Identification and characterization of genes and signaling pathways involved in proliferation and differentiation of mammary epithelial cells [Elektronische Ressource] / by Marcin Jankiewicz

Marcin Sobieszczanski

Sujets

• Gesundheit

Informations

• Publié par: goethe_universitat_frankfurt_am_main
• Publié le: 01 janvier 2008
• Nombre de lectures: 23
• Langue: English
• Poids de l'ouvrage: 4 Mo

Extrait

Identificationandcharacterizationof
genesandsignalingpathwaysinvolvedin
proliferationanddifferentiationof
mammaryepithelialcells


Dissertation

Thesissubmittedinfulfillmentofthe
requirementsforthedegree

DoctorofPhilosophy
at
JohannWolfgangGoetheUniversity
inFrankfurtamMain

By
MarcinJankiewicz
borninPoznan,Poland



FrankfurtamMain
2006

ACKNOWLEDGMENTS

I would like to thank Professor Dr. Bernd Groner for giving me the opportunity to
cometoGermanyandtoworkonaveryimportantandinterestingprojectinhisgroup.
I greatly acknowledgeourfruitfulldiscussionsandhissupportduringcompletionof
mythesis.

IwouldliketothankProfessorDr.AnnaStarzinski-Powitzforherinterestinmywork
andhersupervisionduringcompletionofmythesis.

Many thanks to all members of the Groner group: Corinna Bähr, Nadja Bednorz,
Andrea Belaus, Nadine Böcher, Boris Brill, Claudia Bürger, Natalia Delis, Sylane
Desrivieres, Maresa Eck, Christina Gewinner, Corina Heinz, Sabrina Kraemer,
ChristianKunz,NahomiCastro-Palomino-Laria,CarrieShemanko,VidaVafaizadeh,
AstridWeiss,IlkaWittigandKerstinNagel-Wolfrum.Ienjoyedtheextraordinarytime
wespendtogetherinthelab,inourmeetingsandatcrazyparties.

IwouldliketomentionDr.SylaneDesrivieresandthankherforourlongdiscussions
onmyresultsandhelpfulcriticismofmythesismanuscript.

I also would like to thank Dr. Corina Heinz for her good advice and especially for
criticalreadingofmythesismanuscript.

Idon’twanttoforgetmyfriendsworkinginothergroupsattheGSHandthankthem
forthejoyfultimeandnicecollaborationsthatIhadwiththem.

Many thanks to my parents and Alicja. They always supported me in good and bad
timesduringmystudy.

Intheend,IwouldlikethanktoProf.Dr.hab.AnnaC.Majewska,ProfDr.hab.Jan
Sadowski,Prof.Dr.hab.ArturJarmołowski,Dr.WojciechG.Musiałfortheirhelpand
supporttofindagraduatestudentposition.

2
ABSTRACT
The mammary gland is a perfect system to study the pathways regulating
organogenesis during development of an individual. The proper development of the
mammaryglandrequiresatightcoordinationofexpressionofmanygenesinvolvedin
proliferationanddifferentiation.Theaimofthisworkwastoidentifynovelgenesand
pathways involved in the development of the mammary gland and to find possible
correlations between the signaling pathways and their downstream targets that are
activated during proliferation and functional differentiation of mammary epithelial
cells.InthisstudyrapamycinhasbeenusedtoinhibitthemTORproteintoanalyzeits
role during mammary gland development. Further a genomic approach was used to
identifygenesdifferentlyexpressedduringthisprocess.
TheanalysisoftheeffectscausedbytheinhibitionofthemTORsignalingpathwayby
usingrapamycinonmammaryepithelialcellsforthefirsttimedemonstratethatmTOR
plays central role in the coordination of pathways governing the proliferation and
differentiationofepithelialcellsduringmammaryglanddevelopment.Moredetailed
analysisledtotheidentificationofId1andId2astwomajordownstreameffectorsof
themTORsignalingpathwayregulatingproliferationanddifferentiation,respectively.
Thegenomicsanalysisrevealedseveralinterestinggenesinvolvedintheregulationof
aproliferativeorsecretoryphenotypeofnormalepithelialcellsin vitro.Variousgenes
identified by microarray analysis are of high interest and to determine their role in
mammaryglanddevelopment.Amongtheidentifiedgenessomecontributetoprocess
of proliferation like Nol5 and Kpna2, whereas other genes are required for proper
functional differentiation such as Nkd2 and Cited4. Importantly, the mentioned
candidategenesarealsointerestingregardingcancerdevelopment,sincederegulation
oftheirexpressionmigthcontributetotumorformation.
Thefindingsdescribedinthisworkclearlycontributetoourbetterunderstandingof
the mTOR signaling pathway regulating expression of the genes involved in the
development of mammary gland. In additon, the presented results should allow
broadening our view of the events that contribute to breast cancer development and
helptodesignbetteranticancertherapiesinthefuture.


