-populationsintheirradiatedsamplescomparedtothecontrolcultures.
Figure3.21presentsthekaryotypesof300analyzedmetaphasesforpassageandsample.
Independentoftheradiationqualitythereductionto10%survivingcellsdidnotinduce
theformationofdifferentsub-populationscomparedtocontrolcells.Inallcasestwo
sub-populationswithsimilarmeanchromosomenumbersdevelopedandtheamountof
tetraploidcellswaslow.Thesmallvariationsbetweenthecasesareduetostatistical
reasons.

Infigure3.22theweightedchromosomenumber(WCN)andthemitoticindex(MI)are
givenforeachpassageandsampleatwhichthedatacorrespondstothechromosomeanal-
ysisinfigure3.21.AsexpectedfromthechromosomehistogramstheWCNincreasedall
threesampleswithcultivationtime.InparalleltheMIincreasedwithongoingpassage
numbersaswell.TheslightdecreaseorstagnationofMIinP26/P8mightbeduetore-
ducedcellgrowthwhichwasdetectedinfigure3.20ofthetotalcellnumbers.Inaddition,
figure3.22showsanoverviewovertheexistenceofIEC-6cellswiththenormal(rattus
norvegicus)karyotypeof42,morethan42orlessthan42chromosomes.Thenumber
ofcellswiththenormalkaryotype(2n=42)decreasedwithongoingpassagenumbers.
Thisprocessseemstobeslowerinunirradiatedcontrolcellscomparedtoirradiatedcells,
whereasnodifferencesbetweenx-rayorcarbonionirradiationcanbedetected.Atthe
endofthecultivationperiod,inallthreeculturesthepercentageofcellswith42chro-
mosomeswasbelow5%.TheIEC-6cellbatchA3seemedtoundergodifferentiationor
senescence.Butwiththeseresultstheobservedincreasingradio-resistanceincellbatch
A2andA6cannotbeexplained.

55

3

Results

eFigur

carbon

3.21:

Karyotype

histograms

of

IEC-6

control

cells

ionirradiation.Analysiswasperformedatseveral

realpassageandthesecondPmeansthepassageafter

of

300

56

analyzed

metaphases/

passages

and

samples.

and

after

passages

irradiation.

250

kVp

whereas

eryEv

x-ray

or

firstthe

small

panel

11.4

MeV/u

themeansP

is

the

result

3.4Identificationofsub-populationsandclonesinIEC-6cellculture

(a)WCNandMI[%](b)42,<42or>42chromosomes
Figure3.22:Leftpanel:WCN(square)andMI(circle)ofIEC-6cells.Controlcells,x-rayand
carbonionirradiatedsamplesinpassageP19/P1,P20/P2,P22/P4,P24/P6,andP26/P8.WCNwas
calculatedin300IEC-6cellsandMIwasdeterminedin6000IEC-6cells.Bothvaluesaregiven
withtheirstandarddeviation.Rightpanel:ProgressionofIEC-6controlcells,x-rayandcarbon
ionirradiatedcellswith42,lessthan42,andmorethan42chromosomes.

3.4.3AnalysisofIEC-6singlecellcolonies

TheanalysisofIEC-6cellsexposedtox-raysandcarbonionswhichreducedthesur-
vivalrateto10%couldnotclarifywhetherthereareradio-resistantsub-populationsor
not.TheisolationandanalysisofIEC-6clonesshouldanswerthequestionifthereare
singlecloneswithahigherradio-resistance.ThecloneswereisolatedinP23(cellbatch
A3)andP17(cellbatchA4)andwerecharacterizedfortheirkaryotypeandmitoticindex.
FromcellbatchA3itwaspossibletoisolateandpropagate16clonesfromwhichtwelve
cloneshad53chromosomes,oneclonewith44,45,54,orstable91chromosomes,re-
spectively.FromIEC-6cellbatchA4tenclonescouldbeculturedwhereasfourclones
with42chromosomes,fourcloneswith43chromosomes,andoneclonewith45or88,
respectively,wereisolated.TheIEC-6cultureageaccompaniedbythechangesinchro-
mosomeprofilewasthecrucialfactorforthedifferentkaryotypesoftheisolatedclones.
Sixcloneswereanalyzedovereightweeksconcerningtheirkaryotype,mitoticindex,PE,
radio-resistance,andgrowthrate.Anoverviewovertheisolationcharacteristicsofthe
analyzedclonesisgivenintable3.7.

Fromthetotalcellnumbersperpassageadoublingtimewasdetermined.Figure3.23
clearlyshowsthatanincreasedchromosomenumberleadstoafastercelldoubling.But
cloneswithidenticalchromosomenumbersandespeciallycloneswithnormalkaryotype
didnotshowsimilarcelldoublingtimes.
X-raysurvivalcurvewasperformedineverysecondweektodeterminethePEandthe
radio-resistanceandwereplottedinfigure3.24.Fornoneoftheclonesachangingradio-

57

Results3

Table3.7:OverviewoveranalyzedIEC-6cellclones.
ClonecodeCloneNo.KaryotypeMI[%]
8.024511B35.565321C3423IB311.743.62424ID314.095352A39.434464B2

Figure3.23:CellgrowthofsixIEC-6clones:Ineverypassage4x105cellswereseeded.The
totalcellnumberoftheharvestcellsweredeterminedandaddedonthetotalcellnumberofthe
weekbefore.Theslopofthelinearfitcouldbeconsideredtobeacelldoublingtime.

sensitivitywithageisdetectableoutofthesurvivalcurveprogression.Bycomparing
thedosesfor10%survivalaslightlydecreasingradio-resistanceforclone2andaslowly
increasingradio-resistanceforclone1,5and6weredetermined.TheD10stayedrather
constantforclone3and4.Thesmallchangesinradio-resistancewerenotrecognizablein
αandβvalues.ThePEofclone1-4deviatedwhilethatofclone5strictlyincreasedfrom
44%to59%infourpassages.Clone6showednochangeinPE.Thedataaresummarized
.3.8tablein

Inparalleltothex-raycurves,chromosomepreparationsweredonetocontrolthestabil-
ityofthekaryotype,themitoticindex,andtobringpossiblechangesinradio-resistance
intocontext.Comparedtoisolationcharacteristicsoftheanalyzedclones,presentedin
table3.7,escalatedtheMIofclone2from5.6%to12.0%butduringtheperiodofin-
vestigationtheMIwasstable.ThehugedifferencesintheMIvaluesindicatethatat
thecharacterizationpointthecellcultureofclone2wasnotintheexponentialphaseof
growth,andtheproliferationwasreduced.TheMIofclone3stayedstable,whilethe

58

3.4Identificationofsub-populationsandclonesinIEC-6cellculture

Figure3.24:IEC-6clones:survivalcurvesafter250kVpx-rayirradiation.Thepassagenumber
indicatestherealpassageaswellasthepassageafterisolationandisvalidforbothpanelsina
.wro

aTvableerages3.8:withIEC-6theirclones:standardPE,deα/βviationratio,asandD10percentage.of250ClonekVp1-4:x-rayn=3,survivclonealcurv5+6:es.n=4Thevaluesare

ClonePE[%]ΔPE[%]α/β[Gy]Δα/β[%]D10
125.952.590.7168.44.8
242.142.213.687.44.4
327.846.431.5110.84.3
427.219.027.1116.34.9
550.612.99.257.06.4
639.33.919.778.06.3

[%]DΔ1019.928.319.12.36.09.8

59

Results3

Tcellsablewith3.9:itsIEC-6standardclones:deviationanalysisasaofpercentagekaryotype.(ΔTheMI[%])mitoticandindethex(MI)weightedwaschromosomedeterminedinnumber6000
(WCN)wascalculatedin150cellswithitsstandarddeviationasapercentage(ΔWCN[%]).
Clone1-4:n=3,clone5+6:n=4
CloneMI[%]ΔMI[%]WCNΔWCN[%]
0.946.024.710.513.352.26.612.022.644.29.910.635.845.620.98.440.955.118.610.850.445.739.18.16

WCN[%]MIΔ46.024.752.26.644.29.945.620.955.118.645.739.1

[%]WCNΔ0.93.32.65.80.90.4

MIofclone1and4increased.Clone5and6wereisolatedwitharelativelyhighMIbut
directlyafterdefrostingtheMIwasdramaticallyreducedto8.5%and4.0%.Withongo-
ingpassagesbothclonesreachedtheMIoftheisolation.Itispossiblethatthegrowth
wasrestrictedbyrecoveryfromthefrostingprocess.TheMIcouldbedeterminedinall
sampleswithadeviationof8.6%.
Fortheanalysis,onlycloneswereusedwhichhadstablechromosomenumbersatthe
characterizationpointsothatthedeterminedvaluescouldbeconsideredas100%values.
Aftersixweeksinculturethekaryotypeofallclonesexceptclone2hadchanged.Only
clone2kepttheoriginalchromosomeprofilewithslightlylowerchromosomenumbers.
Thiscouldbeduetosamplepreparation.Theotherclonesincreasedtheirchromosome
numbers.Inconclusion,themajorityofIEC-6cellstendedtoincreasetheirchromosome
numbersandpopulationdoublingtimes.ThecellschangetheMIandPE,also.

eCo-cultur3.5

Clonogenicsurvivalassayswithco-culturedIEC-6cellsandRAT-1cellsweregrown
togetherinonecultureask.Thecolonyformingassayprotocolforbothcelllinesis
identicalsothatnochangeshadtobedone.Thediscriminationbetweenthetwocell
typescausesnoproblemssincecellandcolonymorphologydiffersessentiallyaswell
asthestainingpropertieswithmethyleneblue.Inthemajorityofcasesthecolonies
havenodirectcelltocellcontactinthecultureask.Theco-culturecolonyexamplesin
figure3.25showthatacelllinedifferentiationwasstillpossiblewhencoloniesoverlap
orintermingle.RAT-1cellcolonieswerenotaffectedbyco-culturingwhilethecolony

60

eCo-cultur3.5

Figure3.25:Co-culturecolonies:inallpanelsareIEC-6cellsontheleftsideandRAT-1cellson
therightside,ethanolfixatedandmethylenebluestained,magnification≈10x

Table3.10:Thetablepresentsthenumberofperformedexperimentswhichwerecarriedoutwith
methodPandmethodTaswellasserumIandserumII.Ifnotfurtherspecified,thecellswere
irradiatedwith1,3or6Gy(RBE)of250kVpx-raysorcarbonions(100MeV/uor11.4MeV/u).
MethodSerum250kVpx-rays100MeV/u12C11.4MeV/u12C
batch005IPTI2,(only3Gy)2,(only3Gy)2,(only3Gy)
115IIT

sizeofIEC-6cellsincreased,especiallyintheunirradiatedcontrols,asfoundbyvisual
xamination.e

3.5.1Survivalexperiments

Inthefollowingparagraph,thequestionshouldbeansweredifco-cultureconditions
changetheradio-sensitivityoftheusedcelllinesRAT-1andIEC-6.Thesurvivalex-
perimentswereperformedwithtwodifferentset-ups(methodPandmethodT,seesub-
section2.1.3)andtwofetalcalfserumbatches(serumIandserumII).Table3.10shows
anoverviewoftheperformedexperiments.Independentexperimentsweresummarized
whenserumandmethodsareidentical.Ifonlyoneexperimentwasperformed,anerror
calculationwasdoneasdescribedundersubsection2.7.1andthecolumnsweremarked
withacross.Inthefollowingallexperimentaldataareplottedasapercentagetothe
PE.eragevacorresponding

Ingeneral,theco-culturesurvivaldatadidnotshowreproducibleresultsformethodP
andmethodTaswellasforserumIandserumII.Inaddition,methodPseemstobemore

61

Results3

Figure3.26:Co-cultureserumI+II:0GyIEC-6cells+irradiatedRAT-1cells.
Strippedcolumns:methodP,fullcolumns:methodT,cross:errorcalculation,nomark:mean
valueswithstandarddeviation,ontop:PE:red,250kVpx-ray:blue,12C100MeV/u:cyan,12C
11.4MeV/u:green,onbottom:PE:black,250kVpx-ray:orange,12C100MeV/u:pink,12C
violetMeV/u:11.4

subjectedtoerrorsandtheresultsarethereforequestionable.Overall,RAT-1cellsmight
notbeasmuchinuencedbyco-culturingastheIEC-6cellsare.Nevertheless,forsome
co-culturecombinationsatrendcouldbeextracted.First,independentoftheserumthe
PEcoforIEC-6cellsisabove100%.ThiseffectwasespeciallynoticeablewithmethodT.
WithmethodPtheeffectwasnotsopronouncedbutstillhigherthanthePEmono.ThePEco
forRAT-1cellsisnotinuencedbytheco-culturewithunirradiatedIEC-6cells.ThePEco
isaroundorbelow100%,independentoftheserumorthemethod.Second,unirradiated
IEC-6cellshaveserumindependentwithmethodTnofurtherbenefitwhenthecellsare
combinedwithirradiatedRAT-1cells.Moreover,thesurvivalratewasdecreasedfor
lowerdoses.TheRAT-1cellsinthiscombinationareunaffectedexceptcellsirradiated
with100MeV/uandculturedinserumI(seefigures3.26).Thesecellsslightlyincreased
theirsurvivalratewithincreasingdose.Third,intheinversecombinationtheunirradiated
RAT-1cellsshowedaLET-anddose-dependentbenefitoftheirradiatedIEC-6cellsin
serumIIbutRAT-1cellsgrowninserumIdidnotshowaninuence.IrradiatedIEC-6
cellsinserumIshowedacorrelationbetweendoseandsurvivalfor100MeV/ucarbon
ionsbutaninversecorrelationfor11.4MeV/ucarbonions.Forcarbonirradiatedcells
growninserumIInocleartendencycanbedetected.Forbothserumsthex-rayirradiation
especiallywith3Gyseemstoenhancesurvival(seefigure3.27).Fourth,whenbothcell
linesareirradiatedtheRAT-1cellsshowednoinuencebuttheIEC-6cellsincreased

62

eCo-cultur3.5

Figure3.27:Co-cultureserumI+II:irradiatedIEC-6cells+0GyRAT-1cells.
Strippedcolumns:methodP,fullcolumns:methodT,cross:errorcalculation,nomark:mean
valueswithstandarddeviation,ontop:PE:red,250kVpx-ray:blue,12C100MeV/u:cyan,12C
11.4MeV/u:green,onbottom:PE:black,250kVpx-ray:orange,12C100MeV/u:pink,12C
MeV/u:11.4violet

(a)Bothcelllines3Gy(RBE)ofx-rayor(b)Bothcelllines1,3or6Gyofx-rays
ionscarbonFigure3.28:Co-cultureserumbatchI:irradiatedIEC-6cells+irradiatedRAT-1cells.Stripped
columns:methodP,fullcolumns:methodT,cross:errorcalculation,nomark:meanvalueswith
standarddeviation,PE:red,250kVpx-ray:blue,12C100MeV/u:cyan,12C11.4MeV/u:green

63

Results3

Figure3.29:Co-cultureserumII+methodT:irradiatedIEC-6+irradiatedRAT-1cells.Cross:er-
12rorcalculation,nomark:12meanvalueswithstandarddeviation,PE:black,250kVpx-ray:orange,
C100MeV/u:pink,C11.4MeV/u:violet

theirsurvivalrateinadose-dependentmannerinbothserums.ALETdependencyisnot
detected(seefigure3.28and3.29).

measurCytokine3.5.2ements

Thecytokinemeasurementsincellculturesupernatantsshouldexplainthedetectedef-
fectsinco-culturesurvivalexperiments.Herethecellshavealmostnodirectcelltocell
contactsothattheeffectinducingsubstanceshouldbeasolublefactorwhichismediated
viatheculturemedium.Sincethesurvivalexperimentsarecarriedoutinmediumwith
serumthecytokinemeasurementsareconductedinserumcontainingmediumaswell.
DuetothelimitedaccessibilityofserumIthecytokineexperimentscouldonlybeper-
formedinmediumwithserumII.The6-wellplate/insertsystemwasusedforcytokine
detectingexperiments.HereonecelllinewasgrowninthePetridishandthesecondcell
lineintheinsert.Bytheuseofthissystemthedirectcelltocellcontactsareexcluded.
Thesecontactsareimportantforcelltocellcommunicationwithmoleculesmuchsmaller
thancytokines.Ifthecytokineproductionisinducedbysmallmolecules,thesedirectcell
tocellcontactswouldbeimportantfortheco-cultureeffectaswell.Toinvestigatethe
inuenceofthedirectcontact,theexperimentwascarriedoutwithbothcelllinesgrowing
dish.PetrisametheinTheamountofmeasuredcytokineinthecellculturesupernatantshastoberelatedtothe
numberofcells.Thisallowsaconclusionaboutthesecretedcytokineamountpercelland
acomparisonofdifferentexperiments.Whenbothcelllineswereinonecellsuspension,
itwasnotpossibletocountthecellnumberofeachpopulationseparatelybecauseofcon-
gruentcellhistogramsinthecoultercounter,sothatonlythetotalcellnumbercouldbe
determined.ThishasbeencircumventedbyuorescentstainingwithPKH67ofonecell
lineinvolved.Inaowcytometerthepercentageofstainedcellstounstainedcellscould
bedetermined,andtheobtainedpercentagecouldbeconvertedintorealcellnumberswith

64

eCo-cultur3.5

Figure3.30:TGF-βmeasurements:InsertversusPetridish.Mono-cultureof6Gy(RBE)irradi-
atedIEC-6cellsgrownininserts(ontop)orPetridishes(onbottom).250kVpx-rayexperiment:
red,11.4MeV/ucarbonionexperiment:blue,cross:PKH67stainedcells,horizontalline:TGFβ
contentoftheculturemedium.Leftpanels:TotalamongofTGF5βinthesampleadjustedforthe
serumlevel;Rightpanels:TGFβcontentnormalizedto1x10cells.

