Table Of ContentXENOBIOTIC-DEPENDENTREGULATIONOFGENEEXPRESSIONINHUMAN
PLACENTALCELLLINES
BY
LIYANZHANG
ADISSERTATIONPRESENTEDTOTHEGRADUATESCHOOL
OFTHEUNIVERSITYOFFLORIDAINPARTIALFULFILLMENT
OFTHEREQUIREMENTSFORTHEDEGREEOF
DOCTOROFPHILOSOPHY
UNTVERSrTYOFFLORIDA
1996
ThisdissertationisdedicatedtoYukun,andalsotomyparents,brothersandsisters.
ACKNOWLEDGEMENTS
I would like to sincerely thank my mentor, Dr. Kathleen Shiverick, for her
guidanceandpatiencethroughoutthecourseofthecompletionofthiswork. WhatIhave
gained in knowledge and capability under her direction, I will carry with me into a
hopefullylong-lastingcareerinscience.
I also wishtorespectfully thankthe members ofmydissertationcommittee,Dr.
Stephen Baker, Dr. Thomas Rowe, Dr. Jeffrey Harrison, Dr. Margaret James and Dr.
RosaliaSimmen, fortheiradviceandsupport. Iwishtoalsothankthepastandpresent
membersofDr. Shiverick's laboratory, includingTerryMedrano,Grantly Charles, Paul
Saunders,Dr.ScottMasten,Dr.MaryVaccarello,Dr.YaraSmit,andDr.PhyllisConliffe
for their help and for providing a pleasant working atmosphere. I would also like to
expressmyappreciationtomyfellowgraduatestudentsandallthefacultymembersinthe
DepartmentofPharmacologyfortheirhelpandfriendship. Mysincerethanksalsogoto
the administrative staffof the Department ofPharmacology, especially Judy Adams,
BarbaraReichert,CookieMundorff,DonnaDesmond,andPatsyMessinger.
In addition, I would like to acknowledge the following people for providing
materialsorinstrumentsessentialtothecompletionofthisresearch:Dr.ThomasRowe,Dr.
PaulKlein, Dr. WilliamGreenlee,Dr. OliverHankinson,Dr. ChristopherBradfield, and
Dr.StephenSafe.
Finally,Ithankmyhusband,Yukun,forstandingbymeasIfollowedmydream,
andmyparentsandparents-in-lawfortheirloveandsupport.
TABLEOFCONTENTS
ACKNOWLEDGEMENTS
iii
KEYTOABBREVIATIONS vii
ABSTRACT ix
CHAPTER INTRODUCTION
1: 1
HumanPlacenta:APrimaryTargetOrganofCYP1A1 Inducers 1
TheObjectiveofThisStudy 2
CulturedPlacentalCells:AnInVitroModelfortheStudyofFetoplacental
Toxicity 3
GeneralCharacterizationoftheMechanismsofActionofTCDDandBaP 4
AhReceptor 4
MetabolicActivation 5
EGF Receptor: A Possible Marker of Placental Toxicity 5
TCDD-responsive Growth Control Genes 7
Trophoblast Growth Control Factors 10
TGF-a 10
TGF-pi 11
c-Myc 12
PAI-2 13
CHAPTER2:MATERIALSANDMETHODS 15
Materials 15
Chemicals and Bioreagents 15
RecombinantcDNAClones 16
AntibodiesandELISAKits 16
Methods 17
CellCultureandChemicalTreatment 17
EGF Binding Assay 17
Western Immunoblot Analysis 18
GeneralProcedure 18
CYP1A1 Protein 18
EGFReceptorProtein 18
c-MycProtein 19
RNAIsolationandNorthernBlotAnalysis 19
NuclearRun-offAssay 20
mRNAStabilizationAssay 21
ELISAforSecretedProteins 21
TGF-pi Assay 21
hCGAssay 22
CellProliferationAssay 22
