RARβ [RAR-beta] trans-repression of AP-1 transcription factor in HeLa cervical cancer cells [Elektronische Ressource] : consequences on transcription of viral and cellular AP-1 controlled genes / presented by Johanna De Castro Arce

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Introduction 3Dissertationsubmitted to theCombined Faculties for the Natural Sciences and for Mathematicsof the Ruperto-Carola University of Heidelberg, Germanyfor the degree ofDoctor of Natural Sciencespresented byJohanna De CastroArce, MSc.BogotÆ, ColombiaOral examinationIntroduction 4RARbbb trans-repression ofAP-1 transcription factorbbin HeLa cervical cancer cells: Consequences on transcription ofviral and cellularAP-1 controlled genesReferees: Prof. Dr. Christine ClaytonProf. Dr. Claus-Hobe Schr derIntroduction 5AcknowledgmentsIwouldliketothankmysupervisorProf.Dr.FrankR slfortheopportunitytojoinhisgroup,forhishelp,guidanceandsupport.Thank to Prof. Dr. Harald zur Hausen for his continuous interest in the development of thiswork.I would like to thank Prof. Dr. Christine Clayton for her support and help in order to reach myPhD.degree.ThankstoProf.Dr.Claus-HobeSchr derforthecorrectionofthismanuscript.IamgratefulltoDr.UbaldoSotoforthetimethatheemployedtohelpmenotonlyintechnicalmatters but also in scientific discussions, thanks for his friendship that was very important formeinaforeigncountry.Iliketothankallthecolleagueswhohelpandsupportmealongthis3years.I want specially thanks my children Daniella andAndrØs Felipe, for all the time that this workdisabled me to be with them. Thanks for their love, patience and confidence. Thanks to myhusbandwhopostponedhisdreamstoallowminescometrue.

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Introduction 3
Dissertation
submitted to the
Combined Faculties for the Natural Sciences and for Mathematics
of the Ruperto-Carola University of Heidelberg, Germany
for the degree of
Doctor of Natural Sciences
presented by
Johanna De CastroArce, MSc.
BogotÆ, Colombia
Oral examinationIntroduction 4
RARbbb trans-repression ofAP-1 transcription factorbb
in HeLa cervical cancer cells: Consequences on transcription of
viral and cellularAP-1 controlled genes
Referees: Prof. Dr. Christine Clayton
Prof. Dr. Claus-Hobe Schr derIntroduction 5
Acknowledgments
IwouldliketothankmysupervisorProf.Dr.FrankR slfortheopportunitytojoinhisgroup,for
hishelp,guidanceandsupport.
Thank to Prof. Dr. Harald zur Hausen for his continuous interest in the development of this
work.
I would like to thank Prof. Dr. Christine Clayton for her support and help in order to reach my
PhD.degree.
ThankstoProf.Dr.Claus-HobeSchr derforthecorrectionofthismanuscript.
IamgratefulltoDr.UbaldoSotoforthetimethatheemployedtohelpmenotonlyintechnical
matters but also in scientific discussions, thanks for his friendship that was very important for
meinaforeigncountry.
Iliketothankallthecolleagueswhohelpandsupportmealongthis3years.
I want specially thanks my children Daniella andAndrØs Felipe, for all the time that this work
disabled me to be with them. Thanks for their love, patience and confidence. Thanks to my
husbandwhopostponedhisdreamstoallowminescometrue.
Thankstomymotherwithoutherlove,supportandfaithinme,Iwouldnotbeabletoreachthe
aim.
ThankstoallmyfamilyinColombiafortheirlove,onlytheyknowhowmucheachonecontributes
tomystabilityhereinGermany.