thetotalcellnumberofthecoultercounterresult.
Thecellswereirradiatedwith11.4MeV/ucarbonionsand250kVpofx-rays.Theana-
lyzedcytokinesTGFβ,TNFα,andIL-2wereselectedduetotheirinammatoryeffects
invivoandinvitro(seesection1.5and4.3.4).
IEC-6cellsshowedanoticeableincreaseinadhesionandgrowthinPetridishescom-
paredtoinsertswhileRAT-1cellswerelessaffectedbythegrowthsupport.Unirradiated
IEC-6cellsincreasedtheirgrowthinPetridishesbyafactorof1.62,irradiatedIEC-6
cellsbyafactorof1.26,andunirradiatedRAT-1cellsaswellasirradiatedRAT-1en-
hancedthegrowthbyafactorof1.09and1.05,respectively.
Inthecellculturesupernatantsofallco-culturecombinationsandintheusedcellculture
mediumwithserum(withoutcells)noTNFαorIL-2couldbemeasured.Thepossible
sourceofTNFαandIL-2wouldhavebeentheIEC-6cellswhichareabletosecretboth
cytokineswhichwasshownwithLPS-stimulation[87].Obviouslytheco-culturecondi-
tionsdidnotactivatethesamepathwayastheLPSstimulianddidnotinduceTNFαor
secretion.IL-2Thecytokineexperimentswerecarriedoutinserumcontainingmedium.Serumisknown
tocomprehendhighamountsofTGFβ.Therefore,itwasexpectedtodetecthighlevels
ofTGFβinthemedium.TheanalysisoftheTGFβamountinserumcontainingmedium
(withoutcells)overtheexperimentperiodofmaximumfivedaysweredeterminedtobe
stable460±18pg/ml.ThismeansthatTGFβisnotdegradedbyincubationat37°Cand

65

3Results

thatchangesinthemeasuredTGFβamountarecellinduced.
TheTGFβinthesupernatantsneedstobeactivatedbyincubation5partssupernatantwith
onepart1NHCl(ELISAprotocol,manufacturersinstruction).Withoutactivationactive
TGFβwasonlyfoundinsampleswhichweregrownforfourormoredaysandreached
cellnumbersabove≈3x105cells.UndertheseconditionsthemediumpHchanged
tolowervaluesindicatedbycolorshiftofpHindicatorphenolredinthemedium.Itis
assumedthatthroughthecultivationtimeoffourdaysandtheincreasedcellnumbers
theaccumulationofmetabolitesinthemediumwasenhanced.Theincreasedamountof
metabolitesdecreasedthepHwhichinducedapartialactivationofTGFβ.Themeasured
activeTGFβisthereforeareactiononthecultureconditionsandnotontheco-cultureor
irradiation.theThefigures3.30and3.31showintheleftpanelsthemeasuredTGFβamountsincellcul-
turesupernatantsandintherightpanelstheTGFβamountadjustedto1x105cells.The
measuredTGFβamountssuggesthugedifferencesbetweentheexperimentsinwhichthe
measuredTGFβlevelsincreasedwithtimeallexperiments.TheadjustmentoftheTGFβ
amountto1x105cellsrevealsthatamoreorlessconstantamountofTGFβisproduced
bythecells.Thedetecteddifferenceswerecausedbythedifferingcellnumbersseeded.
Therefore,nodifferencesbetweencellsgrowninPetridishesorinsertsandnodifferences
betweenirradiatedandunirradiatedcellsweredetected.Furthermore,mono-cultureor
co-cultureconditionshadnoinuenceontheTGFβsecretion.ThePKH67stainedcells
didnotchangetheirTGFβsecretionintothemediumandco-cultureswithdirectcellto
cellcontacthadnoinuenceontheexpressionofTGFβ.ToconcludeneitherTNFαnor
IL-2weremeasuredandthedetectedTGFβamountdidnotalterduetotheco-cultureso
thattheobservedeffectsinthesurvivalexperimentsareprobablynotcausedbyoneof
ytokines.canalyzedthe

ItwasremarkablethatinsomesamplesthetimedependentsecretionofTGFβstrongly
uctuated.Itwasnotexplainablebysamplehandlingorperformanceofmethodwhy
somemeasurementpointsdepartfromtheremainingdataorfurther,donotshowamea-
surableTGFβlevelatall.But,sincethebehaviorappearedespeciallyinexperimentswith
lowcellnumberstheconclusionwasmadethatthesecretedTGFβamountwascloseto
orunderthedetectionlimitoftheassay.

66

cHypoxia3.6hamber

Figure3.31:TGF-βmeasurements:mono-cultureversusco-culture.Ontop:Mono-cultureof6
Gy(RBE)irradiatedRAT-1cellsgrowninPetridishes.Onbottom:Co-cultureof6Gy(RBE)
blue,irradiatedopenRATsymbol:-1cellsgroandwthinIEC-6inserts,cells.closed250kVpsymbol:x-rayegrowthxperiments:inPetrired,dish,11.4cross:MeV/uPKH67carbonstainedions:
cell,horizontallines:TGFβcontentoftheculturemedium.Leftpanels:TotalamongofTGFβ
inthesampleadjustedfortheserumlevel;Rightpanels:TGFβcontentnormalizedto1x105
cells.

chamberHypoxia3.6

ForexperimentsunderhypoxicandoxicconditionstheRAT-1cellsweregrownonbio-
Folie25with3x104cellsover48h.Thegasmixtureof95%N2and5%CO2ushed2h
with200ml/mintroughfivein-lineconnectedchambersatroomtemperaturetoachieve
hypoxicconditions.Theoxiccellcultureswerekeptoutsidetheincubatorfor2htosim-
ulatethegassing.Afterirradiationwith250kVpx-raysorcarbonionswithaLETof
100keV/µmthecellsweretrypsinizedandseededinacolonyformingassay.Thesur-
vivalwascalculatedaccordingtoEq.2.3andplottedinfigure3.32.X-rayexperiments
werecarriedoutsixtimeswhilethecarbonionirradiationwasconductedonlyoncewith
hypoxicandtwicewithoxiccellsduetolackofcarbonionbeamtimeswithsuitable
conditions.Theerrorcalculationasdescribedundersection2.7wasnotappliedtothe
survivaldataofcarbonionirradiationunderhypoxicsincetheamountofαandβvalues
forcarbonionirradiationinhypoxiachamberwastolow.Itissuggestedthattheerrorof
carbonionexperimentsperformedinthehypoxiachambersiscomparabletothatofx-ray
xperiments.eirradiationThecellsurvivalafterx-rayirradiationwasashoulderedcurve,asexpected,whilethe
survivalaftercarbonionirradiationfollowsanexponentialdecline.Therefore,forx-ray

67

3Results

Figure3.32:RAT-1cellsurvivalunderhypoxicconditionscomparedtocellsurvivalunderoxic
ionsconditions.irradiation250withkVpx-raySOBPofLEirradiation:T100meanMeV/surviµm:valoxicvaluescurvewithmeanstandardsurvivdealvviationalueswith(n=6),standardCarbon
deviation(n=2),hypoxiccurven=1

irradiationαandβvaluescouldbecalculatedwhileforthecarbonionexperimentonly
anαvaluewasdetermined.Intable3.11theαandβvaluesweresummarizedforthe
experimentsperformedinthehypoxiachambers.Thecomparisonofoxicsurvivalcurves
ofcarbonionsandx-raysaswellashypoxicsurvivalcurvesforcarbonionsandx-rays
identifycarbonirradiationasmoreeffectiveincellinactivation.Thedeectionofthetwo
oxicorhypoxiccurvesistheRBE,respectively.TheRBEwascalculatedforhypoxicand
foroxicconditionsbasedonthecumulativecurvesaccordingtoEq.1.3.Thecellsurvival
underhypoxicconditionswasenhancedcomparedtocellsurvivalunderoxicconditions.
ThedifferencebetweenthetwocurvesisdefinedastheOER.Onthebasisofthecumu-
lativecurvestheOERvaluesweredeterminedaccordingtoEq.1.6.Table3.11showsthe
RBEandOERvaluesbesidethedosesfora10%survivalratewhichwereneededforthe
calculation.

68

cHypoxia3.6hamber

Table3.11:MeanαandβvaluesandD10withthestandarddeviationforsixindependent250kVp
linearx-rayefit.Bloxperiments.wtheForOERcarbonandeRBEofxperimentsexperimentsaretheαinvhalueypoxiaandthechamberD10giarevengivwithen.theh:herrorypoxic,oftheo:
oxicExp.α-1Δα-1β-2Δβ-2α/βΔα/βD10ΔD10
[Gy][Gy][Gy][Gy][Gy][Gy][Gy][Gy]
X-rayh0.0680.0350.0050.00115.90.7816.00.78
X-rayo0.1440.0540.0300.0066.61.076.70.82
Carbonh0.5480.0494.20.38
Carbono0.8190.2192.80.75
OER10,x-rayΔOEROER10,carbonRBE10,hRBE10,oRBEα,hRBEα,o
2.350.131.53.82.48.05.7

RBE10,o2.4

69

outlookandDiscussion4

linesCell4.1

4.1.1RAT-1Dunningprostateadenocarcinomacellline

Theco-cultureexperimentsshouldyieldresultsrelevantfortherapybutalsocomparable
topreviousanimalexperimentsperformedbyDr.P.Peschke(Germancancerresearch
center,DKFZ).ThecooperationwithDr.P.Peschkedefinedratsastheoptimalmodel
foranimalexperimentsandtherebythecellsystemforthisthesis.Peschkeassessesthe
ratDunningR-3327systemasanexcellentmodelforhumanprostatecancerprogression
andthedifferentcelllinesallowaRBEdeterminationrelatedtodifferentiationlevelof
thetumor[100].TheenzymeprofileoftheDunningR-3327systemisclosetohumans,
butnotallcelllinesofthesystemgrowinvitro.TheinvivogrowthislimitedtoFisher
andCopenhagenrats[63,65]whicharebothonlyavailableintheUSAandtherefore
expensivecomparedtootherratstrains.Aftertumorfragmentimplantationinthehind
limp,thesub-lineR-3327-AT-1(RAT-1)growsreliablewithaconstanttumordoubling
time[63].Carbonionirradiationofhindlimbtumorbearingratscauseddose-dependent
sideeffectslikeskinredness,dryand/orwetsites,desquamation,necrosis,hairloss,and
pigmentationabnormality[100].Throughthesmallirradiationfieldandtheexactanimal
positioningsideeffectsappearedonlyinthehindlimbandnotintheinnerorgans.Im-
plantationsoftumorfragmentsintheprostatearepossible[150],butconsiderationsto
simulatethehumanprostatecancerirradiationwithorthotropicimplantedRAT-1tumors
inratsfailthroughtechnicalproblemse.g.organmotion.Afteranimaginaryirradiation
oforthotropicimplantedRAT-1tumorsPeschkewouldsuggesttofindinammatoryside
effectsinthehighlyproliferatingintestine[100].

AsapartofthisthesistheRAT-1cellswerecharacterizedconcerningtheirPE,doubling
time,MI,andkaryotypeinvitro(seesection3.1).ThePEwasdeterminedtobe44.6
±11.3%whiletheRAT-1cellsdoubleincultureevery21±2h(seefigure3.1).The
karyotypedidnotchangeduringthecultureperiod(58.9±9.5)andtheMIwasconstant
(11.7±1.1%)aspresentedintable3.1andinfigure3.4.InvitrodataofRAT-1cells
arerareincomparisontoinvivodata.Isaacsetal.characterizedtheRAT-1cellswith
aninvitrogrowthtimeof32.5±3.7h,aPEof16.7±0.9%andachromosomeprofile
with60±7chromosomes[65].ThevariationsinPEanddoublingtimeofthisthesisin

71

4outlookandDiscussion

comparisontoIsaacsetal.arecertainlyduetothedifferingcellcultureserumswhereas
thechromosomeprofilewasidentically.Furthermore,acelldoublingtimeof13.1hwas
reportedinCorban-Wilhelmetal.whichindicatesahugeintra-celllinevariationwith
regardstocellgrowthtime[23].
Afterirradiationwithx-raysor11.4MeV/ucarbonionstheRAT-1cellsarrestinG2/M-
phaseduringthefirst12hbutnotinG1-phase(seefigure3.2).Inreference[43]thehuman
prostatecancercelllinePC-3accumulateinG2/M-phaseafterirradiationwithphotons,
too.Theauthorsindicateacontextofmutatedp53proteinwhichisacrucialfactorofthe
G1/S-phasecheckpointarrestinresponsetoDNAdamage.TheanalysisofRAT-1cellsin
vivoshowsincreasedp53levelsincomparisontonormalprostatetissuewhichsuggests
thatp53isnotthereasonforthemissingG1/S-phasecheckpointarrestinvitro[22].

4.1.2Developmentofaco-culturesystem-asuitablecelllinetoRAT-1cellsis
needed

Toestablishaco-culturesystem,asuitablecelllineincombinationtoRAT-1cellshadto
befound.Thesecondcelllinehadtofulfillthefollowingcriteria:a)establishedratcell
line,b)male,c)normaltissue,d)biosafetylevel1,e+f)adherentgrowthwithoutoronly
withfewadditionalgrowthfactors,g)originatedtissuenormalprostate,bladder,urethra
orintestineforthesimulationoftheeffectintheentrancechannelofprostatecancerir-
radiationwithcarbonions,andh)contacttoprostatecancercellsintheorganism.The
criteriaaredeterminedbythefollowingreasons.Theworkwithratprostatecellsdefines
thesexandthespeciesofthesecondcellline.Bytheuseofanestablishedcelllinethe
probabilitytoachievereproducibleresultswithdifferentcellbatchesshouldbeenhanced
incomparisontoprimarycells.TheRAT-1tumorcellsshouldbecombinedwithanormal
celllinesincethecommunicationbetweenthetumorandthesurroundingtissueandnot
thecommunicationbetweenthetumoranditsmetastasesshouldbeanalyzed.Inaddition,
theGSIhasonlyastatutoryapprovalforcellswithbiosafetylevel1whichexcludesall
celllineswithahigherbiosafetylevelbylaw.Becausethecellshavetobeirradiatedina
verticalpositionatGSIthecellsshouldgrowadherentinculturevessels.Growthfactors
intheculturemediumofthesecondcelllinewouldcomplicateaco-culturesincetheef-
fectofthegrowthfactorsonRAT-1cellshastobetested.Forprostatecancerirradiation
atGSIthecarbonionsaredeliveredintwoopposingfieldswhichincludethehipbones
andsmallpartsoftheradio-sensitivetissuesintestineandbladder.Thepossibletissue
originsofthesecondcelllinecouldthereforebenormalprostate,bladder,urethraand
intestinewithspecialregardtotherectum.Furthermore,allmentionedtissuetypescan
haveIntestinalepitheliumcellline6(IEC-6cells)hadthehighestintersectionwiththe

72

linesCell4.1

criteria.Thisestablishedcelllinefromtheratssmallintestineisdescribedasmaleand
normal,hasabiosafetylevel1categorization,growsadheredandonlyinsulindependent
[106].ThecombinationofRAT-1cellswithIEC-6cellssimulatesastateIII-IVprostate
tumorwhichspreadstootherorgans,heretheintestine.TheRAT-1cellsareanaplastic
andthereforehistologicalgradedwiththehighestGleasongradof5.Thissupportsthe
simulatedinvivosituationofanaggressivetumor.

linecellIEC-6The4.1.3

TheusedIEC-6cellbatchunderwentsignificantchangesduringitstimeinculturee.g.
increasingPE(figure3.17)andradio-resistance(figures3.14and3.15),changesinkary-
otype(figure3.9andtable3.2),anddecreasingdoublingtime(figure3.5).Onlyinone
cellbatchanindicationforalimitedlifespanwasdetected.Inalltheotherbatchesthe
cellsgrewwithoutdecreasingtD.Incontrasttotheseresults,theIEC-6celllineisde-
scribedwithanormalandstableratkaryotype(2n=42;rattusnorvegicus),aconstanttD
of20h,aPEof2.3%andalimitedlifespanof30-40passages[106].Oninquiry,ATCC
reportedthatthechangesofthecelllinewerenotknownbefore.ButATCCreferredto
thepotentialofmurinecellstospontaneouslyimmortalize[4].Toavoidtheselectionof
alteredcellstheusageofIEC-6cellswaslimitedtopassage15-20.
AcloserlookintoIEC-6relatedliteraturegivesonlyonehintthatage-dependentalter-
ationsofIEC-6cellsareknown.ThegrouparoundJian-YingWangpointedoutthatthere
werenosignificantchangesofbiologicalfunctionsandcharacterizationfrompassage15-
20[99,139].Inreverse,thiscouldmeanthatchangeswereobservedatlaterpassages
butnotinvestigatedincloserspecifications.Otherauthorswhichusethecelllinewithin
passage15-20refertoWangspapers[88]orWangiscoauthor[108].Therearefew
publicationswhichnotetheusageofIEC-6cellsbeforethe20thpassage[129]orwithin
passage16-20tominimizethepassageeffect[109]butwithoutgivingmotivationsorref-
erences.InthemajorityofpapersIEC-6cellswereutilizedasdescribedinQuaroniet
al.withoutcommentingonapassageeffect[106](forexamplessee:passage15-22[9],
passage17-22[62],passage16-30[48],passage17-27[47],passage17-30[132],and
]).59[20-26passage

4.1.4IEC-6cellsub-populationsandclones

ToanalyzetheextrachromosomesintheIEC-6cellmetaphasesstainingtechniquesfor
ratcellswereexamined.Fluorescentstainingproductsagainstratchromosomesarerare

73

outlookandDiscussion4

butsomecompaniesofferkitsagainstsinglechromosomes(e.g.chromosome12,Y,
andX;CambioLtd,Cambridge,UK,).Butwithoutlimitingthepossibleextrachromo-
somesthestainingofarandomlychosenchromosomeisofminorsignificance.Onlyone
publicationwasfoundwhereamulticolorspectralkaryotypingofratchromosomeswas
describedwithselfmadeow-sortedchromosome-specificpaintingprobes[15].Butthis
assaycouldnotbeappliedtotheexistingmicroscopeandsoftwaresystematGSI.Inthe
absenceofasuitablewholegenomewidestainingkitthechromosomeanalysiswasdone
withmousemFISHprobesonratcells(seefigure3.19).Itwasexpectedthatpartsof
theratchromosomesarestainedlikeabandinginthechromosomesectionsinwhichthe
chromosomesofratsandmicematch.TheresultinguorescencesignalofthemFISH
stainingdidnotallowachromosomeidentificationordifferentiation.Comparableex-
perimentswereperformedwithrainbowcross-speciesuorescenceinsituhybridization
withdifferentiallylabeled(Cy3,Cy5,FITC)gibbonchromosomesonhumanlympho-
cyteschromosomeswhichresultin90bands[29].Probablythebiologicalrelationship
betweenmouseandratisnothighenoughtoachievesimilarresultslikeseeningibbon
andhumanmixedsamples.Anotherapproachtoidentifytheextrachromosomescouldbe
theG-bandingtechniquewithwhichthekaryotypeanalysisinDanielpouretal.andSong
etal.ofratepithelialcelllineshasbeendone[128,26].Theanalysisrequiresexperience
andisusuallydonebyspecializedlaboratories.

