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Dysregulation of Wnt-β-catenin [Wnt-beta-catenin] pathway in pituitary adenomas and its stimulation by CRH and inhibition by SRIF in ACTH-secreting pituitary adenomas [Elektronische Ressource] / vorgelegt von Muhammad Nasir Khan Khattak

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138 pages
Aus der Neurochirurgische klinikderFriedrich-Alexander-Universität Erlangen-NürnbergDirektor: Prof. Dr. M BuchfelderDysregulation -catenin pathway in pituitary adenomas and its stimulation by CRH and inhibition by SRIF in ACTH-secreting pituitary adenomasInaugural-Dissertation zur Erlangung der Doktorwürde der Medizinischen Fakultätder Friedrich-Alexander-Universität Erlangen-NürnbergVorgelegt vonMuhammad Nasir Khan Khattak Aus Karak, Pakistan(2010)I:RWQGedruckt mit Erlaubnis der Medizinischen Fakultät der Friedrich-Alexander-Universität Erlangen-NürnbergDekan: Prof. Dr. J SchüttlerReferent: Privat Doz. Dr Andrea KleindienstKorreferent: Prof. Dr. M. BuchfelderProf. Dr. J. H. BrandstätterTag der Mündlichen prüfung: 09 Dezember 2010To my parents and sisterJamanah KhattakAcknowledgementThis work was performed at the Research Laboratory of the Department of Neurosurgery (Director Prof. Dr. Michael Buchfelder), Friedrich-Alexander University of Erlangen-Nuremberg, Germany under the supervision of Prof. Dr. Michael Buchfelder.Higher Education Commission (HEC) of Pakistan granted the fellowship for the work and Deutsche Akademische Auslandamt Dienst (DAAD) provided all possible help for earlier settlement and during the whole study period. My sincere thanks go to officials of HEC and DAAD, especially Ashfaq Anwar (HEC) and Monika Osman (DAAD) for their heartily cooperation.Professor Dr.
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Aus der Neurochirurgische klinik
der
Friedrich-Alexander-Universität Erlangen-Nürnberg
Direktor: Prof. Dr. M Buchfelder
Dysregulation -catenin pathway in pituitary
adenomas and its stimulation by CRH and inhibition by
SRIF in ACTH-secreting pituitary adenomas
Inaugural-Dissertation
zur Erlangung der Doktorwürde
der Medizinischen Fakultät
der
Friedrich-Alexander-Universität
Erlangen-Nürnberg
Vorgelegt von
Muhammad Nasir Khan Khattak
Aus Karak, Pakistan
(2010)
I:RWQGedruckt mit Erlaubnis der
Medizinischen Fakultät der Friedrich-Alexander-Universität
Erlangen-Nürnberg
Dekan: Prof. Dr. J Schüttler
Referent: Privat Doz. Dr Andrea Kleindienst
Korreferent: Prof. Dr. M. Buchfelder
Prof. Dr. J. H. Brandstätter
Tag der Mündlichen prüfung: 09 Dezember 2010To my parents
and sister
Jamanah KhattakAcknowledgement
This work was performed at the Research Laboratory of the
Department of Neurosurgery (Director Prof. Dr. Michael Buchfelder),
Friedrich-Alexander University of Erlangen-Nuremberg, Germany under
the supervision of Prof. Dr. Michael Buchfelder.
Higher Education Commission (HEC) of Pakistan granted the
fellowship for the work and Deutsche Akademische Auslandamt Dienst
(DAAD) provided all possible help for earlier settlement and during the
whole study period. My sincere thanks go to officials of HEC and DAAD,
especially Ashfaq Anwar (HEC) and Monika Osman (DAAD) for their
heartily cooperation.
Professor Dr. Michael Buchfelder gave me an opportunity to work
under his supervision, and that was an excellent experience for me. He
not only helped me with my studies but he taught me understanding
German culture and assisted me in many occasion in my private problems.
His help, guidance and brotherhood are unforgettable.
