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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Publié par
Publié le
01 janvier 2003
Nombre de lectures
17
Langue
English
Poids de l'ouvrage
15 Mo
Publié par
Publié le
01 janvier 2003
Nombre de lectures
17
Langue
English
Poids de l'ouvrage
15 Mo
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......................