BesidethechromosomeanalysisofIEC-6cellmasscultures,singlecloneswereanalyzed
(seetable3.7).Fromtwounirradiatedcellbatchescloneswereisolatedandanalyzedac-
cordingtotheirradio-resistance,doublingtime,andkaryotypeovereightpassages.Inthe
IEC-6cloneexperimentnocorrelationbetweenincreasingchromosomenumbersandin-
creasingradio-resistancecouldbefound.Bothcloneswith42chromosomeshadastable
radio-resistance(figure3.24).Cloneswithhigherchromosomenumbersdividedfaster
thancloneswithlowerchromosomenumbers.Butcloneswithidenticalchromosome
numberdidnotshowsimilardoublingtimes(figure3.23).
TheincreasinggrowthratesoftheIEC-6cellscouldbeinterpretedasafirststepinto
tumorigenesis.Insulinisanimportantgrowthfactorofcolonicepithelialcellsandis
mitogenicfortumorcellgrowthinvitro[44].Giovannuccihypothesizedthathyperinsu-
linemapromotescoloncarcinogenesis[44].FollowingthegeneralinstructionofATCC
theculturemediumofIEC-6cellswasenrichedwithinsulin.Itmightbe,thatremoval
ofinsulinfromthecellculturemediumwouldslowdowntheprocessofdecreasingcell
doublingtime.Co-cultureexperimentsshowedthatIEC-6cellssurviveandproliferate
inaninsulin-freemedium.Whetheralong-termcultivationininsulin-freemediumover
severalweeksispossibleandifthecelllinealterationissuppressed,hadtobetested.

74

Cell4.1lines

Inthecourseoftheidentificationofradio-resistantsub-populationsthesurvivalofan
IEC-6cellbatchwasreducedthroughirradiationwithx-raysorcarbonionsto10%(sub-
section3.4.2).TheirradiatedIEC-6cellswerecomparedwithunirradiatedIEC-6cellsof
thesamecellbatchandjudgedaccordingtotheirdoublingtime(figure3.20),karyotype
(figure3.21),andmitoticindex(figure3.22)overeightpassages.Inthelong-termcultur-
ingdifferenceswereidentifiedbetweenirradiatedandcontrolsamples.Inirradiatedcell
samplesthenumberofcellswithmorethan42chromosomesdevelopedfasterincompar-
isontounirradiatedIEC-6cellsofthesamebatchbutinallthreecasesthegrowthrateand
theMIdecreasedattheendoftheobservationperiod.Theseresultsindicatethatthrough
irradiationanearlysenescencecouldbeinduced.TheX-Galstainingforsenescencewas
performedinunirradiatedcellsofanothercellbatch(section3.2).Here,nopositivesignal
couldbemeasured.Butitispossiblethatthroughtheheterogeneityofthecelllinenot
allIEC-6batchesageinthesameway,anditmightbethatpositivestainedcellscouldbe
samples.cellirradiatedinfoundItcouldbedemonstratedthatSA-β-galisnotrequiredforsenescence[82].Toinvestigate
senescenceinmoredetailtheexpressionofhTERT(telomereshortening)aswellasp53
andp16(senescencepathwayproteins)shouldbemeasuredinIEC-6cells[45].

Afterirradiationwith250kVpx-rays,theIEC-6cellsstoppedinG2/M-phasefor12h.At
laterpointsintimenodifferencestocontrolsamplesweredetected(seefigure3.7).Cell
cycleexperimentswithcarbonionirradiatedIEC-6cellswerenotperformed.Through
thechangesinkaryotypewithtimeinculturetheDNAamountinthecellsvariesessen-
tially.Adifferentiationofthesub-populationsincellcycleexperimentswasthereforenot
).3.6figure(seepossibleIngeneral,thecellgrowthiscontrolledbyseveralcheckpointsincellcycleprogression.
TheG1/S-phasecheckpointandtheintra-S-phasecheckpointpreventthecellsfromun-
faithfulgenomereplication[77].ThecellarrestinG1/S-phasecheckpointappearssoon
afterdamageinductionthroughionizingradiation[81].TheG2/M-phasecheckpointis
initiatedtoallowrepairofDNAdamagespriortomitosis[77].Itcouldbedemonstrated
thatforinitiatingofcheckpointarrestinG2/M-phaseandforcellreleasefromthecheck-
pointarrestathresholdoftwentydoublestrandbreaksexists[81].Consequently,the
incompleterepairofthecellsgenerateschromosomeaberrations.
Afterirradiation,theIEC-6cellsarrestedinG2/M-phasewhichindicateagenomerepli-
cationwithoutdamagerepairandacellrepairbeforeenteringmitosis.Itispossiblethat
theIEC-6celllinehaslostafunctionalG1/S-phasecheckpointarrest.Oneofthemost

75

outlookandDiscussion4

importantfactorsforcellcycleprogressioncontrol,apoptosis,andDNArepairisp53
[86].IftheIEC-6cellshavelostfunctionalp53,thecelllinewouldaccumulateDNA
mutationswhichmightcontributetogenomicinstabilities.
Inalong-termcultivationofanIEC-6cellbatch,whichwasdividedinthreeparts(x-rayor
carbonionirradiatedandcontrolcells),developedidenticalsub-populations(seesubsec-
tion3.4.2).ThisdemonstratesthatthewholeIEC-6cellpopulationdevelopsaneuploidy.
Aneuploidyisassociatedwithadefectinthemitoticspindlecheckpointandtumorigene-
sis.Thespindlecheckpointcandelaythemitoticprogressionbytransientlyinhibitingthe
anaphase-promotingcomplexinresponsetodefectivekinetochore-microtubuleattach-
ment[74].Iftheattachmentisfaulty,thechromosomeswouldnotbedividedcorrectly
tothedaughtercellsandananeuploidyappears.Toprovethishypothesisthespindle
checkpointproteinscouldbeanalyzedfortranscriptionandfunction[74].Insteadofa
conclusionthequestionisraisedwhethertheIEC-6celllineshouldcontinuallybecalled
modelfornormalhumanintestinalepithelialbiology.

4.1.5AlternativecelllinestoIEC-6cells

ThewidevariabilityinPE,radio-resistance,andgrowthtimeoftheIEC-6cellsisanun-
certainelementandasourceoferrorinallperformedexperiments.Therefore,thesearch
foralternativecelllineswasextended.Ingeneral,celllinesoftheurethraorbladder
werenotfound.ButthesearchidentifiedtwonewcelllinesfromthegroupofDavid
Danielpour.Outofthedorsal-lateralprostateofLobund/WistarratsDanielpouretal.
establishedthecelllineNRP-152whichareofepithelialorigin[26].Theanalysisof
thekaryotypeidentifiesananeuploidywithamostlyhypertriploidchromosomaldistribu-
tion.InSongetal.thedevelopmentoftheepithelialcelllineDP-153fromthedorsal
prostateofLobund/Wistarratsisreported[128].Thekaryotypeisaneuploidwithmost
chromosomesinthediploidrange.Buttrisomy(chromosome7and12)andmonosomy
(chromosome14,15,and18)wereobserved,too.Bothcelllinesarenon-tumorigenic
inathymicmiceandepidermalgrowthfactor,insulin,dexamethason,andcholeratoxin
areneededforoptimalgrowths.ThecomparisonofIEC-6cellswiththetwoprostatecell
linesshowsnobenefitsinceDP-153cellsandNRP-152cellshaveananeuploidkaryotype
andarehighlygrowthfactordependent.Twoendothelialcelllines,YPEN-1andYPEN-
2,wereisolatedbyYamazakietal.outoftheprostateofaCopenhagenrat.Thecells
wereimmortalizedbyahybridvirus[148].Throughtheimmortalizationthecelllines
wereclassifiedwithbiosafetylevel2andarenotaccreditedatGSI.Fromthefetalrat
smallintestineNegreletal.isolatedthecelllineIRD-98whichwaseliminatedbecause
ofthefetaloriginandtheexpecteddifferencestoadulttissue[96].

76

linesCell4.1

BesidetheIEC-6celllineQuaronietal.establishedonefibroblastcelllineandthree
furtherepithelialcelllinesfromtheratintestinenamelyRIFandIEC-14[106]aswell
asIEC-17andIEC-18[105].IEC-14andRIFaresparselycharacterizedwhereasIEC-14
showednogrowthinhibition,alifespanover100passages,andsoftagargrowthwhich
isclearlytumorigenicbehavior.RIFcellsweredescribedasslowlygrowing(tD=25h)
withapoorPE(2.4%)[106].Bothcelllinesseemtobenotcommerciallyavailable.IEC-
17cellswereisolatedfromtheduodenumwhileIEC-18cellsoriginatedfromtheileum.
Bothcelllinesarenon-tumorigenicinsyngeneicanimals,donotgrowinsoftagar,and
haveadiploidkaryotype.ForIEC-17cellsprogressivemorphologicalchangesarepub-
lishedwhichseemednottobespontaneoustransformationbutadifferentiationtodefined
cellpolarity[116].ThiscelllinealterationdisqualifiedtheIEC-17cellsforfurtherinves-
tigations.Although,theIEC-18celllineisnotwelldocumentedinliteratureitcouldbe
analternativetoIEC-6cellssincetheiroriginisclosertotherectum,theyarecommer-
ciallyavailable(ATCCNo.CRL-1589),anduptonownoage-dependentchangeswere
published.

4.1.6Changingthecellsystemtohumancells

TheDunningprostatecancercelllinesystemisunique.Forhumancelllinesacom-
parablesystemisnotavailable.Thewelldocumentedhumanprostatecancercelllines
PC-3(gradeIVadenocarcinoma,epithelium,ATCCNo.:CRL-1435,[72]),LNCaP(car-
cinoma,epithelium,ATCCNo.:CRL-1740,[58]),andDU-145(adenocarcinoma,ep-
ithelium,ATCCNo.:HTB-81,[130])offerinterestingoptionsduetotheirdifferentcell
characteristics.ButtheycannotreplaceatumorprogressionmodelliketheDunning
systemwithmorethantencelllines[64].Furthermore,thepossibilitytoperforminvivo
experimentsundercontrolledconditionsandcomparetheresultstoinvitroexperiments
isnotgiveninahumansystem.
Inco-culturethefavoriteoriginforthesecondcelllineisthenormalprostate.Theavail-
abilityofnormalhumanprostatecellsisverylimitedaswellasforratcells.ATCC
offere.g.onlycelllineswithbiosafetylevel2becauseofvirusimmortalization.Via
BioWhittakertheorderofprimarynormalprostatecellswithdifferingoriginispossible
(PrEC(epithelium),PrSMS(Smoothmusclecells),PrSC(stromalcells)).Thevariability
betweendifferentdonorsoftheprimarycellscouldbehigh,andtheco-culturesystem
neededtobetestedwitheverynewcellbatch.Toconclude,achangeofthespeciesform
rattohumanisundesirableforco-cultureexperiments.

77

outlookandDiscussion4

4.2Mono-culturesurvivalexperiments

Thepilotprojectforcarboniontherapyofprostatecancerpatientswasstartedwiththe
goaltocompare30patientstreatedwithacombinedIMRTandacarbonboostwith30
patientsexposedtoIMRTalone.Thecarbonionsfortreatmentweredeliveredwiththe
rasterscanningtechniqueusingapencilbeamthatisguidedover3x105voxelofthe
tumorvolume.Thistechniqueguaranteesanutmosttumorconformtreatment,butneeds
inprinciple,foreveryvoxeltheappropriateRBEvalue.Thesevaluesarecalculatedwith
thelocaleffectmodel(LEM)whichisbasedonphysicalparametersdescribingthebeam
qualityandonthebiologicalsidethecharacterizationofthecellularrepaircapacity.For
theRBEcalculationsthecellularrepairtoionizingradiationdamagesisassessedinx-
rayexperimentsmeasuringclonogenicsurvival.There,theratioα/βofthelinearand
thequadratictermofthedoseresponsecurve,respectively,canbeusedfortheRBE
calculations.Although,intherapytheα/βratiohastobetakenfromtheclinicalsituation
i.e.fromconventionaltherapy,itisinterestingtocomparethecellulardataobtainedin
ourmeasurementswiththeclinicaldata.Inaverycomprehensiveresearchα/βratios
between1.5Gyand6Gyhavebeenreportedbutqualityweightedevaluationsconverged
atavalueof2Gy[13].Therefore,anα/βratioof2Gyisusedinthebiologicaltreatment
planningphaseforprostatecanceratGSI[31].Theα/βratioforconventionalexternal
beamradiationorlow-andhigh-dose-ratebrachytherapyisunderdiscussion.Values
between1.5Gy[95]and3.1Gy[140]areassumed.
TheRAT-1celllineismoderatelyradio-resistantagainst250kVpx-rayswithanα/βratio
of6.8±0.5Gy(figure3.11).ExperimentswithirradiatedRAT-1cellsinvitrocannot
befoundinscientificliterature,andinpublishedinvivoirradiationexperimentsnoα/β
ratiowasdetermined.Thus,itwasnotpossibletocomparetheα/βratioofRAT-1cells
toothercelllinesinvitro.Forsixhumanprostatecancercellsanα/βratiorangewas
estimatedtobebetween1.09to6.29Gy[16].Inallplanningandtreatmentahighradio-
resistanceofprostatecancersispresumed.Incontrast,theRAT-1cellsareonlymoderate
radio-resistant.ThesurvivaldataofcarbonionirradiatedRAT-1cellswascomparedtocalculationsof
LEMinitsversionIIandIV(calculationsperformedbyThiloElsässer,GSI,Biophysics
group).LEMIcalculatedthebiologicaleffectaccordingtothedoseresponseofmea-
suredx-raysurvivalcurves[120].However,radicaldiffusionandtheinteractionofsingle
strandbreaksformingdoublestrandbreakswerefirsttakenintoaccountbyLEMII[33].
ThefurtherversionLEMIIIintegratedamorerealisticcharacterizationoftheradialdose
distribution[32].ThelatestversionLEMIVconsidersinadditionclusteredDNAdouble
strandbreaks[31].TheLEMcalculationforRAT-1cellsurvivalaftercarbonionirra-

78

4.2Mono-culturesurvivalexperiments

diationwithdifferentenergiesisbasedontheaverageαandβvaluesmeasuredfor250
kVpx-rayirradiation(seesubsection3.3.1).Infigure4.1theLEMcalculationsareplot-
tedtogetherwiththemeasuredsurvivalcurves.LEMIVcalculationswereinagreement
withtheexperimentaldatawhileLEMIIcalculationswereonlysuitablefor11.4MeV/u
xperiments.eioncarbon

(a)SurvivalcalculationswithLEMII(b)SurvivalcalculationswithLEMIV
Figure4.1:SurvivalcurvesofRAT-1cellsaftercarbonionirradiationinsolidlinescomparedto
LEMcalculationsindashedlines.Theusedcarbonionenergieswere11.4MeV/u(green),100
MeV/u(red),and270MeV/u(black).

Theradio-resistanceofIEC-6cellsagainst250kVpx-rayschangedwithtimeinculture
anddifferedbetweencellbatches(figures3.14and3.15).Becauseofthissystematic
changemeanαandβvaluesarenotappropriated.Table3.4presentsallαandβvaluesfor
theperformedx-rayexperiments.TheIEC-6cellsaremoreradio-sensitiveincomparison
toRAT-1cellsindicatedbylowerdoseswhichwereneededtoreducethecellsurvival
to10%(D10).TheD10forRAT-1cellswasdeterminedtobe7.0±1.4Gywhilethe
D10forIEC-6cellsuctuatedependentonagebetween5.0and6.0Gy(seetable3.4).
TheIEC-6cellswereusedbyotherauthorsinseveralirradiationexperimentswithx-rays
orneutronsbutαandβvaluesorRBEvalueswerenotpublished.Intheabsenceof
otherdatathedeterminedRBEvaluesforIEC-6cellswerecomparedtoaninvivomouse
study[39].InthemousestudytheRBEvalueoftheintestineaftercarbonionirradiation
(SOBP,differentLETs)and137Crγ-rayirradiationweremeasured.Thecomparisonofthe
RBE10valuesforIEC-6cellsandthemouseintestineisplottedinfigure4.2.Essentially,
theplottedRBEvaluesagainsttheLETdidnotdiffer.AsteepercurvefortheIEC-6
cellsthanfortheinvivomousedataisshown.Thisdemonstratesthataninvitro/invivo
comparisonofexperimentaldataispossible.

ThesurvivalcurvesofIEC-6cellsaftercarbonionirradiationwerecomparedwithcal-

79

outlookandDiscussion4

(greenFigure4.2:circle).RBEThe10ofRBEIEC-610valuescellsfor(redIEC-6square)cellsarecomparedavtoerageinvvivoaluesRBEof10fouroftheindependentmurineintestinecarbon
ionexperimentsandsixteenindependent250kVpx-rayexperimentswhilethedataformurine
intestinearepublishedin[39].

(a)SurvivalcalculationswithLEMII(b)SurvivalcalculationswithLEMIV
Figure4.3:SurvivalcurvesofIEC-6cellsaftercarbonionirradiationinsolidlinescomparedto
LEMcalculationsindashedlines.Theusedcarbonionenergieswere11.4MeV/u(green),100
MeV/u(red),and270MeV/u(black).

culationsoftheLEMinversionIIandIVwhichwereperformedinadvancebyThilo
Elsässer(GSI,Biophysicsgroup).FormoredetailsonLEMI-IVseetwoparagraphs
above.ThecalculationsarebasedonαandβvaluesofIEC-6cellx-raycurves.As
mentionedpreviouslymeanαandβvaluescouldnotbedeterminedduetothechanging
radio-resistanceofthecellline.Tocircumventthisproblem,anestimationformeanα
andβvalueswasperformedasdescribedundersection2.7.Thedeterminedvalueswere
forα0.35±0.02whileβwas0.022±0.003(n=16).Thecomparisonoftheexperimen-
talIEC-6celldatawithLEMcalculationsshowsthatLEMIVpredictedtheIEC-6cell
survivalratemoreexactlythanLEMII.Especiallyfor270MeV/ucarbonions,which
representtheenergyintheentrancechannel,thedifferencesbetweenLEMIVandthe
measureddataareverysmall.