FinallyIwanttothankallthepeoplethatsomehowcontributestothefinalizationofthisworkat
scientific and personal levels. It is imposible citate all of them here, but I am gratefull for the
support,helpandconfidencethattheygavetome.Introduction 6
Index
I.Introduction ................................................................................................................. 10
Abbreviations ........................................................................................................10
1.1Activatorprotein1(AP-1)transcriptionfactor...............................................11
1.1.1GeneralAspects..................................................................................11
1.1.2AP-1familymembersfuctionandregulation.....................................12
1.1.3ModulationofAP-1function-MAPkinasepathway ..........................15
1.1.3.1Signalingthroughextracellularsignal-regulatedprotein
kinase(ERK)pathway..........................................................16
1.1.3.2Signalingthroughthep38pathway........................................17
1.1.3.3theJunN-terminalkinase(JNK)pathway 17
1.1.3.4OtherphosphorylationeventsthatregulateAP-1
activityindependentofMAPK .............................................19
1.1.4AP-1proteinsasmodulators ofneoplastictransformation................19
1.2Retinoicacidreceptor .................................................................................. 20
1.2.1Generalaspects...................................................................................20
1.2.2Structuralorganization........................................................................21
1.2.3ActivationandrepressionbyRAR/RXR............................................22
1.2.4Retinoicacidreceptor b isoforms.......................................................23
1.2.5acid b 2andcancer..................................................24
1.2.6Retinoicacidreceptor b 2re-expression,retinoicacid
treatmentandtumorregression..........................................................24
1.2.7AP-1 trans-repression,thesecondmodeofRAR 2action................25
1.3HumanPapillomavirus18andcervicalcancer...............................................26
1.3.1 Human Papillomavirus .......................................................................26
1.3.2CellularcontrolofHumanPapillomavirusoncogenetranscription ...28
1.3.2.1TranscriptionfactorsinteractingwiththeHPV-18URR:
RoleofAP-1 .........................................................................28
1.3.3Retinoicacidtreatment,HPV-18E6/E7oncogenesdown
regulationandgrowthinhibitionofcervicalcancercells ..................30
Aimsofthisstudy........................................................................................................... 31
II.Materialsandmethods ............................................................................................... 32
2.1Abbreviations ..................................................................................................32
2.2Materials..........................................................................................................34
2.2.1 Plasmids..............................................................................................34
2.2.2Antibodies...........................................................................................34
bbIntroduction 7
2.2.3 PCR primers .......................................................................................36
2.2.4Oligonucleotidesforelectromobilityshiftassays,EMSA..................37
2.2.5Solutionsandbuffers..........................................................................37
2.2.6Celllines.............................................................................................44
2.2.7Chemicalsandreagents ......................................................................44
2.2.8Laboratoryequipment.........................................................................47
2.2.9Others..................................................................................................47
2.2.10Kits....................................................................................................48
2.3Methods...........................................................................................................48
2.3.1DNAprobespreparation.....................................................................48
2.3.1.1Competentbacteriatransformation........................................48
2.3.1.2Plasmid-DNArestrictionanalysis..........................................49
2.3.1.3DNAextractionfromagarosegel QIAquickgel
extractionkit ........................................................................49
2.3.2Techniquesofcellculture...................................................................49
2.3.2.1Cellculture.............................................................................49
2.3.2.2Thawandfreezedowneukaryoticcells .................................49
2.3.2.3Cellcounting..........................................................................49
2.3.2.4Celltreatmentwithretinoicacid,MG-132andTNF ........... 50
2.3.3Proteinanalysis...................................................................................50
2.3.3.1Nuclearandcytoplasmproteinpreparation............................50
2.3.3.2SDS-totalproteinextract........................................................51
2.3.3.3SDS-Polyacrylamidegelelectrophoresis ...............................51
2.3.3.4Westernblot Semidry ...........................................................52
2.3.3.5Kinaseassay SAPK/JNKassaykit ......................................52
2.3.4Nucleicacidanalysis ..........................................................................53
TM2.3.4.1RNAextraction AbsolutelyRNA RT-PCRminiprepkit ..53
2.3.4.2DNAfromeukaryoticcells....................................53
2.3.4.3DNArestrictionanalysis ........................................................54
2.3.4.4DNAelectrophoresisandSouthernblot.................................54
2.3.4.5RNAandNorthernblot55
2.3.4.6Probelabeling Random-priming andhybridization.............55
