Interaction of human papillomavirus 20 and 4-nitroquinoline-1-oxide in carcinogenesis [Elektronische Ressource] / vorgelegt von Elsa Herráez Hernández

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Aus dem Deutschen Krebsforschungszentrum Heidelberg (Geschäftsführender Direktor: Prof. Dr. med. Otmar D. Wiestler) Abteilung für Tumorvirus% Charakterisierung (Abteilungsleiter: Prof. Dr. Ethel%Michele de Villeirs) Interaction of human papillomavirus 20 and 4nitroquinoline1oxide in carcinogenesis Inaugural dissertation zur Erlangung der Doktorwürde Der Naturwissenschaftlich%Mathematischen Gesamtfakultä tder Ruprecht % Karls % Universität Heidelberg vorgelegt von Elsa Herráez Hernández aus Ávila, Spanien 2009 Dissertation submitted to the Combined Faculties for the Natural Sciences and for Mathematics of the Ruperto%Carola University of Heidelberg, Gemr any for the degree of Doctor of Natural Sciences presented by Elsa Herráez Hernández born in Ávila, Spain Oral%examination: ................................................ Referees: (Herr) Prof. Dr. G.J. Hämmerling. (Herr) Prof. Dr. P. Angel. To Lilín Table of contents Table of contents Abbreviations ......................................................................................................vi Summary .............................................................................................................ix Zusammenfassung ......................................................
Publié le : jeudi 1 janvier 2009
Lecture(s) : 18
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Source : ARCHIV.UB.UNI-HEIDELBERG.DE/VOLLTEXTSERVER/VOLLTEXTE/2009/9354/PDF/PHD_ELSA_HERRAEZ.PDF
Nombre de pages : 154
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Aus dem Deutschen Krebsforschungszentrum Heidelberg
(Geschäftsführender Direktor: Prof. Dr. med. Otmar D. Wiestler)
Abteilung für Tumorvirus% Charakterisierung
(Abteilungsleiter: Prof. Dr. Ethel%Michele de Villeirs)





Interaction of human papillomavirus 20 and
4nitroquinoline1oxide in carcinogenesis



Inaugural dissertation
zur
Erlangung der Doktorwürde
Der
Naturwissenschaftlich%Mathematischen Gesamtfakultä t
der
Ruprecht % Karls % Universität
Heidelberg


vorgelegt von
Elsa Herráez Hernández

aus
Ávila, Spanien
2009


Dissertation
submitted to the
Combined Faculties for the Natural Sciences and for Mathematics
of the Ruperto%Carola University of Heidelberg, Gemr any
for the degree of
Doctor of Natural Sciences
















presented by
Elsa Herráez Hernández
born in Ávila, Spain
Oral%examination: ................................................
































Referees: (Herr) Prof. Dr. G.J. Hämmerling.
(Herr) Prof. Dr. P. Angel.





























To Lilín Table of contents

Table of contents
Abbreviations ......................................................................................................vi
Summary .............................................................................................................ix
Zusammenfassung ..............................................................................................xi
1 Introduction ....................................................................................................13
1.1 Papillomaviruses ........................................................................................13
1.1.1 General aspects .......................................................................................................13
1.1.2 HPV types ................................................................................................................14
1.1.2.1 Mucosal types...................................................................................................14
1.1.2.2 Cutaneous types................................................................................................14
1.1.3 HPV20 .....................................................................................................................15
1.1.4 HPV genomic organization .....................................................................................15
1.1.4.1 Early proteins....................................................................................................17
1.1.4.2 Late proteins .....................................................................................................22
1.1.4.3 Upstream regulatory region ..............................................................................23
1.1.5 HPV life cycle ..........................................................................................................23
1.1.6 HPV and cancer.......................................................................................................25
1.1.6.1 Cervical cancer .................................................................................................25
1.1.6.2 Head and neck cancer .......................................................................................26
1.1.6.3 Non melanoma skin cancer...............................................................................27
1.2 4%NQO......................................................................................................28
1.3 p53 gene family..........................................................................................29
1.3.1 p53 ...........................................................................................................................29
1.3.2 p63 ...........................................................................................................................30
1.4 Cell cycle control by cyclin%dependent kinase nihibitors..........................31
1.5 The skin......................................................................................................31
1.5.1 Skin organization.....................................................................................................31
1.5.2 Epidermal layers......................................................................................................32
1.5.3 The skin barrier .......................................................................................................33
1.5.4 Keratins ...................................................................................................................34
1.5.4.1 Keratin expression in simple epithelia..............................................................35
1.5.4.2 Keratin expression in stratified epithelia..........................................................35 Table of contents