80

experimentseCo-cultur4.3

eCo-cultur4.3xperimentse

4.3.1Comparisonofmethods:cellpre-seedingversustrypsintreatment

Theco-culturesurvivalexperimentsofIEC-6cellsandRAT-1cellswereperformedwith
twodifferentset-ups.Inthefollowingbothmethodsarediscussedwithregardtothe
protocolforaclonogenicsurvivalassayandthecytokinesecretionofthecellsintothe
medium.Inaclonogenicsurvivalassaythecellsneededtobetrypsinizedafterirradiation
andreseededindefinednumbers.Notallcellswithhighradiationdamagesdore-attach
afterthetrypsintreatmentandareexcludedfromtheexperiment.Inirradiatedtissueno
eliminationprocesstookplacesothatthesehighlydamagedcellsexist.Themotivation
formethodP,wherecellsarepre-seededinsuitableconcentrations,wastoanalyzethe
communicationbetweenlow,medium,highornotirradiatedcellswithoutexcludingone
ofthesub-population.Thepre-seedingwasonlypossibleforx-rayirradiationandnotfor
carbonionirradiation.AtSISfacilitytheirradiationtimespanislimitedandtheirradi-
ationtimeoftheneededcultureaskswithpre-seededcellswouldhavetakentoolong.
IrradiationatUNILACfacilityisonlypossibleinPetridisheswithadiameterof3cm
whichcorrespondstoagrowthareaofapproximately7cm2.TheamountofPetridishes
foracolonyformingassaywithcomparablecolonystatisticsasincultureaskswould
havebeentoohigh.Besidethelongirradiationprocedure(3hforax-rayexperiment)
methodPhasadditionaldisadvantages:a)therealnumberofirradiatedcellsisunknown,
b)theirradiatedcellswerestainedaftertwelvedaysofgrowinsteadofelevendays,c)
twoPEvaluesareneededforsurvivalcalculation,andd)thebackgroundofcellswhich
starttogrowafterirradiationbutdonotachievefiftydaughtercellsisveryhigh.These
uncertaintiesledtomistakesinthesurvivalcalculationandtheexperimentalset-upwas
changed.InmethodTthecellsweretrypsinizedafterirradiationandreseededincultureasks
fortheclonogenicsurvivalassay.MethodTreducedthex-rayirradiationtimeto30min
whereasthenumberofcellsinthecultureaskwasoflittleimportanceduringtheirradia-
tion.Throughthetrypsination,allcellswereseededonthesamedayandcouldbestained
afterelevendays.TheadditionalsecondPEwasomitted.Furthermore,thebackground
ofcellswasminimized,whichattachedbutlosttheirmitoticabilityafterafewdevia-
tions.Butthetrypsinstepmightexcludehighlydamagedcell.Acentraldisadvantageof
methodTisthemediumexchangeafterirradiation.Ifthecellssecretsubstancesdirectly
afterirradiationintothemedium,thesesubstancescannothaveaninuenceontheother
unirradiatedcellline,andthesesubstanceswillnotbedetectedinacytokineassay.That
thereareveryfastreactionswasdemonstratedinco-cultureexperimentwithunirradiated

81

outlookandDiscussion4

andα-particleirradiatedhumanprostatecarcinomacellsDU-145[142].Abystander
effectintheunirradiatedcellscouldonlybeobservedwhentheunirradiatedcellswere
presentinthemediumoftheirradiatedcellsduringtheirradiation.Ifthecellswerecom-
bined1minafterirradiation,theeffectwasnotdetected.Theauthorsconcludedthatthe
mediatingmoleculehastobeashortliferadicalotherthannitricoxide.WithmethodPas
wellaswithmethodTfastreactionslikedescribedbyWangandCoderre[142]cannotbe
detectedbutfortheanalysisofcytokinesecretionandchangingproteinprofilesmethod
TismorequalifiedthanmethodP.

4.3.2Inuenceofserumonsurvivalexperimentsandcytokinedetection

Theco-culturessurvivalexperimentswereperformedinserum-containingmediumand
withtwodifferentserumbatches(BiochromAG,Berlin,Germany).Bothcellculture
serumsinuencedthePEandthesurvivalasdemonstratedintable3.4andfigure3.15.
ButthemeasuredresultswithserumIcouldnotbereproducedwithserumII.
Cellcultureserumcouldcontainavarietyofcytokineswhicharenotidentifiedandquan-
tifiedbytheproducingcompanies.Oninquiry,theBiochromAG(Berlin,Germany)
declarethatallcytokinesandgrowthfactorswhichareabletopenetratetheplacenta
couldbefound(mostlyinlowconcentrations)inthefetalcalfserum[11].Thedifferent
cytokinecompositioninthetwoserumbatchescausedthedifferingresultsintheco-
culturessurvivalexperiments.
Becauseofthecytokineamountincellcultureserum,themajorityofpublishedcytokine
detectingexperimentswereperformedinserum-freeculturemediumtoobtaincontrol-
lableandreproduciblegrowthconditions.However,inthisthesistheco-culturecytokine
detectingexperimentswereperformedonpurposeinserum-containingmediumtoachieve
identicalconditionstotheco-culturesurvivalexperiments.Acomparativeco-culturesur-
vivalexperimentoverelevendaysinserum-freemediumwasnotperformedsincethe
lackofserumwouldleadtogrowthinhibitionorevencelldeath.

4.3.3Survivalexperiments

Intheperformedco-culturesurvivalexperimentsthenumberofdirectcelltocellcon-
tactswasrareandthecommunicationviagapjunctions(directcelltocellcontacts)could
contributeonlyinasmallpercentagetothedetectedeffectsinsurvival.Therefore,the
co-cultureeffectinducingmoleculewassuggestedtobeasolublefactor,whichisme-
diatedviathecellculturemedium.Itwasassumedthatmorethanonefactorisrespon-
sibleforthedetectedeffectsinco-culture.First,thePECoofIEC-6cellswasenhanced

82

xperimentseeCo-cultur4.3

whichindicatesanirradiation-independentfactor(figure3.26).Second,apositivesur-
vivaleffectforRAT-1cellswasonlydetectedwhenIEC-6cellswereirradiatedwhich
indicatesanirradiation-dependentfactor(figure3.27).Third,theIEC-6cellsurvivalwas
onlyenhancedwhenbothcelllineswereirradiatedwhichindicatesasecondirradiation-
dependentfactor(figure3.29).
Theresponseofunirradiatedcellstosignalsproducedbyneighboringirradiatedcellsis
calledthebystandereffect.Theuseddosesforirradiation(1,3,and6Gy(RBE))were
therapyrelatedandchosendependentonthesurvivallevel.Theirradiationdoseswere
notinthetypicalbystanderrangewhichisbelow1Gyorabove15Gy[103].Neverthe-
less,thebystandereffectwillbediscussedbutwithoutgreatdetail.Theeffectismediated
byafactorreleasedtothemediumorinvolvesdirectcelltocellcontactviagapjunction
whereasthecommunicationviagapjunctionsisgenerallydownregulatedintumorcells.
Inaddition,gapjunctionslimitthesignalmoleculesizeto1,000-1,500Da,whilesoluble
factorsinthemediumcanhaveupto10,000kDawhichincludescytokines.Bystander
signalinghascloseparallelstoinammatoryresponsewhichincludese.g.TGFβ,TNFα,
IL-6,IL-8,ROS,andNO.Correspondingtotheeffectinthewholeorganism,theanalyzed
endpointsaretermedthebystanderresponsedamaging(DNAdamage,mutation,trans-
formation,celldeath)orprotective(terminaldifferentiation,apoptosis,radio-adaptive
response).Theeffecthasnodose-responserelationshipandbecomessaturatedatvery
lowdoses(below1Gy)[103].
Celllinesusedinthisthesis,RAT-1cellsandIEC-6cells,werenotassayedinbystander
experimentsbefore.Thegeneralcombinationofprostatecancercellswithepithelialcells
wasalsonotstudied.Inaddition,theoriginatingtissueoftheRAT-1cellsisunknowndue
totheanaplasticcharacterofthecellline.Therefore,theresultofthisthesiscanonlybe
comparedtootherpublicationswhichcombinedgeneralprostatecancerwithothercells
orepithelialcellswithothercells.
ThemajorityofpublicationswhichshowedabystandereffectonPEorsurvivalcom-
binedirradiatedcellswithunirradiatedcellsofthesamecelllinee.g.increasingPEof
unirradiatedhumanepithelialcellsinco-culturewithirradiatedhumanepithelialcellsaf-
terirradiationwithcarbonionsofdifferingLETandx-rays[124].Shaoetal.reported
thatahigherLETcarbonionirradiationwasmoreefficientininducingthebystanderef-
fect(increasingPEofunirradiatedcells)thanalowerLETcarbonionirradiation.An
LET-dependencycouldnotbemeasuredinthisthesis.Bothcellcombinationsofunir-
radiatedIEC-6cellswithirradiatedRAT-1cellsaswellasunirradiatedRAT-1cellswith
irradiatedIEC-6cells,didnotshowaLET-dependentincreaseofPE,independentofthe
batch.serumusedAnzenbergetal.studiedtheco-cultureeffectofx-rayorα-particleirradiatedhuman

83

outlookandDiscussion4

prostatecancercells,DU-145,withunirradiatedhumanfibroblasts,AG01522[3].They
showedthatthesurvivalofunirradiatedfibroblastsdecreasedwhenthecellswerecom-
binedwithx-rayirradiatedbutnotwithα-particleirradiatedprostatecancercells.The
negativeeffectonthefibroblastsurvivalcouldbeblockedbythenitricoxide(NO)spe-
cificscavengerPTIO,whichindicatestheinvolvementofNOinthebystandereffect[3].
Furthermore,Wangetal.reportedforthesameprostatecancercells,DU-145,aby-
standereffect(micronucleiformation)afterα-particleirradiationinunirradiatedDU-145
cellswhichinvolvesashortliferadicalbutanotherthanNO[142].
Inco-culturesurvivalexperiments,performedinthisthesis,thex-rayorcarbonionirra-
diatedRAT-1cellsinducedadecreaseinsurvivalofco-culturedunirradiatedIEC-6cells.
IfthedecreasingsurvivaloftheIEC-6cellswasinducedbyradicals,theeffectshould
bemorepronouncedwhenhigherdoseswereappliedbecausehigherdosesofx-raysor
carbonionsproducemoreradicalsthanlowerdoses.ButthenegativeeffectontheIEC-6
cellsurvivalwasnotlongerpresentwithincreasingdelivereddosetoRAT-1cells(see
figure3.26).Therefore,itisimplausibletoconsiderradicalsastheonlymoleculeinduc-
ingtheeffectonthesurvivalofIEC-6cells.Itispossiblethattheeffectofradicalsis
superimposedbyothermechanismswhichbecomerelevantathigherdoses.
Gaugleretal.irradiatedprimaryhumanlungendothelialcellswith15Gyof6MVx-
raysandcombinedthecellswithunirradiatedhumancolonepithelialcells.Theinduced
bystandereffectsinepithelialcellsweredecreasingcellnumbersandmitoticcellsaswell
asincreasedapoptoticcells.Thethreeeffectsinepithelialcellscorrespondingeneralto
reducedsurvivalrateandwereenhancedwhenbothcelllineswereirradiated[42].The
detectedeffectsinGaugleretal.werecontrarytotheresultsinthiswork.Intheirradi-
atedsampleRAT-1cellsandIEC-6cellsadose-dependentincreaseinsurvivalratewas
inducedinIEC-6cellswhiletheRAT-1cellswerenotaffected(figure3.29).
Inconclusion,theseresultsindicatethatnotonlyacelltypespecificresponsetoirra-
diationbutalsothecombinationofcelltypeareresponsiblefortheinducedbystander
fect.ef

Cytokines4.3.4

Basedonthedetectedeffectinthesurvivalexperiments(seesubsection3.5.1)substances
werechosenforanalysisinmediumsupernatantswhichcouldbetheaffectingmediator.
Itwasexpectedtofindaninammatoryresponseafterirradiationandthattheinvolved
moleculescouldbeinammatorycytokines.Asmentionedundersubsection4.3.3atleast
threefactorscouldinducetheeffectswhereasonefactorisindependentofirradiationand
dependent.irradiationwereotw

84

xperimentseeCo-cultur4.3

βTGFTGFβcouldbemeasuredintheperformedexperimentsandthemeasuredTGFβamount
wasadjustedforthemediumTGFβlevelaswellasforthecellnumberwhichproduced
theTGFβ.InallcarriedoutexperimentsnoadditionalTGFβsecretionwasfoundthrough
theirradiationortheco-culturing.ItseemsthatthecellskeptthelevelofTGFβinthe
mediumconstanttothecellnumber.Furthermore,noactiveTGFβwasfoundinprolif-
eratingcultures.ActiveTGFβcouldonlybemeasuredinconuentcellculturesatwhich
themediumpHwasshiftedtolowervalues,whichmighthaveactivatedTGFβ.
ThedecisiontoanalyzeTGFβwasmadeonthefollowingworkinghypothesis.Thenum-
berofpublicationsislimitedwhichdealingeneralwithinvitroRAT-1cellexperiments
andinparticularwithcytokineorcytokinereceptorproduction.Butitwasreportedthat
theRAT-1celllineexpressedinvitroelevatedTGFβ1,TGFβRI,andTGFβRIImRNA
levels[145].TheeffectofTGFβonRAT-1cellsisunknown.SincetheRAT-1tumor
celllineisanaplastic,theoriginatingcelltypecannotbeidentified(e.g.basal,luminal,
stromal,glandular,epithelium,smoothmusclecellsoftheprostate).Therefore,direct
conclusionsontheTGFβeffectonRAT-1cellsarenotpossiblebasedonthedifferent
prostatecelltypes.TGFβsecretioncanbeinducedbyirradiation[2]sothattheRAT-1
cellscouldbestimulatedbyirradiationtoproduceandsecretthecytokineaswell.In
co-culturewithIEC-6cells,themediummediatingTGFβ,secretedbytheRAT-1cells,
couldinduceeffectsintheIEC-6cells.PodolskyreportedthatTGFβnegativelyaffects
theproliferationofIEC-6cells[102].ByKoetal.itwasshownthatTGFβarreststhe
IEC-6cellsinG1-phasebyblockingtheinductionofcyclinD1[75].Insummary,the
irradiationshouldinduceTGFβsecretionbytheRAT-1cellsandthismediummediated
TGFβshouldreduceoraffectthesurvivalrateofco-culturedunirradiatedIEC-6cells.
WiththishypothesisthereducedsurvivalrateofunirradiatedIEC-6cellinco-culture
withirradiatedRAT-1cellsshouldbeexplained.Butintheperformedexperimentsno
additionalTGFβwasfoundsothatthehypothesiswasdisproved.

IL-2andαTNFThemotivationtomeasurebothcytokinesTNFαandIL-2wasbasedonthepaperbyLyu
etal.whichdemonstratedthatIEC-6cellsareabletosecretTNFαandIL-2afterLPS
stimulation[87].Foralongtime,IL-2andTNFαwerethoughttobeonlyproducedand
secretedbycellsoftheimmunesystem.Butseveralpublicationsshowedthatalsoother
normalcellsortumorscellsarecapableofIL-2[110]andTNFα[107]productionas
wellassecretion.IL-2hasbeendetectedinnormaltissueslikeendothelialandintestinal

85

outlookandDiscussion4

epitheliumtostimulatetheproliferation[5]whichwasalsopublishedbyShigematsuet
al.fortheIEC-6cells[126].ForTNFαaneffectoncellproliferation,differentiation,
andinductionofothercytokinesinepithelialcellswasreported[107].Inagreementwith
theseresultsTNFαinducedinIEC-6cellsDNAsynthesisandincreasedcellproliferation
[149].TheIEC-6cellswerecombinedwiththeprostatecancercelllineRAT-1.Inhighly
proliferatingtumorslikeprostatetumorsincreasedexpressionofIL-2andIL-2Rhasbeen
reported[41].OnRAT-1cellsIL-2hasinvivoaninhibitorygrowtheffect[70,54].TNFα
inducedslowertumorgrowthinvivoinsomeDunningsub-lines[125].Thisresultwas
notreproducedininvitroexperimentswiththesamesub-lines[135].Untilnowtheef-
fectofTNFαonRAT-1cellswasnotinvestigated,whichmeansthatanegativeeffecton
proliferationcanbutdonotnecessarilyhavetoappear.
TheworkinghypothesisforTNFαandIL-2measurementswasasfollowed.Intheco-
culturesurvivalexperimentsanegativegrowtheffectontheRAT-1coloniesandapositive
growtheffectontheIEC-6colonyformationconcomitantwithincreasedcolonysizewere
detected(seesection3.5.1).IL-2andTNFαcouldmediatethedetectedeffectssinceIL-2
andTNFαinducedproliferationandDNAsynthesisinIEC-6cellsaswellasthecytokines
promotedgrowthinhibitioninvivoinRAT-1cellsatwhichIL-2invitroalsoinhibitthe
RAT-1cellgrowth.ButintheperformedexperimentsneitherTNFαnorIL-2couldbe
detected.ItmightbethatthesensitivityoftheusedELISAwasnothighenough.The
detectionlimitofbothusedELISAkitswas62.5pg/ml.Humanserume.g.containsa
IL-2levelof5-15pg/ml[6].TheusedfetalcalfserumwasnottestedforIL-2andTNFα
levelsbutthemanufacturerassumesthatlowamountofbothcytokinesarepresentinthe
].11[serumInsummary,intheanalyzedcellsystemonlyTGFβcouldbedetectedwhileIL-2and
TNFαwerenotsecretedbythecelllineorbelowthedetectionlimitoftheassay.Since
thewholeTGFβamountinthesupernatantwasdetermined,theorigincelllinecouldnot
named.be

ThethreeanalyzedcytokinesTNFα,TGFβ,andIL-2cannotrepresentthehugeand
complicatedsignalingsystemwhichisinducedincellsafterirradiation.Otherinteresting
cytokinesforanalysisinco-culturewouldbeIL-1α,IL-1β,andIL-6.IEC-6cellshave
largeintracellularIL-1αpoolswhichthecellsdonotsecret.Stadnyketal.suggestedthat
IL-1αpoolsmightbereleasedfromdamagedcellsandmightactonneighboringcells
[129].Itmightbethatthesecretioncanbeinducedwithionizingradiation.
BesideIL-1αdetectionitwouldbeinterestingtoanalyzetheinterplaybetweenIL-1β,
TGFβandIL-6whichcouldpartlyexplaintheresultsoftheco-culturesurvivalexperi-
ments.IL-1βandTGFβstronglymodulatetheIL-6sectionbyIEC-6cells[90].IL-6

86

4.4Hypoxiachamber-measurementoftheoxygeneffect

itselfmodulatestheintestinecellpopulation.Inaddition,IL-1βenhanceepithelialcell
restitutionandenhancetheproductionofactiveTGFβ[102].