MTTAssay 22
3H]ThymidineIncorporationAssay 23
[
InVitroInvasionAssay 23
ProteinAssay 24
DataAnalysis 24
CHAPTER3:EVIDENCETHATTHEHUMANPLACENTALCELLLINES
BEWOANDJEG-3HAVEAFUNCTIONALAHRECEPTORSYSTEM 25
Introduction 25
Results 26
ExpressionofAhReceptorandArntmRNAinBeWoandJEG-3Cells 26
SuperinductionofCYP1A1 mRNAandStructure-ActivitySpecificity 27
InductionofCYP1A1 Protein 27
Discussion 28
CHAPTER4:EFFECTSOFTCDDANDBAPONEGFRECEPTOR
EXPRESSION 34
Introduction 34
Results 35
EffectsofTCDDandBaPonSpecificBindingof125I-EGFtoBeWo
andJEG-3Cells 35
Effects ofTCDD and BaP on EGF Receptor Protein 36
RelationshipbetweenCYP1A1 InductionandEGFReceptorChanges 37
EffectsofActinomycinDandCychloheximideonBaP-Mediated
ChangesinEGFReceptors 38
EffectsofTCDDandBaPonSteadyStateEGFReceptormRNA
Levels 38
Discussion 39
CHAPTER5:EFFECTSOFTCDDANDBAPONTGF-a,TGF-P1,C-MYC
ANDPAI-2GENEEXPRESSION 56
Introduction 56
Results 57
EffectsofTCDDandBaPontheSteadyStatemRNALevelsforTGF-
a,TGF-pi,PAI-2andc-myc 57
EffectsofTCDDandBaPontheRateofTGF-p1 andc-mycGene
Transcription 58
EffectsofBaPon the StabilityofTGF-pl and c-myc mRNA 59
EffectsofTCDDandBaPonTGF-p1 andc-MycProteinLevels 60
Discussion 60
CHAPTER6:EVALUATIONOFTHEEFFECTSOFTCDDANDBAPON
CELLULARGROWTHANDENDOCRINERESPONSESOFBEWOAND
JEG-3 CELLS 77
Introduction 77
Results 77
EffectsofTCDDandBaPonCellProliferationasMeasuredbythe
MTTConversionAssay 77
EffectsofTCDDandBaPonJEG-3CellProliferationasMeasuredby
3H]Thymidine Incorporation and Cell Number Changes 78
Effe[ctsofTCDDandBaPonBeWoCellProliferationasMeasuredby
3H]Thymidine Incorporation 79
[
EffectsofTCDDandBaPonSecretionoftheHormonehCG 79
DifferentialEffectsofTCDDandBaPonJEG-3Cell Invasiveness 80
Discussion 80
CHAPTER 7: CONCLUSIONS AND FUTURE DIRECTIONS 95
LISTOFREFERENCES 103
BIOGRAPHICALSKETCH 115
KEYTOABBREVIATIONS
AD actinomycinD
Ah arylhydrocarbon
AhR arylhydrocarbonreceptor
Arnt arylhydrocarbonreceptornucleartranslocator
oc-NF a-naphthoflavone
BaP benzo(a)pyrene
BPDE benzo(a)pyrene-7,8-diol-9,10-epoxide
BSA bovineserumalbumin
c-myc celluarmycgeneorribonucleicacid
c-Myc c-myconcoprotein
cDNA complementarydeoxyribonucleicacid
CHX cycloheximide
CYP1A1 cytochromeP450 1Al
DMSO dimethylsulfoxide
DNA deoxyribonucleicacid
DRE/XRE dioxinresponsiveelement/xenobioticresponsiveelement
DTT dithiothreitol
ECM extracellularmatrix
EGF epidermalgrowthfactor
EGFR epidermalgrowthfactorreceptor
ELISA enzyme-linkedimmunosorbentassay
FBS fetalbovineserum
HCB 2,2',4,4',5,5'-hexachlorobiphenyl
hCG humanchorionicgonadotropin
hi hours
IGF-II insulin-likegrowthfactorII
IGFBP insulin-likegrowthfactorbindingprotein
kb kilobases
kd dissociationconstant
kDa kilodaltons
MMP
matrixmetalloproteinase
MNF 3'-methoxy-4'-nitroflavone
mRNA messengerribonucleicacid