3Contents
CONTENTS
ACKNOWLEDGMENTS........................................................................2
ABSTRACT...............................................................................................3
CONTENTS ..............................................................................................4
1.INTRODUCTION ..............................................................................10
1.1Developmentofthemousemammarygland .................................................10
1.1.1Embryonaldevelopmentofthemammarygland .........................................10
1.1.2Developmentofthemammaryglandduringpuberty ..................................11
1.1.3Developmentofthesecretorycompartmentduringpregnancy ...................12
1.1.4Lactationinthemammarygland..................................................................13
1.1.5Involutionofthemammarygland................................................................13
1.2 Signaling pathways playing a essential roles during mammary gland
development ............................................................................................................15
1.2.1Prolactin/Stat5signalinginthemammarygland .........................................15
1.2.2Theinsulin/IGFpathwayinmammaryglanddevelopment.........................17
1.3TargetofRapamycin .......................................................................................20
1.3.1DiscoveryofTOR ........................................................................................20
1.3.2StructureofmTOR.......................................................................................20
1.3.3ThefunctionofthemTORprotein...............................................................22
1.3.4RegulationofmTORactivity.......................................................................23
1.3.4.1ThePI3K/AktsignalingpathwayisanupstreamregulatorofmTOR..23
1.3.4.2TSCnegativelyregulatesmTOR ..........................................................24
1.3.4.3RhebapositiveregulatorofmTORissuppressedbyTSC...................24
1.3.4.4AMPKactivatesTSC2andnegativelyregulatesthemTORpathway..26
1.3.4.5RegulationofTSCby14-3-3proteins ..................................................26
1.3.5ComponentsofthemTORcomplex.............................................................27
1.3.5.1mTORiscomplexedwithRaptorandGβL ..........................................27
1.3.5.2RictorisacomponentofthemTORcomplex.......................................29
1.3.6DownstreameffectorsofthemTORpathway..............................................31
1.3.6.1Phosphorylationof4E-BP1bymTORleadstotheactivationofeIF4E
...........................................................................................................................32
1.3.6.2mTORphosphorylatesS6kinasepromotingtranslation......................33
1.3.6.3mTORregulatestheeIF4BthroughS6K..............................................34
1.3.6.4mTORactivatesdegradationofPDCD4throughS6kinase.................34
4Contents
1.3.6.5PhosphorylationloopsinthemTOR/S6Ksignalingpathway ..............35
1.4ThemTORpathwayandcancerdevelopment..............................................36
1.5TheroleofbHLHproteinsinmammaryglanddevelopment......................37
1.5.1TheroleofId1inproliferationandinvasion ...............................................39
1.5.2TheroleofId2duringpregnancyandlactation ...........................................40
1.6RNAinterferenceasatoolfortheanalysisofgenesinvolvedinmammary
glanddevelopment..................................................................................................41
1.6.1EndogenousexpressionofsiRNA................................................................42
1.6.2 The use of lentiviral vectors for stable siRNA expression in mammalian
cells........................................................................................................................43
1.7Systemsforcultivatingmammaryepithelialcells.........................................44
1.8Theaimsofthiswork.......................................................................................46
2MATERIALSANDMETHODS............................. ...........................47
2.1MATERIALS....................................................................................................47
2.1.1Chemicals.....................................................................................................47
2.1.2Commercialkitsandreagents ......................................................................48
2.1.3Enzymes .......................................................................................................48
2.1.4Materialusedincellculture.........................................................................48
2.1.5Markers.........................................................................................................49
2.1.6Plasmids .......................................................................................................49
2.1.7Antibodies ....................................................................................................50
2.1.8Primers .....................................................................................................