Afurtherresultoftheco-culturewastheincreasingPEofIEC-6cellsinco-culturewith
unirradiatedRAT-1cells(seefigure3.26).AnincreasingPEcanbecausedbyenhanced
celladhesionorproliferation.Theadhesion-mediatingmoleculesbetweencellsorcells
andmatrixareintegrins.IntegrinsregulatestheadhesioninG1-phaseandtheentryinS-
phaseofthecellcycle.Inaddition,integrinsareconnectedtotheactincytoskeletonand
controlproliferationaswellasmigration[60].Asecondgroupofadhesionmoleculesare
cadherins.ThemajorexpressedcadherininepitheliumisE-cadherinwhichisimportant
incell-cellcontacts[49].
Independentoftheco-culturetheIEC-6celllineunderwenthugechangesinPE,cell
doublingtime,andradio-resistance(seesections3.2and3.4).Anintegrinwhichisis
involvedinallthreeprocessesisintegrinβ1.Integrinβ1mediatescell-matrixinteractions
(proliferation)andisassociatedwithmalignantprogression(migration).Furthermore,it
ismediatingpro-survivalsignalingaswellasresistancetoradiation[24].Itwouldbe
interestingtoanalyzetheintegrinprofilewithspecialregardtointegrinβ1andtheE-
cadherinappearanceinmono-culturesandco-culturesofIEC-6cellstoseea)ifthemono-
culturemodifytheexpressionintegrinβ1withage,whichcouldexplainthedetected
changesinIEC-6massculturesandb)iftheco-cultureinducedchangesintheadhesion
moleculeexpression,whichcouldstimulatetheadhesionortheproliferationofIEC-6
cells.

4.4Hypoxiachamber-measurementoftheoxygeneffect

Toanalyzetheradiationresponseofcellsunderhypoxicconditionsthecontainerinwhich
theirradiationtakesplacehastofulfillseveralcriteriawhichwerementionedinsec-
tion2.5.Nosuitableapplicationwasfoundtobecommerciallyavailable.Forexample
Billups-RothbergInc.(SanDiego,CA,USA)andBiopherixLtd.(Lacona,NY,USA)
offeredhypoxiachambersmadeofplasticandstainlesssteel.Bothcontainersarecom-
parabletoanincubationwithanadditionalinuxforgasmixtures(seefigure4.4)anddo
irradiation.samplewallonot

Todeterminetheoxygeneffect,RAT-1cellswereirradiatedinthehypoxiachamberwhich
wasdevelopedfortheirradiationwithx-raysandionsunderoxicandhypoxicconditions.
Therefore,thecellswereseededonbioFolie25insteadofinPetridishesorinculture
asks.Detailsonconstructionandsamplehandlingaregiveninsection2.5whileinsec-

87

outlookandDiscussion4

FigureBillups-Rothber4.4:CellgInc.,culturerightsystempanel:forhHypoxiaypoxicChamberconditions.fromLeftBiopherixpanel:Ltd.Hypoxiachamberfrom

tion3.6theobtainedresultsarepresented.

4.4.1OERforx-rayirradiation

TheincubationatroomtemperaturedidnotfurtheraffectthePEofcellsgrowingonbio-
Folie25atwhichingeneralthePEonbioFolie25waslowerthanthePEincultureasks.
In250kVpx-rayexperimentsperformedunderoxicconditionsthestandarddeviations
ofsurvivalincreasedcontinuouslyfrom12%to65%(0Gyto10Gy,r2=0.96)whilethe
standarddeviationforhypoxicsurvivalexperimentswas29±9%.Thedetectederrors
inthehypoxiachamberwerehigherforoxicexperimentsthantheerrorofexperiments
performedunderoxicconditionsincultureasks.Herethestandarddeviationincreased
continuouslyfrom10%to51%(0Gyto10Gy,r2=0.87).Butthesurvivaldataunder
oxicconditionsobtainedincultureasksorinsampleringswerecomparableasshownin
figure4.5.Itseemsthatthesurvivalunderoxicconditionsismorestrongelyinuenced
thanthesurvivalunderhypoxicconditionssincetheoxicexperimentshadhighererror
valuesindependentofthegrowthsupport.Itmightbethattheoxicsurvivalisaffected
morebysub-lethaldamagesbecauseofthedamagefixationwithoxygenthanthehypoxic
survival.Underhypoxiathepurelylethaldamagesweremeasured.

Thestrongerincreaseoftheerrorathigherdosesindicatesdifferentradio-sensitivitiesin
thecellculture.Areasonforthedifferentradio-resistancemaybethedifferentsituation
duringthegassingtime.Whereasthehypoxicsampleswheregassedfor2hwithamix-
tureofnitrogenand5%CO2atroomtemperature,theoxicchambersstayedforthesame
timeatroomtemperaturebutwithoutadditionalCO2.Duringthe2hatroomtemperature
thepHoftheoxicsampleschangedtoalkalinevaluesopticallyevaluatedbycolorchange
ofpHindicatorphenolredfromlightredintodeepred.Itmightbethattheslightlyen-
hancedpHinuencedtheradio-sensitivityofthecells.Jayanthetal.demonstratedthat

88

4.4Hypoxiachamber-measurementoftheoxygeneffect

Figure4.5:RAT-1cells:Comparisonofcellsurvivalafter250kVpx-rayirradiationunderoxic
conditionsincultureasksandsamplerings.

cellsurvivalafterx-rayirradiationisreducedwhenthecellsareculturedinmediumwith
apHbeyondtheoptimum(6.6)whereasalowermediumpH(6.0)affectthesurvivalmore
thanthehighermediumpH(7.6)[69].Infutureexperimentstheoxicsamplewillbeaer-
atedwithagasmixturecomposedofsynthesizedairwith5%CO2toavoidpossibleerrors.

Asecondreasonforthechangingradio-sensitivitymaybethatdifferencesincellcycle
phasesareexpressedmoreunderoxicthanunderhypoxicconditions.Hypoxia,withoxy-
genlevelsunder0.5%,inducesanarrestinS-phasewhileoxygenlevelsabove0.5%have
littleeffectsoncellproliferation[8].Withtheusedsystemoxygenlevelsbelow0.5%are
achievedafter≈70minofgassing.Sincethetotalgassingtimeis2h,intheadditional
50minapartialsynchronizationofthecellsinS-phasecanappear.Thereoxygenation
afterhypoxiaactivatesATMwhichinducesG2-phasearrest[8].IshowedthatRAT-1
cellsunderwentG2/M-phasearrestafterirradiationindependentofthereoxygenationaf-
terirradiation.TheinducedG2/M-phasearrestafterreoxygenation,isthereforeofminor
importanceintheperformedexperiments.
Asmentionedpreviously,themediumpHofoxicsamplesinhypoxiachambershifted
towardalkalinevalueswithin2h.Musgroveetal.demonstratedthattheintracellularpH
hasaninuenceonthecellcyclephase[94].HigherpHisassociatedwithenrichmentof
cellsinS-,G2-,andM-phase.IftheextracellularpHofthemediuminuencetheintra-
cellularpHwithin2h,isnotknown.Butthepossiblecellcycleshiftintheoxicsamples
wouldbecomparabletothatofthehypoxicsamples.

TheOER10,calculatedwiththecumulativecurves,forx-rayirradiationwas2.35±0.13

89

outlookandDiscussion4

inthisthesis.ButadosedependentchangeoftheOERwasdetected.Forhighersurvival
levelstheOERdecreasedto2.18(90%survival).ThishasalsobeenreportedbyFreyer
etal.whichanalyzedtheOERasafunctionofcellcycle[36].TheOERwashigherfor
cellsinS-phase(2.8-2.9)comparedtocellsinG2-phase(2.6-2.7)orG1-phase(2.3-2.4).
TheyconcludedthattheOERofcellsinonecellcyclephaseisapurelydose-modifying
effect.InasynchronousproliferatingcellsincreasedtheOERwithdosagebecauseofthe
changingradio-resistanceofthecellsindifferentcellcyclephases.ThechangingOER
inthisthesisdemonstratedthatnopartlysynchronizationtookplacethroughthehypoxia
orthepHshiftintheoxicsamples.Theoxicsurvivalcurvesuctuatedmorethanthe
hypoxicsurvivalcurveswhichisindicatedbyhigherstandarddeviations(seethreepara-
graphsabove).Withthemostsensitiveandthemostresistantoxicsurvivalcurveaswell
asthecumulativehypoxicsurvivalcurveaminimumOER10of2.01andanmaximum
OER10of2.84wascalculated.
AcomparisonofthedeterminedOERvalueswithliteraturedataispresentedintable4.1.
TheOERforx-raysisinagreementwiththevaluesdeterminedinZölzerandStreffer
[151]whileHirayamaetal.measuredahigherOER[56].Thedifferencesmaybeex-
plainedbytheusedcelllines.InthecaseofthisworkaswellasinZölzerandStreffer
studytumorcellswereused[151]whileHirayamaetal.workedwithnormalcells[56].
Furthermore,theirradiationmodalitiesinthisworkweremoresimilartoZölzerandStr-
effer[151]thantoHirayamaetal.[56].ButconcerningthehypoxicgassingHirayamaet
al.performedtheirexperimentsusingacomparableset-up[56].Intheitemizedliterature
(seetable4.1)theoxicsamplesweregassedwithair/5%CO2whichwasnotdoneinthis
work.Asmentionedaboveinfutureexperimentstheoxicsampleswillbegassedaswell.
Asexpected,OERvaluesobtainedinmodelcalculationoftheclinicaldataonprostate
cancerpatientsarelower,as100%hypoxiawillnotbereachedintumorsorotherwise
tissue.necrotictoleads

4.4.2OERforcarbonionirradiation

TheRBEvaluesofcarbonionexperimentsdeterminedunderoxicconditionsinthehy-
poxiachambercanbecomparedwithRBEvaluesdeterminedincultureasksunderthe
sameirradiationconditions.Figure4.6presentsthecomparison.ForRAT-1cellsgrow-
ingonbioFolie25theRBE10,oxicwas2.5whiletheRBEα,oxicwas6.0.Infigure4.6it
isshownthattheRBE10,oxicvaluefitsperfectlytotheRBEvaluesdeterminedinculture
askswhiletheRBEα,oxicishigher.Thedeviationmightbeduetothefactthattheun-
derlyingfitparameterαofthesurvivalcurvesisverysensitivetouctuationsespecially

90

4.4Hypoxiachamber-measurementoftheoxygeneffect

Table4.1:ComparisonofOERvaluesfoundinliterature(Lit)andOERvaluesdeterminedinthis
ork.w

LitThisorkw]151[

]56[Thisorkw]40[]56[[]141

IrradiationlineCellGassing95%N2+5%CO2RAT-1tumor,rat250kVpx-ray
/air95%N2+5%CO2Be11tumor,human240kVpx-ray
/air+5%CO2,MeWotumor,human
3hunder4197tumor,human
hypoxiccondition4451tumor,human
95%N2+5%CO2/CHOnormal,hamster200kVpx-ray
COair+5%295%N2+5%CO2RAT-1tumor,ratcarbonion,LET
/air100keV/µm
95%N2+5%CO2/salivaryglandtumor,carbonion,LET
air+5%CO2human100keV/µm
hamsternormal,V7995%N2+5%CO2/CHOnormal,hamstercarbonion,LET
air+5%CO280keV/µm
clinicaldata,prostatecancerpatients,modelcalculation

OER0.12.2±0.52.1±0.32.1±0.12.3±0.72.5±2.80.2±1.52≈2≈0.01.8±1.2-(CI1.41.8)

91

4outlookandDiscussion

Figure4.6:RBEα(circle)andRBE10(square)determinedwithRAT-1cellsincultureasks
(closedsymbols)comparedwithRAT-1cellsonbioFolie25inthehypoxiachamber(opensym-
bols)plottedagainsttheLET.

inthecaseoflowstatisticslikethatforsurvivalcurvesirradiatedwithcarbonions(oxic:
n=2)inthehypoxiachamber.Inprinciple,themeasuredRBEvaluesindicatesthatthe
cellsurvivalratewasnotaffectedthroughthecellgrowthonbioFolie25andthesample
method.preparation

TheOERobtainedforcarbonirradiationwithaSOBPandaLETof100kev/µmwas1.5.
Thisvalueiscomparedtoliteraturedataintable3.11aswell.Despitethelowstatistics
ofcarbonionexperimentsperformedinhypoxiachamber(oxic:n=2,hypoxic:n=1)the
obtainedOERvaluewasinagreementwithpublisheddata.Additionalexperimentswill
beperformedanddetermineamoreprecisevalue.

4.4.3Gassingmodalities:acuteorchronichypoxia

Itwasdemonstratedthatthedevelopedhypoxiachamberissuitableforx-rayandcarbon
ionirradiationunderhypoxicandoxicconditions.Thehypoxiawasachievedbygassing
with200ml/minover2hwith95%N2/5%CO2asmeasuredwiththeopticalO2-sensor
andthethermalmassowmeter.
Thenormaloxygensupplyintissueisrangingfromatmosphericlevelsofnearly21%O2
(159mmHg)to2-8%O2(15-60mmHg)[84].Tumortissueoftenischaracterizedbya
partialloweroxygenpressureof≈10mmHg(1.32%O2)[14]whichcausesalteredgene
expressionandradio-resistance[18].Thislowertissuesupplyoccurswhenthesupporting
bloodvesselismorethan100-150µmawayfromthecells.Studiesshowthathypoxiaisa
verydynamicprocessintissue.Anacutehypoxicstatusinvivoisresolvedwithin20min
whilechronichypoxiacanretainforhoursorevendays[14].Withthisdefinitionthe
performedexperimentsweredoneunderchronichypoxia.Tomeasurecellsunderacute
hypoxia,thechamberhastobedeoxygenated,irradiatedandreoxygenatedwithin20min.

92

4.4Hypoxiachamber-measurementoftheoxygeneffect

Thisistechnicallynotpossiblewiththeexistingchamberandsamplering.Itisdifficult
toevaluateifthehypoxiadefinitionsmadeinvivoshouldbeadaptedtoinvitroexperi-
ments.Therearepublicationsinwhichhypoxiaisachievedin1h[40,56]butshorter
timeperiodstoreachhypoxiathroughgassingcouldnotbefoundinscientificliterature.
Todevelopnewstrategiesfordeoxygenating,theoxygencontentinthechamberwascal-
culated.Thehypoxiachambersampleringhasacapacityof1.4mlmedium.Theoxygen
contentin1.4mlmediumcanbecalculatedwiththeassumptionthattheoxygenamount
solvedinmediumisequaltothatsolvedinwater.Theroomtemperatureisassumed
tobe25°Cwhichcorrespondsto8mg/lsolvedoxygeninwater.Themolecularweight
ofO2is32g/mol.Takentogetherin1.4mlwater,respectivelymedium,0.35µmolof
oxygenissolved.TheoxygenexchangeisproceedviathewholesurfaceofthebioFolie
25(6.3µmol/cm2h)whichisincontacttomedium(7.6cm2).Duringthe2hofgassing
anexchangeof≈95µmolO2ispossible.ThismeansthatthepermeabilityofbioFolie
25foroxygendoesnotlimitthetimeforreachinghypoxia.Thedeoxygenatingprocess
ofthemediumcanbemodulatedthroughthegassingasshownundersubsection2.5.3.
Thehigherinuxenhancesthegascross-owalongthemedium/membrane-interfaceand
ensuresaconsequentlylowoxygenlevelalongthemembrane-interface.Thismighthave
hadaninuenceontheoxygenconcentrationgradientinthemedium.Inaddition,the
oxygenconsumptionofcellsgrowingonthemembraneisimportantfortherapidde-
creaseoftheO2.TypicalO2uptakeratioforCHOcells,whichweregrownfor48hin
culture,weredeterminedtobe1.8∙10-13molO2/h∙cell[27].AssumingthatoneRAT-1cell
hasasimilarO2uptakeratioandthat2x105RAT-1cellsweregrownonthemembrane,
thesecellswouldconsume0.128µmolO2within2hofgassing.Itseemsthatthediffu-
sionofoxygenmoleculesfromdeepermediumlayerstothemedium/membrane-interface
definethetimeforachievinghypoxiaconditions.In1955WilkeandChangdevelopeda
correlationfordiffusioncoefficientinliquids[146]whichisbasedonStockes-Einstein
equation.

5.0DO2−H2O=7.4∙10−8T∙(ΨH2O∙0.M6H2O)(4.1)
VηO2∙WithEq.4.1thediffusioncoefficientforoxygeninwaterDO2−H2O[cm2/s]wascalculated
tobe2.26∙10−5cm2/swhereTistheabsolutetemperature[25°C=298.15K],ΨH2Oan
associationparameterforthesolventwater[2.26;[111]],MH2Othemolecularweightof
water[18g/mol],ηtheviscosityofwater[0,8904cP=g/cm∙s∙10-2;[143]],andVO2the
molarvolumeofoxygen[25.6cm3/mol;[144]].During2hofgassinganO2molecule
candiffusethrough0.163cm2.ThistimeistheminimumneededforanO2moleculeto
diffusefromthecenterofthemediumlayeroverthemembranewhenassumingthatthe

93

Discussion4outlookand

moleculemoveswithadirectedmotiontothemembranecausedbytheoxygenconcen-
trationgradientinthemedium.Tomeasurecellsunderacutehypoxiatheamountmedium
andtheamountofO2hastobedecreased.Thisisonlyfeasiblewithreducedthicknessof
thesampleringfrom3mmtolowervalues.

chamber:Hypoxia4.4.4outlook

Besidethegassingprocedurethereareotherstepswhichneedtobeoptimized.The
siliconpasteforconnectingthefoilswiththeringisatimeconsumingmethodandisnot
suitableforhighsamplenumbers.Testswithdifferentgluesareinprogresstosimplify
thetechnique,reducethepreparationtime,andprovideopportunitiesforthesterilization
process.Forupcomingresearchthequestionoftheirradiationofseveralsamplesin
onechamberisofinteresttomeasuree.g.acompletedepthdoseprofileunderhypoxic
conditions.Withregardtosimulatingatumorwithitssurroundingtissueachamberwill
bedevelopedinwhichsampleswithdifferentoxygenstatuscanbemeasured.

ThecooperationwithDr.P.PeschkefromtheDKFZallowsaninvivo/invitrocomparison
ofalldata.Concerninghypoxia,adirectcomparisondoesnotseemtobepossible.Halin
etal.exposedRAT-1cellsinaBillups-RothbergInc.chamberover24htoahypoxic
gasmixture(1%O2,5%CO2,94%N2)andanalyzedthestatusofangiogeneticfactors
andchemokinesafterward.Theinvitrodatawerecomparedtoinvivoexperiments.The
authorsreportedthatnomajordifferencesbetweeninvitroRAT-1cellsunderhypoxic
ornormoxicconditionsweredetectedbutthecomparisontotheinvivodatashowed
largedifferencestotheinvitrodata.Theyconcludedthatthechangedexpressionprofile
betweentheRAT-1tumorsandtheRAT-1cellswasduetothesurroundingtumorhost
].51[cells

94

yBibliograph

[1]B.Alberts,D.Bray,A.Johnson,J.Lewis,M.Raff,K.Roberts,andP.Walter.
LehrbuchderMolekularenZellbiologie,2.korrigierteAuage.Wiley-VCHVerlag
GmbH,Weinheim,Germany,2001.