PAI plasminogenactivatorinhibitor
PBS phosphate-bufferedsaline
PCB polychlorinatedbiphenyl
PCDF polychlorinateddibenzofurans
PDGF platelet-derivedgrowthfactor
PMSF phenyl-methylsulfonylfluoride
RNA ribonucleicacid
SE standarderror
SDS-PAGE sodiumdodecylsulfatepolyacrylamidegelelectrophoresis
TCB 3,3',4,4'-tetrachlorobiphenyl
TCDD 2,3,7,8-tetrachlorodibenzo-p-dioxin
TGF transforminggrowthfactor
TIMP tissueinhibitorofmetalloproteinases
t-PA tissueplasminogenactivator
u-PA urokinaseplasminogenactivator
AbstractofDissertationPresentedtotheGraduateSchool
oftheUniversityofFloridainPartialFulfillmentofthe
RequirementsfortheDegreeofDoctorofPhilosophy
XENOBIOTIC-DEPENDENTREGULATIONOFGENEEXPRESSIONINHUMAN
PLACENTALCELLLINES
By
LiyanZhang
December1996
Chairman: KathleenT.Shiverick
MajorDepartment: PharmacologyandTherapeutics
This study evaluated the usefulness of human placental trophoblastic
choriocarcinoma cell lines BeWo and JEG-3 to study the effects of the prototype
environmental chemicals on the expression ofimportant trophoblast growth regulatory
genes. The environmental chemicals studied are the aryl hydrocarbon (Ah) receptor
agonists, 2,3,7,8-tetrachlorodibenzo-/>-dioxin (TCDD) andbenzo(a)pyrene (BaP). The
placentalgenesunderstudyincludethegrowthfactors,transforminggrowthfactor(TGF)-
a, TGF-fjl and the epidermal growth factor (EGF) receptor, the proteinase inhibitor,
plasminogenactivatorinhibitor(PAI)-2,andtheprotooncogene,c-myc. Thisstudy first
demonstratedthatbothcelllinespossessafunctionalAhreceptorsystemasevidencedby
CYP1A1 inductionfollowingTCDDandBaPexposure. Secondly,inductionofCYP1A1
does not appearto be directly linked with loss ofEGF receptor. BaP decreased EGF
receptorbinding and protein in both cell lines, without affecting EGF receptor mRNA
level. TCDD,however,wasnotfoundtoaltertheEGFreceptorexpressionineithercell
line. Thirdly, significant dysregulation of TGF-p"1 and c-myc gene expression was
observedwithBaPbutnotTCDDinJEG-3cells. BeWocellsprimarilyshowedeffectsof
both chemicals on hCG secretion, BaP on TGF-[}1 and TCDD on TGF-a expression.
Finally, BaPhasbeen shown toinhibitchoriocarcinomacell proliferation andinvasion,
whereasTCDDincreasedcellinvasionwithoutaffectingcellproliferation.
In summary, BaP-mediated changes in EGF receptor, TGF-pM and c-myc
expressionwerecorrelatedwithalteredtrophoblastproliferationandinvasiveness. These
alteredprocessesmayunderliemechanismsbywhichxenobioticssuchasthosefoundin
cigarettesmokedisruptplacentalfunctionandleadtofetalgrowthretardation. Dataalso
indicatethatTCDDandBaPproduceplacentaltoxicitythroughdifferentmechanisms. In
addition, this study supports the feasibility ofusing the BeWo and JEG-3 cell lines to
investigatebiomarkersandmechanismsofplacentaltoxicity.