2.3.4.7Reversetranscriptionandpolymerasechainreaction:
Semi-quantitativeRT-PCR....................................................55
2.3.5Protein-DNAinteraction.....................................................................56
2.3.5.1Electrophoresismobilityshiftassay(EMSA)........................56
2.3.6Transienttransfectionanalysis............................................................57
2.3.6.1Transienttransfectionprotocol Effectene ............................57
2.3.6.2Fireflyluciferasereportergeneanalysis.................................58
2.3.6.3 -galactosidasenormalization High-sensitivity
-Galactosidaseassaykit ......................................................58
2.3.7Retroviralvectorconstruction59
babIntroduction 8
2.3.7.1CloningofFra-1cDNAonpLXINvector .............................59
TM2.3.7.2TransfectionofthevirusproducingcelllineRetroPack
PT67......................................................................................59
2.3.7.3Viruscollectionandstorage...................................................61
2.3.7.4InfectionofHeLacells...........................................................61
III.Results....................................................................................................................... 62
3.1RAR trans-repressionofAP-1transcriptionfactor ......................................62
3.1.1RAR expressioninHeLaRAR transfectedcells ..........................62
3.1.2EctopicRARb expressionleadstoaselectivereductionofAP-1......64
3.1.2.1EffectofRARb expressiononindividualAP-1family
members................................................................................64
3.1.2.2Mechanismofc-Jundownregulation....................................65
3.1.3Increaseofc-JunproteinleadstoAP-1/DNA-binding
reconstitutioninHeLaRARb clones .................................................67
3.1.3.1Overexpressionofexogenousc-JuninHeLaand
HeLaRAR clones...............................................................67
3.1.3.2Tumornecrosisfactor treatmentinHeLaand
HeLaRAR clones68
3.1.3.3ProteasomeinhibitortreatmentofHeLaandHeLaRAR
clonesactivatestheJNKpathway.........................................70
3.1.3.4Serumstarvationandstimulationtreatmentresultsin
c-JuninductionandAP-1binding reconstitutionin
HeLaRAR clones...............................................................72
3.1.3.5ConstitutivelyactiveproteinsthatbelongtotheJNK
pathwaycausec-JuninductionandAP-1binding
reconstitutioninHeLaRARb clones....................................74
3.1.4RoleofRAR onproteindegradation................................................79
3.1.4.1RoleofPOH1inc-JunstabilizationinHeLaRAR clones..80
3.2EffectofRARb expressiononHPV-18oncogenesE6/E7 .............................83
3.2.1HPV-18oncogenesE6/E7expressioninHeLaRAR clones............83
3.2.2AnalysisoftheupstreamregulatoryregionofHPV-18:
Protein-DNAinteractionattheenhancerandpromoterelements
intheHeLaRARb clones ..................................................................84
3.2.3AP-1bindingreconstitutionandHPV-18expression.........................86
3.2.4RoleofFra-1asnegativeregulatoroftheHPV-18expression ..........90
3.2.4.1AntisensesilencingofFra-1duringAP-1binding
reconstitutionbyMEKK1 inHeLaRAR clones.............90
3.2.4.2RetroviralinfectionofHeLacellstoreachstableand
constitutiveexpressionofFra-1............................................91
IV.Discussion................................................................................................................. 93
bDabbbbbbbb
b
bIntroduction 9
4.1RAR trans-repressionofAP-1transcriptionfactor ......................................93
4.1.1RAR trans-repressionofAP-1transcriptionfactor..........................93
4.1.2MechanismofAP-1downregulation.................................................93
4.1.2.1Mechanismofc-Jundownregulation....................................96
4.1.2.2Increaseofc-JunproteinleadstoAP-1/DNA-binding
reconstitutioninHeLaRAR clones96
4.1.3RoleofRAR onproteindegradation................................................98
4.1.4RoleofPOH1inc-JunstabilizationinHeLaRAR clones ..............98
4.2EffectofRARb expressiononHPV-18oncogenesE6/E7 .............................99
4.2.1AP-1bindingreconstitutionandHPV-18expression.........................99
4.2.2RoleofFra-1asnegativeregulatoroftheHPV-18expression ........104
4.2.2.1RetroviralinfectionofHeLacellstoreachstableand
constitutiveexpressionofFra-1..........................................104
V.Abstract .................................................................................................................... 106
VI.References .............................................................................................................. 107
bbb
b
bIntroduction 10
Abbreviations
AF-1 Activationfunction1
AF-22
AP-1 Activatorprotein1
atRA all-trans-retinoicacid
CDK Cyclindependentkinase
CoA Co-activator
DBD DNA-bindingdomain
ERK Extracellularsignal-regulatedproteinkinases
FCS Fetalcalfserum
Ha-ras Harveyratsarcomavirusoncogene
HAT Histoneacetyltransferase
HDACde-acetylase
GAPDH Glyceraldehyde-3-phosphatedehydrogenase
GSK Glycogensynthasekinase
HPV Human papilloma virus
JNK JunN-terminalkinase
LBD Ligand-bindingdomain
MAPK Mitogen-activatedproteinkinases
MEKK1 MAPKkinasekinase
MKPphosphatase
MKK MAPKkinase
MMP Metalloproteinase
N-CoR Nuclaerreceptorco-repressor
NR Nuclear
NLSlocalizationsignal
P Phospho
POH1 Human Pad1 homolog
RAR Retinoicacidreceptor
RAREacidresponseelement
Rb Retinoblastomaprotein
Skp-2 S-phasekinaseassociatedprotein2(p45)
SMRT SilencingmediatorforRXRandTR
TNF Tumornecrosisfactor
TR Thyroidreceptor
TRE TPAresponsiveelement
URR Upstreamregulatoryregion
a
aIntroduction 11
I. INTRODUCTION
Thepresentworkanalyzestheinteractionbetweentheretinoicacidreceptorbeta(RARb )with
the transcription factorAP-1 in the context of HPV-induced carcinogenesis. Before providing
evidencehowconstitutiveRAR expressionabrogatesAP-1activityincervicalcarcinomacells,
I shall give a brief overview about the individual key regulatory proteins and their function in
normaleukaryoticcells.