[2]K.L.Andarawewa,J.Paupert,A.Pal,andM.H.Barcellos-Hoff.Newrationales
forusingTGFbetainhibitorsinradiotherapy.InternationalJournalofRadiation
Biology,83(11-12):803–811,Dec.2007.PMID:18058368.

[3]V.Anzenberg,S.Chandiramani,andJ.A.Coderre.LET-dependentbystanderef-
fectscausedbyirradiationofhumanprostatecarcinomacellswithxraysoralpha
particles.RadiationResearch,170(4):467–476,Oct.2008.PMID:19024654.

[4]ATCC.E-mailcommunication:.IEC-6cellline,September2007.

[5]B.Azzarone,C.Pottin-Clemenceau,P.Krief,E.Rubinstein,C.Jasmin,M.Scud-
eletti,andF.Indiveri.Areinterleukin-2andinterleukin-15tumorpromotingfac-
torsforhumannon-hematopoieticcells?EuropeanCytokineNetwork,7(1):27–36,
8704093.PMID:1996..Mar

[6]M.Balasoiu,A.Turculeanu,C.Avramescu,V.Comanescu,C.Simionescu,and
L.Mogoanta.Cytokineslevelsinprostateadenocarcinomas.RomanianJournalof
MorphologyandEmbryology=RevueRoumaineDeMorphologieEtEmbryologie,
16444302.PMID:2005.46(3):179–182,

[7]P.L.Beck,I.M.Rosenberg,R.J.Xavier,T.Koh,J.F.Wong,andD.K.Podol-
sky.Transforminggrowthfactor-betamediatesintestinalhealingandsusceptibility
toinjuryinvitroandinvivothroughepithelialcells.TheAmericanJournalof
Pathology,162(2):597–608,Feb.2003.PMID:12547717.

[8]Z.Bencokova,M.R.Kaufmann,I.M.Pires,P.S.Lecane,A.J.Giaccia,andE.M.
Hammond.ATMactivationandsignalingunderhypoxicconditions.Molecular
andCellularBiology,29(2):526–537,2009.PMID:18981219.

[9]S.Bhattacharya,R.M.Ray,andL.R.Johnson.Integrinbeta3-mediatedsrcac-
tivationregulatesapoptosisinIEC-6cellsviaaktandSTAT3.TheBiochemical
Journal,397(3):437–447,Aug.2006.PMID:16669788.

95

aphygrBiblio

[10]K.Bühling.Intensivkurs-AllgemeineundspeziellePathologie,3.Auage.Urban
,Fischer&2004.

[11]BiochromAG.Telefonecommunication:.aboutcellcultureserum,2009.

[12]V.A.Bourke,D.Zhao,J.Gilio,C.Chang,L.Jiang,E.W.Hahn,andR.P.Mason.
Correlationofradiationresponsewithtumoroxygenationinthedunningprostate
R3327-AT1tumor.InternationalJournalofRadiationOncology,Biology,Physics,
67(4):1179–1186,Mar.2007.PMID:17336219.

[13]S.Brons.Privatecommunication:.alpha/betaratioforprostatecancertreatment,
2009.

[14]J.M.Brown.Tumormicroenvironmentandtheresponsetoanticancertherapy.
CancerBiology&Therapy,1(5):453–458,Oct.2002.PMID:12496469.

[15]A.Buwe,C.Steinlein,M.R.Koehler,I.Bar-Am,N.Katzin,andM.Schmid.
Multicolorspectralkaryotypingofratchromosomes.CytogeneticandGenome
Research,103(1-2):163–168,2003.PMID:15004481.

[16]D.J.Carlson,R.D.Stewart,X.A.Li,K.Jennings,J.Z.Wang,andM.Guerrero.
Comparisonofinvitroandinvivoalpha/betaratiosforprostatecancer.Physicsin
MedicineandBiology,49(19):4477–4491,Oct.2004.PMID:15552412.

[17]C.Cepko,E.Ryder,D.M.Fekete,andS.Bruhn.Detectionofß-galactosidaseand
aljalinephosphataseactiitiesintissue,2009.

[18]N.Chan,M.Milosevic,andR.G.Bristow.Tumorhypoxia,DNArepairand
prostatecancerprogression:newtargetsandnewtherapies.FutureOncology(Lon-
don,England),3(3):329–341,June2007.PMID:17547528.

[19]A.D.ChristandR.S.Blumberg.Theintestinalepithelialcell:immunological
aspects.SpringerSeminarsinImmunopathology,18(4):449–61,1997.PMID:
9144864.

[20]R.J.Cohen,T.M.Wheeler,H.Bonkhoff,andM.A.Rubin.Aproposalonthe
identification,histologicreporting,andimplicationsofintraductalprostaticcar-
cinoma.ArchivesofPathology&LaboratoryMedicine,131(7):1103–1109,July
17616999.PMID:2007.

[21]S.E.Combs,A.Nikoghosyan,O.Jaekel,C.P.Karger,T.Haberer,M.W.Mün-
ter,P.E.Huber,J.Debus,andD.Schulz-Ertner.Carbonionradiotherapyfor

96

aphygrBiblio

pediatricpatientsandyoungadultstreatedfortumorsoftheskullbase.Cancer,
115(6):1348–1355,Mar.2009.PMID:19156905.

[22]D.B.Cooke,V.E.Quarmby,D.D.Mickey,J.T.Isaacs,andF.S.French.Oncogene
expressioninprostatecancer:Dunningr3327ratdorsalprostaticadenocarcinoma
system.TheProstate,13(4):263–272,1988.PMID:3217275.

[23]H.Corban-Wilhelm,W.E.Hull,G.Becker,U.Bauder-Wüst,D.Greulich,and
J.Debus.Cytosinedeaminaseandthymidinekinasegenetherapyinadun-
ningratprostatetumourmodel:absenceofbystandereffectsandcharacterisa-
tionof5-uorocytosinemetabolismwith19F-NMRspectroscopy.GeneTherapy,
12424609.PMID:2002.Dec.9(23):1564–1575,

[24]N.CordesandC.C.Park.ß-1integrinasamoleculartherapeutictarget.Inter-
nationalJournalofRadiationBiology,83(11-12):753–760,Dec.2007.PMID:
18058364.

[25]D.Danielpour.Functionsandregulationoftransforminggrowthfactor-ßinthe
prostate.EuropeanJournalofCancer(Oxford,England:1990),41(6):846–57,
15808954.PMID:2005..Apr

[26]D.Danielpour,K.Kadomatsu,M.A.Anzano,J.M.Smith,andM.B.Sporn.De-
velopmentandcharacterizationofnontumorigenicandtumorigenicepithelialcell
linesfromratdorsal-lateralprostate.CancerResearch,54(13):3413–3421,July
8012960.PMID:1994.

[27]R.R.DeshpandeandE.Heinzle.On-lineoxygenuptakerateandcultureviabil-
itymeasurementofanimalcellcultureusingmicroplateswithintegratedoxygen
sensors.BiotechnologyLetters,26(9):763–767,May2004.PMID:15195979.

[28]G.P.DimriandJ.Campisi.Molecularandcellbiologyofreplicativesenescence.
ColdSpringHarborSymposiaonQuantitativeBiology,59:67–73,1994.PMID:
7587128.

[29]M.Durante,K.George,andF.A.Cucinotta.Chromosomeslackingtelomeresare
presentintheprogenyofhumanlymphocytesexposedtoheavyions.Radiation
Research,165(1):51–8,2006.PMID:16392962.

[30]R.Eckert,D.Randall,W.Burggren,andK.French.Tierphysiologie.George
ThiemeVerlagStuttgart,2002.

[31]T.Elsässer.Privatecommunication:.Localeffectmodel,2009.

97

aphygrBiblio

[32]T.Elsässer,M.Krämer,andM.Scholz.Accuracyofthelocaleffectmodelfor
thepredictionofbiologiceffectsofcarbonionbeamsinvitroandinvivo.Interna-
tionalJournalofRadiationOncology,Biology,Physics,71(3):866–872,July2008.
18430521.PMID:[33]T.ElsässerandM.Scholz.Clustereffectswithinthelocaleffectmodel.Radiation
Research,167(3):319–29,Mar.2007.PMID:17316069.
[34]P.Erbar.Onkologie,4.Auage.SchattauerStuttgart,2002.
[35]C.A.FeghaliandT.M.Wright.Cytokinesinacuteandchronicinammation.
FrontiersinBioscience:AJournalandVirtualLibrary,2:d12–26,1997.PMID:
9159205.[36]J.P.Freyer,K.Jarrett,S.Carpenter,andM.R.Raju.Oxygenenhancementratio
asafunctionofdoseandcellcyclephaseforradiation-resistantandsensitiveCHO
cells.RadiationResearch,127(3):297–307,Sept.1991.PMID:1886986.
[37]K.Fu,P.Rui-yun,G.Ya-bing,W.De-wen,L.Qing-liang,D.Bo,andM.Jun-jie.
Effectofil-2onthegrowthandapoptosisofintestinalepithelialcellsradiatedby
neutronandmechanismsofil-2ontheinjurediec-6.ChineseJournalofCellular
andMolecularImmunology,23(8):723–726,Aug.2007.PMID:17618564.
[38]K.Fu,R.yunPeng,Y.bingGao,D.wenWang,Q.liangLuo,Y.Yang,and
B.Dong.Anexperimentalstudyoftheeffectofil-2onthegrowthofirradiated
intestinalepithelialcells.ChineseJournalofCellularandMolecularImmunology,
21(2):250–253,Mar.2005.PMID:15766418.
[39]K.Fukutsu,T.Kanai,Y.Furusawa,andK.Ando.Responseofmouseintestineafter
singleandfractionatedirradiationwithacceleratedcarbonionswithaspread-out
braggpeak.RadiationResearch,148(2):168–174,Aug.1997.PMID:9254736.
[40]Y.Furusawa,K.Fukutsu,M.Aoki,H.Itsukaichi,K.Eguchi-Kasai,H.Ohara,
F.Yatagai,T.Kanai,andK.Ando.Inactivationofaerobicandhypoxiccellsfrom
threedifferentcelllinesbyaccelerated(3)He-,(12)C-and(20)Ne-ionbeams.Ra-
diationResearch,154(5):485–496,Nov.2000.PMID:11025645.
[41]I.García-Tuñón,M.Ricote,A.Ruiz,B.Fraile,R.Paniagua,andM.Royuela.
Interleukin-2anditsreceptorcomplexalpha,betaandgammachainsininsitu
andinfiltrativehumanbreastcancer:animmunohistochemicalcomparativestudy.
BreastCancerResearch:BCR,6(1):R1–7,2004.PMID:14680494.

98

grBiblioaphy

[42]M.Gaugler,M.Neunlist,S.Bonnaud,P.Aubert,M.Benderitter,andF.Paris.In-
testinalepithelialcelldysfunctionismediatedbyanendothelial-specificradiation-
inducedbystandereffect.RadiationResearch,167(2):185–193,Feb.2007.PMID:
17390726.

[43]A.A.Geldof,M.A.B.D.Plaizier,I.Duivenvoorden,M.Ringelberg,R.T.Ver-
steegh,D.W.W.Newling,andG.J.J.Teule.Cellcycleperturbationsandradiosen-
sitizationeffectsinahumanprostatecancercellline.JournalofCancerResearch
andClinicalOncology,129(3):175–182,Mar.2003.PMID:12684891.

[44]E.Giovannucci.Insulinandcoloncancer.CancerCauses&Control:CCC,
6(2):164–179,Mar.1995.PMID:7749056.

[45]V.C.Gray-Schopfer,S.C.Cheong,H.Chong,J.Chow,T.Moss,Z.A.Abdel-
Malek,R.Marais,D.Wynford-Thomas,andD.C.Bennett.Cellularsenescencein
naeviandimmortalisationinmelanoma:aroleforp16?BritishJournalofCancer,
16880792.PMID:2006.Aug.95(4):496–505,

[46]V.C.Gray-Schopfer,J.K.Soo,andD.C.Bennett.Commenton:Absenceof
senescence-associatedß-galactosidaseactivityinhumanmelanocyticneviinvivo.
JInvestDermatol,128(6):1581,2008.

[47]A.Grishin,H.Ford,J.Wang,H.Li,V.Salvador-Recatala,E.S.Levitan,and
E.Zaks-Makhina.Attenuationofapoptosisinenterocytesbyblockadeofpotas-
siumchannels.AmericanJournalofPhysiology.GastrointestinalandLiverPhys-
iology,289(5):G815–821,Nov.2005.PMID:16020659.

[48]A.V.Grishin,J.Wang,D.A.Potoka,D.J.Hackam,J.S.Upperman,P.Boyle,
R.Zamora,andH.R.Ford.Lipopolysaccharideinducescyclooxygenase-2inin-
testinalepitheliumviaanoncanonicalp38MAPKpathway.JournalofImmunology
(Baltimore,Md.:1950),176(1):580–588,2006.PMID:16365453.

[49]X.Guo,J.N.Rao,L.Liu,T.Zou,D.J.Turner,B.L.Bass,andJ.Wang.Regulation
ofadherensjunctionsandepithelialparacellularpermeability:anovelfunctionfor
polyamines.AmericanJournalofPhysiology.CellPhysiology,285(5):C1174–
1187,Nov.2003.PMID:12853285.

[50]T.Haberer,W.Becher,D.Schardt,andG.Kraft.Magneticscanningsystemfor
heavyiontherapy.NuclearInstrumentsandMethods,A330:296–365,1993.

[51]S.Halin,S.H.Rudolfsson,N.V.Rooijen,andA.Bergh.Extratumoral
macrophagespromotetumorandvasculargrowthinanorthotopicratprostate

99

aphygrBiblio

tumormodel.Neoplasia(NewYork,N.Y.),11(2):177–186,Feb.2009.PMID:
19177202.

[52]E.J.Hall.Radiobiologyfortheradiologist,ThirdEdition.JBLippincottCompany,
1988.USA,Philadelphia,

[53]S.Hautmann,E.Huland,A.Wullbrand,M.Friedrich,andH.Huland.Treatmentof
metastatichormone-refractoryprostateadenocarcinoma(MatLyLu)incopenhagen
ratswithmicro-osmoticinterleukin-2pumps.AnticancerResearch,20(6B):4495–
11205294.PMID:2000.Dec.4498,

[54]S.H.Hautmann,E.Huland,andH.Huland.Localintratumorimmunotherapyof
prostatecancerwithinterleukin-2reducestumorgrowth.AnticancerResearch,
10470215.PMID:1999.19(4A)(4A):2661–3,

[55]R.Henriksson,A.Widmark,A.Bergh,andJ.E.Damber.Interleukin-2-induced
growthinhibitionofprostaticadenocarcinomadunningR3327inrats.Urological
Research,20(3):189–91,1992.PMID:1615579.

[56]R.Hirayama,Y.Furusawa,T.Fukawa,andK.Ando.RepairkineticsofDNA-DSB
inducedbyx-raysorcarbonionsunderoxicandhypoxicconditions.Journalof
RadiationResearch,46(3):325–332,Sept.2005.PMID:16210789.

[57]N.M.Hoosein.Neuroendocrineandimmunemediatorsinprostatecancerprogres-
sion.FrontiersinBioscience:AJournalandVirtualLibrary,3:D1274–1279,Dec.
9851913.PMID:1998.

[58]J.S.Horoszewicz,S.S.Leong,T.M.Chu,Z.L.Wajsman,M.Friedman,L.Pap-
sidero,U.Kim,L.S.Chai,S.Kakati,S.K.Arya,andA.A.Sandberg.TheLNCaP
cellline–anewmodelforstudiesonhumanprostaticcarcinoma.ProgressinClin-
icalandBiologicalResearch,37:115–132,1980.PMID:7384082.

[59]C.J.Hunter,V.K.Singamsetty,N.K.Chokshi,P.Boyle,V.Camerini,A.V.Gr-
ishin,J.S.Upperman,H.R.Ford,andN.V.Prasadarao.Enterobactersakazakii
enhancesepithelialcellinjurybyinducingapoptosisinaratmodelofnecrotiz-
ingenterocolitis.TheJournalofInfectiousDiseases,198(4):586–593,Aug.2008.
18588483.PMID:

[60]S.Huveneers,H.Truong,andH.J.Danen.Integrins:signaling,disease,andther-
apy.InternationalJournalofRadiationBiology,83(11-12):743–751,Dec.2007.
17852562.PMID:

100

Biblioaphygr

[61]IAEA.Cytogeneticanalysisforradiationdoseassessment.InternationalAtomic
EnergyAgency.,amanualtech.rep.405edition,2001.

[62]M.Iizuka,K.Sasaki,Y.Hirai,K.Shindo,S.Konno,H.Itou,S.Ohshima,Y.Horie,
andS.Watanabe.Morphogenicproteinepimorphinprotectsintestinalepithelial
cellsfromoxidativestressbytheactivationofEGFreceptorandMEK/ERK,PI3
kinase/Aktsignals.AmericanJournalofPhysiology.GastrointestinalandLiver
Physiology,292(1):G39–52,2007.PMID:16891298.

[63]J.T.Isaacs,W.D.Heston,R.M.Weissman,andD.S.Coffey.Animalmodels
ofthehormone-sensitiveand-insensitiveprostaticadenocarcinomas,dunningR-
3327-H,R-3327-HI,andR-3327-AT.CancerResearch,38(11Pt2):4353–4359,
Nov.1978.PMID:698976.

[64]J.T.IsaacsandB.Hukku.Nonrandominvolvementofchromosome4inthe
progressionofratprostaticcancer.TheProstate,13(2):165–188,1988.PMID:
3174494.

[65]J.T.Isaacs,W.B.Isaacs,W.F.Feitz,andJ.Scheres.Establishmentandcharac-
terizationofsevendunningratprostaticcancercelllinesandtheiruseindevelop-
ingmethodsforpredictingmetastaticabilitiesofprostaticcancers.TheProstate,
3774632.PMID:1986.9(3):261–81,

[66]H.Ishikawa,H.Tsuji,T.Kamada,N.Hirasawa,T.Yanagi,J.Mizoe,K.Akakura,
H.Suzuki,J.Shimazaki,andH.Tsujii.Riskfactorsoflaterectalbleedingaftercar-
boniontherapyforprostatecancer.InternationalJournalofRadiationOncology,
Biology,Physics,66(4):1084–1091,Nov.2006.PMID:16979840.

[67]K.Itahana,J.Campisi,andG.P.Dimri.Methodstodetectbiomarkersofcellular
senescence:thesenescence-associatedß-galactosidaseassay.MethodsinMolecu-
larBiology(Clifton,N.J.),371:21–31,2007.PMID:17634571.

[68]C.Janeway,P.Travers,M.Walport,andM.Shlomchik.Immunologie,5.Auage.
SpektrumAkademischerVerlagGmbH,Heidelberg,Germany,2002.