My due thanks are for Dr. Natalia Kremenevskaya, who not only
kept eye on my research work but was a dear friend, supported and
helped me continuously. Her valuable advice in all the steps of this study
is praiseworthy.
Professor Dr. Christof Schöfl helped me in completing this project to
an end. I would like to thank him for all his cooperation.
All my colleagues extended their heartily cooperation and provided
me a nice company throughout the study. I learnt much from all of them
and they helped me in adjusting to a new environment. Thanks to: Crina
Ramona Rus and Brigitte Gillner (for their unforgettable company and encouragement throughout the study), Frank Bittner and Steffie Kreckel
(for the help in photography, scanning, preparation of presentations,
printing and also very nice company), Irene Emtmann, Nadine Scheufler,
Marc Schwarz, Saskia, Ines Hunsicker, Ilker Yasin Eyüpoglu, Phillip
Rummel, Bernhard Mayr for providing a friendly environment in the
laboratory through their smiling faces throughout the study period. It was
nice to work and spend time with all of them.
Anja Völzke helped me much in my experiments and she remained
friendly enough to make me feel at home.
Dr. Andrea Kleindienst supported me much during my stay at
Göttingen and also helped much during my thesis completion. Her
cooperation is unforgettable. Dr. Ramin Naraghi, proved to be a nice friend
and colleague, he encouraged and helped me much during my stay in
Erlangen. My friends Aamer Imran, Shahbaz Awan, Azizullah and Khwaja
Basharat also provided friendly environment during my stay at Erlangen.
Thanks to all of them.
Encouragement and cooperation of my beloved Shama Khan are
unforgettable. Many heartily thanks for her.
Last but not the least, Miss Silke Speck and Karina Herpich,
secretary to Professor Buchfelder always extended their cooperation
whenever needed. Thanks to them for all.- I -
Table of contents
No Title Page
No
-- Zusammenfassung 1
-- Summary 4
1 Introduction 7
1.1 Pituitary gland 7
1.1.1 Anatomy and physiology 7
1.1.2 Vascularisation of the anterior pituitary 11
1.1.3 Pituitary adenomas 13
1.1.4 Cell cycle and tumor development-general overview 16
1.1.5 Pituitary tumor genesis 20
1.2 -catenin signaling pathway 22
1.2.1 -catenin signaling pathway itself 22
1.2.2 -catenin signaling in development of various tissues 29
1.2.3 -catenin signaling in tumors 30
1.2.4 -catenin signaling in pituitary adenomas 32
1.3 Somatostatin (SRIF) and corticotrophin releasing 33
hormone (CRH) pathways
2 Aims of the present study 38
3 Material and Methods 39
3.1 Reagents 44
3.2 Solutions 46
3.3 Human tissues 46
3.3.1 RNA isolation 48
3.3.2 Reverse Transcriptase-polymerase chain reaction (rtPCR) 50
3.3.3 Western immunoblotting 50
3.3.3.1 Principle 50
3.3.3.2 Procedure 50
3.3.4 Primary pituitary cell culture of growth hormone (GH) secreting 52
adenomas
3.4 Rat lactosomatotroph GH3 cell line culture and treatment 53
3.5 ACTH-secreting pituitary cell line AtT-20 culture 53
3.5.1 RNA-extraction from AtT-20 cells 54
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3.5.2 Protein extraction from AtT-20 cells 55
3.5.3 Western blotting using AtT-20 cells proteins 56
3.6 Immunoflourescence Microscopy 57
3.6.1 Principle 57
3.6.2 Procedure 57
3.7 Immunoprecipatation of TCF- -catenin complex 58
3.7.1 Principle 58
3.7.2 Procedure 58
3.8 Cloning of dnTCF-4 plasmid 60
3.9 Cell transfection and Cell proliferation Assay 61