1.1Activatorprotein 1 (AP-1) transcription factor
1.1.1Generalaspects
ThetranscriptionfactorAP-1(activatorprotein1)iscomposeofheterogeneousdimericproteins
consistingofmembersofJun,FosandATF-2families.OnceboundtoDNA,thetrans-activation
function of a given dimeric complex can vary, which can be explained by differences in
phosphorylation by upstream protein kinases as well as protein/protein interaction with other
cellularfactors.Asaconsequence,distinctsetsofgenesaretargeted,suggestingthatindividual
membersoftheAP-1familyhavespecificfunctionsinAP-1regulatedcellularprocesses(Angel
et al.,2001).
In vitro, dimers formed by Fos and Jun bind with the highest affinity to an asymmetric
heptanucleotiderecognitionsequenceTGA(C/G)TCA(TRE)andwithslightlysloweraffinityto
asymmetricoctanucleotideTGACGTCA(CRE)(Figure1).TheTREelementisfoundinawide
rangeofpromoterandenhancerregionsembeddedindifferentregulatorycontexts.Variationin
recognition sequences may contribute to the differential functions of different Fos-Jun family
dimersatvariousregulatoryelements(ChinenovandKerppola,2001).
The repertoire of Fos-Jun proteins in a given cell is subject to changes in response to various
extracellular stimuli. Through dimerization mediated by the leucin zipper, the seven family
memberscanform18differenthomoandheterodimers.ThenumberofdetectableFos-Jundimers
variesamongdifferentcelltypes(ChinenovandKerppola,2001).Table1
c-Jun JunB JunD c-Fos FosB Fra-1 Fra-2
c-Jun +++++++
JunB ++++++
JunD +++++
c-Fos ----
FosB - - -
Fra-1 - -
Fra-2 -
Table 1: Dimer combinations of Fos and Jun family proteins capable of forming stableAP-1 transcription factor
complexes.
bIntroduction 12
The functions of Fos-Jun family proteins depend on the specific cell type in which they are
expressed and are also affected by the specific signals that elicit their expression. Thus, the
functionsofAP-1mustbemediatedbymechanismsthatdependonthecellularcontextinwhich
they are expressed. The mechanisms that give cell specificity include selective dimerization,
interaction with other regulatory proteins and post-translational modifications. Moreover
extracellular stimuli can modulate the abundance of the AP-1 proteins by controlling the
transcription of their genes, the stability of their mRNAs and the activity and stability of the
specificFos-Junproteins(Karin et al.,1997;ChinenovandKerppola,2001).
1.1.2AP-1familymembersfunctionandregulation
ThemaincomponentsofAP-1transcriptionfactorscanbedividedintothreemajorfamilies:
Fos,JunandATF.
The Fos family contains the following members, all of them characterized as immediately early
genesbecausethisresponseoccursintheabsenceofnewproteinsynthesis:
i. c-Fos:c-fosproteinandmRNAareundetectableinmostquiescentcellsandrequirestimu-
lation by hormones, serum mitogens or other ligands to reach easily detectable levels
(Distel and Spiegelman, 1990). Stimulation of the cells induces c-fostranscription very
rapidlyandtransiently.Severalelementsmediatec-fosinduction:CRE(cAMPresponseele-