[69]V.R.Jayanth,M.T.Bayne,andM.E.Varnes.Effectsofextracellularandintracel-
lularpHonrepairofpotentiallylethaldamage,chromosomeaberrationsandDNA
double-strandbreaksinirradiatedplateau-phasea549cells.RadiationResearch,
8052690.PMID:1994.Aug.139(2):152–162,

101

aphygrBiblio

[70]S.Johansson,M.Landström,K.Hellstrand,andR.Henriksson.Theresponseof
dunningR3327prostaticadenocarcinomatoIL-2,histamineandradiation.British
JournalofCancer,77(8):1213–9,Apr.1998.PMID:9579825.

[71]S.Johansson,M.Landström,andR.Henriksson.Alterationsoftumourcells,
stromaandapoptosisinratprostaticadenocarcinomafollowingtreatmentwithhis-
tamine,interleukin-2andirradiation.AnticancerResearch,19(3A):1961–1969,
10470141.PMID:1999.June

[72]M.E.Kaighn,K.S.Narayan,Y.Ohnuki,J.F.Lechner,andL.W.Jones.Es-
tablishmentandcharacterizationofahumanprostaticcarcinomacellline(PC-3).
InvestigativeUrology,17(1):16–23,July1979.PMID:447482.

[73]K.Kimura,C.Bowen,S.Spiegel,andE.P.Gelmann.Tumornecrosisfactor-alpha
sensitizesprostatecancercellstogamma-irradiation-inducedapoptosis.Cancer
Research,59(7):1606–1614,Apr.1999.PMID:10197636.

[74]K.KitagawaandY.Niikura.Caspase-independentmitoticdeath(CIMD).Cell
Cycle(Georgetown,Tex.),7(8):1001–1005,Apr.2008.PMID:18414023.

[75]T.C.Ko,W.Yu,T.Sakai,H.Sheng,J.Shao,R.D.Beauchamp,andE.A.Thomp-
son.TGF-beta1effectsonproliferationofratintestinalepithelialcellsaredue
toinhibitionofcyclind1expression.Oncogene,16(26):3445–3454,July1998.
9692552.PMID:

[76]G.Kraft,H.Daues,B.Fischer,U.Kopf,H.Leibold,D.Quis,H.Stelzer,J.Kiefer,
R.Schoepper,E.Schneider,U.Weber,H.Wulf,andH.Dertinger.Irradiation
chamberandsamplechangesforbiologicalsamples.NuclearInstrumentsand
1980.168:175–179,,Methods

[77]A.Krempler,D.Deckbar,P.A.Jeggo,andM.Löbrich.AnimperfectG2M
checkpointcontributestochromosomeinstabilityfollowingirradiationofSandG2
phasecells.CellCycle(Georgetown,Tex.),6(14):1682–1686,July2007.PMID:
17637566.

[78]M.Krämer,O.Jäkel,T.Haberer,G.Kraft,D.Schardt,andU.Weber.Treatment
planningforheavy-ionradiotherapy:physicalbeammodelanddoseoptimization.
PhysicsinMedicineandBiology,45(11):3299–317,Nov.2000.PMID:11098905.

[79]D.J.KuhnandQ.P.Dou.Theroleofinterleukin-2receptoralphaincancer.
FrontiersinBioscience:AJournalandVirtualLibrary,10:1462–74,2005.PMID:
15769637.

102

aphygrBiblio

[80]D.J.Kurz,S.Decary,Y.Hong,andJ.D.Erusalimsky.Senescence-associated
(beta)-galactosidasereectsanincreaseinlysosomalmassduringreplicativeage-
ingofhumanendothelialcells.JournalofCellScience,113(Pt20):3613–3622,
11017877.PMID:2000.Oct.

[81]M.LöbrichandP.A.Jeggo.TheimpactofanegligentG2/Mcheckpointonge-
nomicinstabilityandcancerinduction.NatureReviews.Cancer,7(11):861–869,
Nov.2007.PMID:17943134.

[82]B.Y.Lee,J.A.Han,J.S.Im,A.Morrone,K.Johung,E.C.Goodwin,W.J.
Kleijer,D.DiMaio,andE.S.Hwang.Senescence-associatedbeta-galactosidase
islysosomalbeta-galactosidase.AgingCell,5(2):187–195,Apr.2006.PMID:
16626397.

[83]C.Lee,S.M.Sintich,E.P.Mathews,A.H.Shah,S.D.Kundu,K.T.Perry,J.S.
Cho,K.Y.Ilio,M.V.Cronauer,L.Janulis,andJ.A.Sensibar.Transforming
growthfactor-betainbenignandmalignantprostate.TheProstate,39(4):285–90,
10344218.PMID:1999.June

[84]A.S.E.Ljungkvist,J.Bussink,J.H.A.M.Kaanders,andA.J.vanderKogel.
Dynamicsoftumorhypoxiameasuredwithbioreductivehypoxiccellmarkers.Ra-
diationResearch,167(2):127–145,Feb.2007.PMID:17390721.

[85]R.Lüllmann-Rauch.Histologie.ThiemeVerlagStuttgart,2003.

[86]A.Luch.Cellcyclecontrolandcelldivision:implicationsforchemicallyin-
ducedcarcinogenesis.Chembiochem:AEuropeanJournalofChemicalBiology,
12325006.PMID:2002.June3(6):506–516,

[87]S.LyuandW.Park.Mistletoelectinmodulatesintestinalepithelialcell-derived
cytokinesandbcellIgAsecretion.ArchivesofPharmacalResearch,32(3):443–
451,Mar.2009.PMID:19387590.

[88]B.S.Marasa,L.Xiao,J.N.Rao,T.Zou,L.Liu,J.Wang,E.Bellavance,D.J.
Turner,andJ.Wang.InducedTRPC1expressionincreasesproteinphosphatase2A
sensitizingintestinalepithelialcellstoapoptosisthroughinhibitionofNF-kappaB
activation.AmericanJournalofPhysiology.CellPhysiology,294(5):C1277–1287,
18322138.PMID:2008.May

[89]L.Marignol,M.Coffey,M.Lawler,andD.Hollywood.Hypoxiainprostatecan-
cer:apowerfulshieldagainsttumourdestruction?CancerTreatmentReviews,
18334284.PMID:2008.June34(4):313–27,

103

aphygrBiblio

[90]D.W.McGee,T.Bamberg,S.J.Vitkus,andJ.R.McGhee.Asynergisticrela-
tionshipbetweenTNF-alpha,IL-1beta,andTGF-beta1onIL-6secretionbythe
IEC-6intestinalepithelialcellline.Immunology,86(1):6–11,Sept.1995.PMID:
7590883.

[91]J.E.McNeal.Thezonalanatomyoftheprostate.TheProstate,2(1):35–49,1981.
7279811.PMID:

[92]C.Michaloglou,M.S.Soengas,W.J.Mooi,andD.S.Peeper.Commenton"Ab-
senceofsenescence-associatedbeta-galactosidaseactivityinhumanmelanocytic
neviinvivo".TheJournalofInvestigativeDermatology,128(6):1582–1583;au-
thorreply1583–1584,June2008.PMID:18478016.

[93]T.R.Munro.Therelativeradiosensitivityofthenucleusandcytoplasmofchi-
nesehamsterfibroblasts.RadiationResearch,42(3):451–70,June1970.PMID:
5463516.

[94]E.Musgrove,M.Seaman,andD.Hedley.RelationshipbetweencytoplasmicpH
andproliferationduringexponentialgrowthandcellularquiescence.Experimental
CellResearch,172(1):65–75,Sept.1987.PMID:3653258.

[95]A.E.Nahum,B.Movsas,E.M.Horwitz,C.C.Stobbe,andJ.D.Chapman.In-
corporatingclinicalmeasurementsofhypoxiaintotumorlocalcontrolmodelingof
prostatecancer:implicationsforthealpha/betaratio.InternationalJournalofRa-
diationOncology,Biology,Physics,57(2):391–401,Oct.2003.PMID:12957250.

[96]R.Négrel,P.Rampal,J.L.Nano,C.Cavenel,andG.Ailhaud.Establishmentand
characterizationofanepithelialintestinalcelllinefromratfetus.Experimental
CellResearch,143(2):427–437,Feb.1983.PMID:6131830.

[97]A.Nikoghosyan,D.Schulz-Ertner,B.Didinger,O.Jäkel,I.Zuna,A.Höss,
M.Wannenmacher,andJ.Debus.Evaluationoftherapeuticpotentialofheavyion
therapyforpatientswithlocallyadvancedprostatecancer.InternationalJournal
ofRadiationOncology,Biology,Physics,58(1):89–97,2004.PMID:14697425.

[98]D.PaduaandJ.Massagué.RolesofTGFbetainmetastasis.CellResearch,
19050696.PMID:2009.19(1):89–102,

[99]A.R.Patel,J.Li,B.L.Bass,andJ.Y.Wang.Expressionofthetransforming
growthfactor-betageneduringgrowthinhibitionfollowingpolyaminedepletion.
TheAmericanJournalofPhysiology,275(2Pt1):C590–598,Aug.1998.PMID:
9688614.

104

aphygrBiblio

[100]P.Peschke.Privatecommunication:.Dunningprostatecarcinomacellsystem,
2009.

[101]P.Peschke,S.Heimburg,G.Wolber,I.Zuna,andE.W.Hahn.Improvedther-
apeuticresponsebydistincttimingofmultipleheattreatmentsduringinterstitial
radiationinthedunningR3327prostatetumormodel.JournalofCancerResearch
andClinicalOncology,124(3-4):172–178,1998.PMID:9619743.

[102]D.K.Podolsky.Reviewarticle:healingafterinammatoryinjury–coordinationof
aregulatorypeptidenetwork.AlimentaryPharmacology&Therapeutics,14Suppl
1:87–93,Apr.2000.PMID:10807409.

[103]K.M.PriseandJ.M.OSullivan.Radiation-inducedbystandersignallingincancer
therapy.NatureReviews.Cancer,9(5):351–360,May2009.PMID:19377507.

[104]T.T.PuckandP.I.Marcus.Arapidmethodforviablecelltitrationandclone
productionwithhelacellsintissueculture:Theuseofx-irradiatedcellstosup-
plyconditioningfactors.ProceedingsoftheNationalAcademyofSciencesofthe
UnitedStatesofAmerica,41(7):432–7,July1955.PMID:16589695.

[105]A.QuaroniandK.J.Isselbacher.Cytotoxiceffectsandmetabolismof
benzo[a]pyreneand7,12-dimethylbenz[a]anthraceneinduodenalandilealepithe-
lialcellcultures.JournaloftheNationalCancerInstitute,67(6):1353–1362,Dec.
6273638.PMID:1981.

[106]A.Quaroni,J.Wands,R.L.Trelstad,andK.J.Isselbacher.Epithelioidcellcul-
turesfromratsmallintestine.Characterizationbymorphologicandimmunologic
criteria.TheJournalofCellBiology,80(2):248–65,Feb.1979.PMID:88453.

[107]M.M.RahmanandG.McFadden.Modulationoftumornecrosisfactorbymicro-
bialpathogens.PLoSPathogens,2(2):e4,Feb.2006.PMID:16518473.

[108]J.N.Rao,O.Platoshyn,L.Li,X.Guo,V.A.Golovina,J.X.Yuan,andJ.Wang.
ActivationofK(+)channelsandincreasedmigrationofdifferentiatedintestinal
epithelialcellsafterwounding.AmericanJournalofPhysiology.CellPhysiology,
282(4):C885–898,Apr.2002.PMID:11880277.

[109]N.N.Raveendran,K.Silver,L.C.Freeman,D.Narvaez,K.Weng,S.Ganta,and
J.D.Lillich.Drug-inducedalterationstogeneandproteinexpressioninintestinal
epithelialcell6cellssuggestaroleforcalpainsinthegastrointestinaltoxicityof
nonsteroidalanti-inammatoryagents.TheJournalofPharmacologyandExperi-
mentalTherapeutics,325(2):389–399,May2008.PMID:18281595.

105

Biblioaphygr

[110]T.E.Reichert,S.Nagashima,Y.Kashii,J.Stanson,G.Gao,Q.P.Dou,andT.L.
Whiteside.Interleukin-2expressioninhumancarcinomacelllinesanditsrolein
cellcycleprogression.Oncogene,19(4):514–525,2000.PMID:10698521.

[111]R.Reid,J.Praunsnitz,andT.Sherwood.ThePropertiesofGasesandLiquieds,
ThirdEdition.McGraw-Hill,NewYork,1977.

[112]A.R.ReynoldsandN.Kyprianou.Growthfactorsignallinginprostaticgrowth:
significanceintumourdevelopmentandtherapeutictargeting.BritishJournalof
Pharmacology,147Suppl2:S144–52,Feb.2006.PMID:16465179.

[113]V.Rohde,A.Katalinic,andJ.Wasern.GesundheitsberichtserstattungdesBundes
"Prostataerkrankungen",Heft36.RobertKoch-Institut,2007.

[114]M.Royuela,M.P.D.Miguel,F.R.Bethencourt,B.Fraile,M.I.Arenas,and
R.Paniagua.IL-2,itsreceptors,andbcl-2andbaxgenesinnormal,hyperplastic
andcarcinomatoushumanprostates:immunohistochemicalcomparativeanalysis.
GrowthFactors(Chur,Switzerland),18(2):135–146,2000.PMID:11019784.

[115]M.Royuela,G.Rodríguez-Berriguete,B.Fraile,andR.Paniagua.TNF-alpha/IL-
1/NF-kappaBtransductionpathwayinhumancancerprostate.Histologyand
Histopathology,23(10):1279–90,Oct.2008.PMID:18712680.

[116]Y.SambuyandI.D.Angelis.Formationoforganoidstructuresandextracellu-
larmatrixproductioninanintestinalepithelialcelllineduringlong-terminvitro
culture.CellDifferentiation,19(2):139–147,Sept.1986.PMID:3757038.

[117]C.Schicker.EntwicklungeinesSystemszurSimulationhypoxischerTumorefür
dieBestrahlungsplanungmitvariablerbiologischerWirksamkeit.Mastersthesis,
HochschuleDarmstadt,FachbereichChemie-undBiotechnologie,2007.

[118]C.Schicker,C.vonNeubeck,U.Kopf,andW.Kraft-Weyrather.Patent:De10
2008010918.5,Zellkultur-Bestrahlungskammer,2008.

[119]C.Schicker,C.vonNeubeck,U.Kopf,andW.Kraft-Weyrather.Patent:Ep09002
2009.-Bestrahlungskammer,Zellkultur402.7,

[120]M.Scholz,A.M.Kellerer,W.Kraft-Weyrather,andG.Kraft.Computationofcell
survivalinheavyionbeamsfortherapy.themodelanditsapproximation.Radia-
tionandEnvironmentalBiophysics,36(1):59–66,Feb.1997.PMID:9128899.

106

grBiblioaphy

[121]B.W.Schuller,A.B.Rogers,K.S.Cormier,K.J.Riley,P.J.Binns,R.Julius,
M.F.Hawthorne,andJ.A.Coderre.Nosignificantendothelialapoptosisinthe
radiation-inducedgastrointestinalsyndrome.InternationalJournalofRadiation
Oncology,Biology,Physics,68(1):205–210,May2007.PMID:17448874.

[122]D.Schulz-Ertner,C.P.Karger,A.Feuerhake,A.Nikoghosyan,S.E.Combs,
O.Jäkel,L.Edler,M.Scholz,andJ.Debus.Effectivenessofcarbonionradiother-
apyinthetreatmentofskull-basechordomas.InternationalJournalofRadiation
Oncology,Biology,Physics,68(2):449–457,June2007.PMID:17363188.

[123]D.Schulz-Ertner,A.Nikoghosyan,H.Hof,B.Didinger,S.E.Combs,O.Jäkel,
C.P.Karger,L.Edler,andJ.Debus.Carbonionradiotherapyofskullbase
chondrosarcomas.InternationalJournalofRadiationOncology,Biology,Physics,
17056193.PMID:2007.67(1):171–177,

[124]C.Shao,M.Aoki,andY.Furusawa.Medium-mediatedbystandereffectsonHSG
cellsco-cultivatedwithcellsirradiatedbyx-raysora290MeV/ucarbonbeam.
JournalofRadiationResearch,42(3):305–316,Sept.2001.PMID:11840647.

[125]M.W.Shaw,P.D.Guinan,A.Dubin,C.F.McKiel,andM.Rubenstein.Ad-
ministrationofrecombinanttumornecrosisfactortoratsbearingthedunning
r3327MAT-LyLuprostaticadenocarcinoma.ClinicalPhysiologyandBiochem-
istry,5(6):315–319,1987.PMID:3446431.

[126]T.Shigematsu,S.Miura,M.Hirokawa,R.Hokari,H.Higuchi,N.Watanabe,
Y.Tsuzuki,H.Kimura,S.Tada,R.C.Nakatsumi,H.Saito,andH.Ishii.Induction
ofendothelin-1synthesisbyIL-2anditsmodulationofratintestinalepithelialcell
growth.TheAmericanJournalofPhysiology,275(3Pt1):G556–563,Sept.1998.
9724269.PMID:

[127]D.H.SmithandJ.J.DeCosse.Radiationdamagetothesmallintestine.World
JournalofSurgery,10(2):189–94,Apr.1986.PMID:3705603.

[128]K.Song,S.C.Cornelius,andD.Danielpour.Developmentandcharacterizationof
DP-153,anontumorigenicprostaticcelllinethatundergoesmalignanttransforma-
tionbyexpressionofdominant-negativetransforminggrowthfactorbetareceptor
typeII.CancerResearch,63(15):4358–4367,Aug.2003.PMID:12907605.

[129]A.W.Stadnyk,G.R.Sisson,andC.C.Waterhouse.IL-1alphaisconstitutivelyex-
pressedintheratintestinalepithelialcelllineIEC-6.ExperimentalCellResearch,
1995.Oct.220(2):298–303,7556437.PMID:

107

aphygrBiblio

[130]K.R.Stone,D.D.Mickey,H.Wunderli,G.H.Mickey,andD.F.Paulson.Isolation
ofahumanprostatecarcinomacellline(DU145).InternationalJournalofCancer.
JournalInternationalDuCancer,21(3):274–281,Mar.1978.PMID:631930.

[131]J.L.Tatum,G.J.Kelloff,R.J.Gillies,J.M.Arbeit,J.M.Brown,K.S.C.Chao,
J.D.Chapman,W.C.Eckelman,A.W.Fyles,A.J.Giaccia,R.P.Hill,C.J.
Koch,M.C.Krishna,K.A.Krohn,J.S.Lewis,R.P.Mason,G.Melillo,A.R.
Padhani,G.Powis,J.G.Rajendran,R.Reba,S.P.Robinson,G.L.Semenza,
H.M.Swartz,P.Vaupel,D.Yang,B.Croft,J.Hoffman,G.Liu,H.Stone,and
D.Sullivan.Hypoxia:importanceintumorbiology,noninvasivemeasurement
byimaging,andvalueofitsmeasurementinthemanagementofcancertherapy.
InternationalJournalofRadiationBiology,82(10):699–757,Oct.2006.PMID:
17118889.