4 Results 63
4.1 -catenin, TCF-4 and cylcin D1 at mRNA 63
and protein level in non-functioning pituitary adenomas
(NFPA’s) and GH-secreting pituitary adenomas (Acro)
4.2 -catenin signaling pathway affects 65
cellular proliferation in GH-secreting pituitary adenomas
primary cultures
4.3 -catenin signaling pathway is upregulated in 66
human ACTH-secreting pituitary adenomas
4.4 -catenin signaling pathway 67
through cAMP/PKA/GSK- -20 cells
4.5 -catenin pathway through 73
dephosphorylation of GSK- -20 cells
4.6 Control of cellular proliferation by CRH and SRIF 76
-catenin pathway in AtT-20 cells
4.7 - 79
catenin in atT-20 cells
4.8 Effects of CRH and SRIF on prolactin and GH-secreting 82
rat lactosomatotrophs GH3 cells
5 Discussion 83
6 References 90
7 Abbreviations 120
8 Publications 124
9 Curriculum Vitae 126
W,IRRSKQ[RLL:V+VIHQUQSW[ZHWUWDQHWOGFXVQW6FH\IWIDWH)V,Y5D$&GQ:DNQ%LROLRUNVKULR))57$QWQD:KDHKWQVHKYOHRDYLQFL75W$W:QHL7QRHDWFQO:IHKRWVHVSW(LEL- III -
List of Tables
Table Title Page
No No
1.1 Cell types in the anterior pituitary 10
3.1 Primers used for rtPCR reaction 49
3.2 Antibodies used in western blotting 52
3.3 Primers used for rtPCR for AtT-20 cells 55
3.4 Antibodies used for western blotting in AtT-20 cells 56- IV -
List of Figures
Figure Title Page
No No
1.1 Model of pituitary cell lineage 10
1.2 Diagrammatic representation of the blood supply and 12
venous drainage of the median eminence and pituitary
gland
1.3 A schematic representation of the mammalian cell cycle 18
Pituitary adenomas formation and growth result from the 1.4 21
combined action of transforming (initiation) and
promoting (promotion) events
1.5 Wnt and its binding partners 22
1.6 Frizzled and its binding partners 23
1.7 Dishevelled (Dsh/Dvl) and its binding partners 24
1.8 Axin and its binding partners 24
1.9 Beta-catenin and its binding partners 25
1.10 Adenomatous Polyposis coli (APC) and its binding 26
partners
1.11 Tcf/Lef and binding partners 27
1.12 Landscape of WNT signaling cascades 28
1.13 SRIF-receptor-mediated modulation of signaling 34
cascades leading to changes in hormone secretion,
apoptosis and cell growth
1.14 Signaling characteristics of the CRH receptors 37
4.1 -catenin, TCF-4 and cyclin D1 in non- 64-65
functioning pituitary adenomas (NFPA,s) and GH-
secreting pituitary adenomas (Acro) patients compared
with normal pituitary tissues
4.2 Effe -catenin blockade on GH-secreting 66
pituitary adenomas tissues proliferation
4.3 -catenin, TCF-4 and cyclin D1 67
proteins in ACTH-secreting pituitary adenomas
4.4 - 69-70
catenin, TCF-4 and cyclin D1 in AtT-20 cells
4.5 Forskolin inhibits while H-89 and SRIF stimulate the 7 1
-catenin in AtT-20 cell line
4.6 Forskolin induces while SRIF inhibits phosphorylation of 7 5
GSK- -20 cell line
4.7 CRH and forskolin increases while SRIF decreases 7 6
proliferation in AtT-20 cells
ZVV\VFRRKDSLWWHWLLSQHS$RWI7YUVHQDIRRXVQSRH(QIR+:5KQRHLVIVHRUWSO[UHKLPHOKLWVUV[WVLUE[L&KRQLI)W,R56QH2OL- V -
4.8 Effects of CRH, SRIF and dnTCF-4 on cyclin D1 78
expression at mRNA level and effects of dnTCF-4 on
AtT-20 cells proliferation
4.9 SRIF decreases while forskolin increases nuclear 80
-catenin
4.10 -catenin-TCF-4 81
complex formation in AtT-20 cell line
4.11 CRH, SRIF and Forskolin do not affect the expression of 8 2
-catenin in GH3 cell line
5.1 -catenin pathway 89
modulation by CRH and SRIF
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