1.5.4.3 Keratin expression in squamous cell carcinoma...............................................36
1.5.5 Differentiation markers ...........................................................................................37
1.5.5.1 Involucrin..........................................................................................................37
1.5.5.2 Loricrin .............................................................................................................37
1.6 Aim of the project ......................................................................................37
2 Materials..........................................................................................................39
2.1 Equipment ..................................................................................................39
2.2 Consumables ..............................................................................................41
2.3 Reagents .....................................................................................................42
2.4 Software .....................................................................................................46
2.5 Commercial kits .........................................................................................46
2.6 Solutions.....................................................................................................47
2.7 Bacteria.......................................................................................................52
2.7.1 Strain .......................................................................................................................52
2.7.2 Medium ....................................................................................................................53
2.8 Mammalian cell lines .................................................................................53
2.8.1 Cell lines and mediums............................................................................................53
2.9 Plasmids .....................................................................................................55
2.10 Primers .....................................................................................................56
2.11 Enzymes ...................................................................................................58
2.12 Antibodies ................................................................................................58
2.13 Markers.....................................................................................................59
3 Methods ...........................................................................................................60
3.1 Bacterial manipulation ...............................................................................60
3.1.1 Bacterial culture......................................................................................................60
3.1.2 Bacterial stocks........................................................................................................60
3.1.3 Bacterial transformation .........................................................................................60
3.2 DNA ...........................................................................................................61
3.2.1 Small scale preparation...........................................................................................61
3.2.2 Large scale preparation ..........................................................................................61
3.2.3 Determination of nucleic acid concentration ..........................................................62 Table of contents

3.2.4 Agarose gel electrophoresis ....................................................................................62
3.2.4.1 DNA isolation from agarose gels .....................................................................62
3.2.5 Polymerase Chain Reaction (PCR) .........................................................................63
3.2.6 Cloning ....................................................................................................................64
3.2.6.1 Digestion of DNA using restriction enzymes...................................................64
3.2.6.2 Dephosphorylation of DNA..............................................................................64
3.2.6.3 Ligation.............................................................................................................64
3.2.6.4 Analysis of transformed clones ........................................................................65
3.3 RNA ...........................................................................................................65
3.3.1 RNA isolation from cultured cells ...........................................................................65
3.3.2 DNAse I treatment ...................................................................................................66
3.3.3 cDNA synthesis by Reverse Transcriptase7Polymerase Chain Reaction ................66
3.4 Protein ........................................................................................................66
3.4.1 Protein extraction....................................................................................................66
3.4.1.1 Passive lysis buffer ...........................................................................................66
3.4.1.2 RIPA buffer .....................................................................................................67
3.4.1.3 Organotypic culture protein isolation...............................................................67
3.4.2 Protein quantification..............................................................................................67
3.4.3 SDS7polyacrilamide gel electrophoresis ................................................................68
3.4.4 Western blot.............................................................................................................68
3.4.4.1 Transfer of proteins onto nitrocellulose membrane..........................................68
3.4.4.2 Immunoprobing of nitrocellulose membranes..................................................68
3.4.4.3 Densitometric analysis......................................................................................69
3.5 Cell culture.................................................................................................69
3.5.1 Culturing of cell lines ..............................................................................................69
3.5.2 Cell lines cryopreservation......................................................................................70
3.5.3 Determination of cell number and viability.............................................................70
3.5.4 Transient transfection..............................................................................................70
3.5.5 Luciferase assay ......................................................................................................71
3.5.6 Retroviral production ..............................................................................................72
3.5.6.1 Transduction of target cells ..............................................................................72
3.5.6.2 Study of life span..............................................................................................73
3.5.7 Organotypic raft cultures ........................................................................................73
3.5.7.1 Preparation of dermal equivalent matrix ..........................................................74 Table of contents