[132]C.ThomasandP.S.Oates.IEC-6cellsareanappropriatemodelofintestinaliron
absorptioninrats.TheJournalofNutrition,132(4):680–687,Apr.2002.PMID:
11925460.

[133]D.C.Tomlinson,S.H.Freestone,O.C.Grace,andA.A.Thomson.Differential
effectsoftransforminggrowthfactor-beta1oncellularproliferationinthedevel-
opingprostate.Endocrinology,145(9):4292–300,Sept.2004.PMID:15192047.

[134]H.Tsujii,T.Kamada,M.Baba,H.Tsuji,H.Kato,S.Kato,S.Yamada,S.Yasuda,
T.Yanagi,H.Kato,R.Hara,Naotakayamamoto,andJ.Mizoe.Clinicaladvantages
ofcarbon-ionradiotherapy.NewJournalofPhysics,10:075009(16pp),2008.

[135]R.J.vanMoorselaar,B.T.Hendriks,P.vanStratum,P.H.vanderMeide,
F.M.Debruyne,andJ.A.Schalken.Synergisticantitumoreffectsofratgamma-
interferonandhumantumornecrosisfactoralphaagainstandrogen-dependentand
-independentratprostatictumors.CancerResearch,51(9):2329–2334,May1991.
1901759.PMID:

[136]C.vonNeubeckandT.Friedrich.Reportonerrorcalculation.Technicalreport,
2009.GSI,

[137]M.Wakatsuki,H.Tsuji,H.Ishikawa,T.Yanagi,T.Kamada,T.Nakano,H.Suzuki,
K.Akakura,J.Shimazaki,andH.Tsujii.Qualityoflifeinmentreatedwithcar-
boniontherapyforprostatecancer.InternationalJournalofRadiationOncology,
Biology,Physics,72(4):1010–1015,Nov.2008.PMID:18495370.

108

aphygrBiblio

[138]M.F.Walsh,D.R.Ampasala,J.Hatfield,R.V.Heide,S.Suer,A.K.Rishi,and
M.D.Basson.Transforminggrowthfactor-betastimulatesintestinalepithelial
focaladhesionkinasesynthesisviasmad-andp38-dependentmechanisms.The
AmericanJournalofPathology,173(2):385–399,Aug.2008.PMID:18583311.

[139]J.Y.Wang,S.A.McCormack,M.J.Viar,H.Wang,C.Y.Tzen,R.E.Scott,and
L.R.Johnson.Decreasedexpressionofprotooncogenesc-fos,c-myc,andc-jun
followingpolyaminedepletioninIEC-6cells.TheAmericanJournalofPhysiol-
ogy,265(2Pt1):G331–338,Aug.1993.PMID:8368314.

[140]J.Z.Wang,M.Guerrero,andX.A.Li.Howlowisthealpha/betaratiofor
prostatecancer?InternationalJournalofRadiationOncology,Biology,Physics,
12504054.PMID:2003.55(1):194–203,

[141]J.Z.Wang,X.A.Li,andN.A.Mayr.Doseescalationtocombathypoxiain
prostatecancer:aradiobiologicalstudyonclinicaldata.TheBritishJournalof
Radiology,79(947):905–911,Nov.2006.PMID:16885177.

[142]R.WangandJ.A.Coderre.Abystandereffectinalpha-particleirradiationsofhu-
manprostatetumorcells.RadiationResearch,164(6):711–722,Dec.2005.PMID:
16296877.

[143]R.C.Weast,editor.HandbookofChemistryandPhysics54thEdition.CRCPress,
1973.

[144]J.Welty,C.Wicks,andR.Wilson.FundamentalsofMomentum,Heat,andMass
Transfer,ThirdEdition.JohnWiley&Sons,NewYork,1984.

[145]P.Wikström,G.Lindh,A.Bergh,andJ.E.Damber.Alterationsoftransforming
growthfactorbeta1andTGFbetareceptorexpressionswithprogressionindunning
ratprostaticadenocarcinomasublines.UrologicalResearch,27(3):185–93,June
10422820.PMID:1999.

[146]C.WilkeandP.Chang.A.E.CH.E.Journal,1:264–270,1955.

[147]R.Wróblewski,M.Jalnäs,G.V.Decker,J.Björk,J.Wroblewski,andG.M.
Roomans.Effectsofirradiationonintestinalcellsinvivoandinvitro.Histol-
ogyandHistopathology,17(1):165–77,2002.PMID:11813866.

[148]K.Yamazaki,J.E.Lehr,J.S.Rhim,andK.J.Pienta.Establishmentofimmor-
talizedcopenhagenratprostateendothelialcelllines.InVivo(Athens,Greece),
8900918.PMID:1995.Oct.9(5):421–426,

109

aphygrBiblio

[149]

[150]

[151]

110

K.Zachrisson,V.Neopikhanov,A.Samali,andA.Uribe.Interleukin-1,
interleukin-8,tumournecrosisfactoralphaandinterferongammastimulateDNA
synthesisbuthavenoeffectonapoptosisinsmall-intestinalcelllines.Euro-
peanJournalofGastroenterology&Hepatology,13(5):551–9,May2001.PMID:
11396536.

C.M.Zechmann,E.C.Woenne,G.Brix,N.Radzwill,M.Ilg,P.Bachert,
P.Peschke,S.Kirsch,H.Kauczor,S.Delorme,W.Semmler,andF.Kiessling.
Impactofstromaonthegrowth,microcirculation,andmetabolismofexperimental
prostatetumors.Neoplasia(NewYork,N.Y.),9(1):57–67,2007.PMID:17325744.

F.ZölzerandC.Streffer.Increasedradiosensitivitywithchronichypoxiainfour
humantumorcelllines.InternationalJournalofRadiationOncology,Biology,
Physics,54(3):910–920,Nov.2002.PMID:12377345.

wledgementAckno

knowledgAcement

First,letmethankProf.Dr.GerhardKraftandProf.Dr.GerhardThielwhoofferedme
thepossibilityforthiswork.
IthankDr.WilmaK.-Weyratherforhermentoringthroughouttheentiredoctorateand
mytimeatGSI.Shealsoassistedmewiththisdoctoratewithheradviceaswellasher
actions.Iwouldliketoexpressmysincereappreciationtoallcurrentandformermembersofthe
biophysicsgroupwhohavecontributed,directlyorindirectly,tothisdoctorateinformof
technicalorothersupport(inalphabeticorder):DanielaBecker(),WolfgangBecher
(technicalsupportatUNILACfacility),Dr.SandroConrad(MayIhaveone?ELISA
instructor),AnnaConstantinescu(Tryitwith200,RT,water+fiveinline.),Prof.Dr.
MarcoDurante(Discussions:Icallitfreakofnature!),Dr.ThiloElsässer(LEMcalcu-
lations,beamtimesupportatSISfacility,Whatdoyouthinkabout...?),Dr.Claudia
Fournier(Letstalkaboutcytokines!),Dr.ThomasFriedrich(Mustbethisterm!Ithink,
Imsure.),EvaGehrmann(4h?!Notbad!15,000intotal?!Respect!),Dr.Alexander
Gemmel(Shouldbepossibletomoveit...Didyoulostsome?),CarolaHartel(Silver!
MayIhavethisrecipe?),PetraHessel(chromosomesandcocktails),Dr.GheorgheIancu
(nightlybeamtimefriend),KerstinKnoop(MikTexInstructor1),GabrieleKragl(ex-
perimentalsupport,wallpainting),Dr.MichaelKrämer(therealdude,TRiPplans),Dr.
RyonfaLee(anegativeresultandgrammarbook),GünterLenz(technicalsupportatUNI-
LACfacilityandwithgasbottles.Doyouthinkthatyoucanproduceitbymeansofmy
drawing?!),JaninaLindemann(Finaldestination?),EvaKehr(technicalsupportincell
labandexperiments,pastasalad),BarbaraMeyer(Itsraining...natch!)Dr.SylviaRitter
(chromosomeworld),Priv.Doz.Dr.MichaelScholz(LEMcalculations,beamtimesup-
portatSISfacility.Knock,knock,justsomequestions...),JörnSpliter(Tomorrow-half
pastseven?Ey,today...herewegoagain!),FrankTobias(MikTexInstructor2),Dr.Jana
Topsch(owcytometerinstructor,teaandchocolatedonor).
Inaddition,Iwouldliketothankmyparentswhoconsistentlysupportedmeduringmy
yearsofstudy.Theymadethisworkpossible.
Finally,Ialsowanttothankmyfamilyandfriendsfortheirencouragement,proof-
reading,understanding,andpatienceduringthiswork.

111

ErklärungEidesstattliche

ErklärungheEidesstattlic

IcherklärehiermitanEidesStatt,dassichdievorliegendeDissertationselbständigund
nurmitdenangegebenenHilfsmittelnangefertigthabe.

HierdurcherkläreichanEidesStatt,dassichbishernochkeinenPromotionsversuchun-
ternommenhabe.

Darmstadt,November17,2009

..............................
CläreHannaFreiinvonNeubeck

113

Annex

solutionsUsed

ufferbensenSoer

➢0.067MNa2HPO4inpurifiedwater(MerckKGaA,Darmstadt,Germany)
➢0.067MKH2PO4inpurifiedwater(MerckKGaA,Darmstadt,Germany)
➢mixinequalvolumes
6.8pH➢

threefoldmethyleneblue

solutionsUsed

➢300mlLöfersmethylenebluesolution(MerckKGaA,Darmstadt,Germany)
➢90ml0.1%potassiumhydroxideinpurifiedwater
➢50mlmethanolforanalysis(MerckKGaA,Darmstadt,Germany)
➢560mlpurifiedwater

ufferbGal

➢40mMNa2HPO4x12H2O
➢40mMC6H8O7xH2O
➢dissolvein1lpurifiedwater
➢adjustpHwithNaOHto6.0
add➢➢5mMK3[Fe(CN)6]
➢5mMK4[Fe(CN)6]
NaClmM150➢➢2mMMgCl2x6H2O

solutionstainingX-Gal

➢5mgX-Gal(5-bromo-4-chloro-3-indolyl-β-D-galactoside)
➢280µlC3H7NO
➢720µlX-Galbuffer
➢dilutebeforeuseto1mgX-GalwithX-Galbuffer

I

esFigurofList

FofListesigur

1.1Depthdoseprofilesofdifferentparticles......................
1.2Relativebiologicaleffectiveness..........................

2.1IEC-6cellsandRAT-1cells:cellcycledistributioninnormalandinvertedmedium
2.2Co-culture:schematicdrawingofcellseeding...................
2.3Co-culture:schematicdrawingof6-wellplate/insertsystem...........
2.4Cellcycle:aschematicdrawing..........................
2.5IEC-6cellsandRAT-1cells:fixationmethodsandPKH67staining........
2.6Hypoxiachamber..................................
2.7Retainingbracketwithfixedcellsample......................
2.8IEC-6cells:errorcalculationforRBE.......................

3.1RAT-1cellsinculture................................
3.2RAT-1cells:cellcycledistributionafterirradiation................
3.3RAT-1cells:metaphases..............................
3.4RAT-1cells:chromosomeanalysis.........................
3.5IEC-6cellsinculture................................
3.6IEC-6cells:cellcycledistributionindifferentpassages..............
3.7IEC-6cells:cellcycledistributionafterirradiation................
3.8IEC-6cells:metaphases..............................
3.9IEC-6cells:chromosomeanalysis.........................
3.10RAT-1cellscolonies................................
3.11RAT-1cells:survivalcurvesafter250kVpx-rayirradiation............
3.12RAT-1cells:survivalcurvesaftercarbonionsirradiation.............
3.13IEC-6cellcolony..................................
3.14IEC-6cells:survivalcurveafter250kVpx-rayirradiation............
3.15SerumtestofIEC-6cells:survivalcurvesafter250kVPx-rayirradiation....
3.16IEC-6cells:survivalcurveaftercarbonionirradiation..............
3.17IEC-6cells:platingefficiency...........................
3.18RBEαandRBE10..................................
3.19IEC-6cells:mFISHstainedmetaphase.......................
3.20IEC-6cells:cellnumbersofirradiatedcultures..................
3.21IEC-6cells:chromosomeanalysisafterx-rayandcarbonionirradiation.....
3.22IEC-6cells:weightedchromosomenumberandmitoticindexafterx-rayandcar-
bonionirradiation..................................
3.23IEC-6clones:cellgrowthinculture........................

34

1718192527282835

3738393941424343454646474848495152525455565758

III

esigurFofList

IV

3.24IEC-6clones:survivalcurveafter250kVpx-rayirradiation...........
3.25IEC-6cellsandRAT-1cells:co-culturecolonies..................
3.26Co-culture:serumI+serumII,0GyIEC-6cells+irradiatedRAT-1cells....
3.27Co-culture:serumI+serumII,irradiatedIEC-6cells+0GyRAT-1cells....
3.28Co-culture:serumbatchI,irradiatedIEC-6cellsandirradiatedRAT-1cells...
3.29Co-culture:serumII,irradiatedIEC-6cells+irradiatedRAT-1cells.......
3.30TGF-βmeasurements:insertversusPetridish...................
3.31TGF-βmeasurements:mono-cultureversusco-culture..............
3.32RAT-1cellsinhypoxiachamber:survivalunderhypoxicandoxicconditionsafter
x-rayandcarbonionirradiation..........................

4.14.24.34.44.54.6

RAT-1cells:comparisonofsurvivalcurvesaftercarbonionirradiationwithcal-
culationsofLEM..................................
IEC-6cells:comparisonofRBEwithliterature.................
10IEC-6cells:comparisonofsurvivalcurvesaftercarbonionirradiationwithcalcu-
lationsofLEM...................................
Commercialcellculturesystemsforhypoxicconditions..............
RAT-1cells:Comparisonofcellsurvivalafterx-raysincultureasksandsample
ring.........................................
RAT-1cells:comparisonofRBEincultureaskswithRBEonbioFolie25...

596162636364656768

798080888992

ablesTofList

ListablesTof

1.1Prostatecancerclassification............................7

2.1Co-culture:useddosesandenergies........................18
2.2Errorpropagation:calculatederrorpercentageforselecteddoses.........33

3.1RAT-1cells:weightedchromosomenumbersandmitoticindexes.........40
3.2IEC-6cells:weightedchromosomenumbersandmitoticindexes.........44
3.3RAT-1cells:αandβvalues,ratioα/β,andD10ofcarbonionsurvivalcurves..47
3.4IEC-6cells:αandβvalues,ratioα/β,andDof250kVpx-raysurvivalcurves50
103.5IEC-6cells:αandβvalues,ratioα/β,andDofcarbonionsurvivalcurves..51
103.6Physicalparametersofthecarbonionbeams....................53
3.7IEC-6clones:anoverview.............................58
3.8IEC-6clones:platingefficiency,α/βratioandD10of250kVpx-raysurvivalcurves59
3.9IEC-6clones:analysisofkaryotype........................60
3.10Co-culture:overviewoverperformedexperiments.................61
3.11RAT-1cellsinhypoxiachamber:αandβvalues,D,OER,andRBEofx-ray
10andcarbonionsurvivalcurves...........................69

4.1OER:comparisonofliteraturedatawithdataofthisthesis............

91

V

ListofAbbreviations

AbbrofListviationse

β-gal.............β-galactosidase
LET.............dose-averagedLET
ATCC............AmericanTissueCultureCollection
DAPI............4,6-diamidino-2-phenylindole
DMEM..........DulbeccosmodifiedEaglemedium
DMSO...........Dimethylsulfoxide
DNA.............Deoxyribonucleicacid
EDTA............Ethylenediaminetetraaceticacid
ELISA...........Enzyme-LinkedImmunoSorbentAssay
Eq...............Equation
FCS.............FetalCalfSerum
I.................Inoculum
IEC-6............IntestinalEpithelialCellline6
IL-2.............Interleukin2
IMRT............IntensityModulatedRadiotherapy
KCl..............Potassiumchloride
LET.............LinearEnergyTransfer
methodP.........Co-cultureset-upatwhichthecellswerePre-seededbeforetheirradiation
methodT.........Co-cultureset-upatwhichthecellswereTrypsinizedafterirradiation
mFISH...........Multicoloruorescenceinsituhybridization
MI...............Mitoticindex
NC...............Colonynumber/cultureaskwhichshouldgrowinacolonyformingassay
N..............Cellnumber/mlincellsuspension
mlN...............Averageofresultingcolonynumber/sample
ROER.............OxygenEnhancementRatio
–PBS.............PhosphateBufferedSalinewithoutcalciumandmagnesiumions
PE...............PlatingEfficiency

VII

viationseAbbrofList

PE.............
co

PE...........
mono

PFA..............

RAT-1............

RBE.............

RPMI............

S................

SIS..............

SOBP............

TGFβ............

TNFα............

UNILAC.........

WCN............

X-Gal............

VIII

Co-culturePEinco-cultureexperiments

Mono-culturePEinco-cultureexperiments

ydearaformaldehP

DunningR-3327-AT-1cellline

RelativeBiologicalEffectiveness

RoswellParkMemorialInstitutemedium

alvSurvi

vyHeasynchrotronion

PeakBraggOutSpread

Transforminggrowthfactorβ

Tumornecrosisfactorα

UniversalIonLinearAccelerator

numberchromosomeeightedW

-D-gβ5-bromo-4-chloro-3-indolyl-alactoside

EATIVMULUCIRRUC

CLÄREHANNAFREIINVONNEUBECK

144gerstraßeHeidelberDarmstadt64285

Address:

1981September25.yGerman(Main),FrankfurtSingleGerman

DateBirth:ofBirth:ofPlacestatus:MaritalNationality:

Since8/2006

1988-19921992-19931993-200110/2001-4/2006

QualificationsandEducation

LinnéElementarySchool,Frankfurt(Main),Germany
HerderMiddleSchool,Frankfurt(Main),Germany
OberurselMiddleandHighSchool,Oberursel,Germany
(2.1)AbiturGraduation:UniversityofAppliedScience(Darmstadt,Germany);
(1.8)Dipl.-Ing.Graduation:,BiotechnologyThesistitle:Aufbaueines3-dimensionalenSystemszur
biologischenVerifikationderBestrahlungsplanungmit
SchwerionenPromotionsupervisedbyProfDrG.Thiel(TUDarmstadt)
andProfDr.G.KraftatGSIDarmstadt,Biophysicsgroup

vitaeCurriculum

IX

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