3.5.7.2 Raft cultures......................................................................................................74
3.5.7.3 Barrier function analyses ..................................................................................75
3.6 Immunohistochemistry...............................................................................75
3.6.1 Haematoxylin7eosin staining ..................................................................................76
3.6.2 Fluorescence microscopy immunohistochemistry...................................................76
3.6.3 Light microscopy immunohistochemistry. Avidin7Biotin7peroxidase Complex ......77
3.6.4 Lipid staining...........................................................................................................77
3.7 Electron microscopy...................................................................................78
3.8 Gas chromatography% mass spectrometry..................................................78
3.9 Flow cytometry ..........................................................................................79
3.9.1 Determination of cell cycle profile ..........................................................................79
3.9.2 GFP measurement ...................................................................................................79
4 Results..............................................................................................................80
4.1 Characterization of HPV20 promoter activity in H1299 cells...................80
4.1.1 Effect of wtp53 and mut p53R248W expression on HPV20 promoter ....................80
4.1.2 Effect of TAp63α and ?Np63α expression on HPV20 promoter .............................81
4.1.3 Effect of HPV20 E6 expression on HPV20 promoter..............................................82
4.1.3.1 E6 protein does not influence the activation of HPV20 promoter by wtp53 ...82
4.1.3.2 E6 protein does not influence the activation of HPV20 promoter by TAp63α 84
4.2 Effect of 4%NQO in immortalized oral keratinoctyes ................................84
4.2.1 Standardization of transfection conditions..............................................................84
4.2.2 Optimization of 47NQO doses for treatment of keratinocytes .................................86
4.2.3 Effect of 47NQO on cell cycle ................................................................................87
4.2.4 Influence of 47NQO on HPV20 promoter activity..................................................88
4.2.5 Influence of 47NQO together with Flag7tagged HPV20 E6 on endogenous
expression of ?Np63α, p53, PCNA and p16.....................................................................89
4.3 HPV20 E6 expression and/or 4%NQO treatment do ont increase the life
span of primary keratinocytes ..........................................................................90
4.4 Organotypic cultures ..................................................................................92
4.4.1 Epidermal morphology of organotypic cultures......................................................93
4.4.2 Verification of Flag7tagged HPV20 E6 expression in organotypic cultures...........95
CIP1
4.4.3 Flag7tagged HPV20 E6 alters the expression of p21 , p53 and ?Np63α proteins
..........................................................................................................................................96 Table of contents

4.4.4 Profiles of epidermal markers differ in Flag7tagged HPV20 E6 and HPV16 E6/E77
expressing organotypic cultures.....................................................................................100
4.4.4.1 Keratin pattern ................................................................................................100
4.4.4.2 Late differentiation markers ...........................................................................103
4.4.5 Proliferation measured by PCNA staining and BrdU incorporation....................106
4.4.6 Electron microscopy..............................................................................................109
4.4.7 Loss of barrier function .........................................................................................111
4.4.7.1 Lipid quantification ........................................................................................111
4.4.7.2 Lipid droplets accumulate in organotypic cell cultures expressing Flag%tagged
HPV20 E6...................................................................................................................112
4.4.8 Skin permeability assay .........................................................................................112
4.4.8.1 The outside%in barrier is not functional i nFlag%tagged HPV20 E6 rafts........113
4.4.8.2 Increased permeability of tight junctions: altered inside%out barrier in Flag%
tagged HPV20 E6 epithelia ........................................................................................113
4.4.9 Cell7cell communication: desmosomal distribution..............................................114
5 Discussion ......................................................................................................116
6 References......................................................................................................127
7 Publications and poster presentations........................................................149
8 Acknowledgements .......................................................................................150





Abbreviations vi


Abbreviations
A Adenin
APS Ammonium persulfate
ß%ME β%Mercaptoethanol
BaP Benzo[a]pyrene
bp Base pair(s)
BPV Bovine papillomavirus
BrdU 5´ bromo%2%deoxyuridine
BSA Bovine serum albumin
C Cytosine
cdk Cyclin%dependent kinase
cDNA Complementary DNA
DAB 3,3´%diaminobenzidine substrate
DAPI 4´, 6% Diamidino%2%phenylindole dihydrochloer id
DEPC Diethylpyrocarbonate
DKFZ Deutsches Krebsforchungszentrum
DMEM Dulbecco´s modified Eagle´s medium
DMSO Dimethyl sulfoxide
DNA Deoxyribonucleic acid
DNase Deoxyribonuclease
dNTP Deoxynocleosidetriphosphate
DOSPA 2,3%dioleyloxy%N%[2(spermine%carboxamido)le]%thy
N,N%dimethyl%1%propanaminiumtrifluoro%acetate
DTT 1,4%dithio%DL%threitol
E. coli Escherichia coli
EDTA Ethylenediaminetetraacetic acid
EGF Epidermal growth factor
EtBr Ethidium bromide
EtOH Ethanol
EV Epidermodysplasia verruciformis
FACS Fluorescence%activated cell sortin g
FCS Fetal calf serum
Fig Figure

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