The MENX syndrome [Elektronische Ressource] : an animal model to study the role of p27 in tumor predisposition and the response of neuroendocrine tumors to therapeutic agents / Mi Su Lee. Gutachter: Jochen Graw ; Angelika Schnieke ; N. S. Pellegata. Betreuer: N. S. Pellegata

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• Biologie

Informations

• Publié par: technische_universitat_munchen
• Publié le: 01 janvier 2011
• Nombre de lectures: 21
• Langue: English
• Poids de l'ouvrage: 3 Mo

Extrait

TECHNISCHE UNIVERSITÄT MÜNCHEN

Lehrstuhl für Entwicklungsgenetik




The MENX syndrome: an animal model
to study the role of p27 in tumor predisposition and the response of
neuroendocrine tumors to therapeutic agents


Mi Su Lee



Vollständiger Abdruck der von der Fakultät Wissenschaftszentrum Weihenstephan für
Ernährung, Landnutzung und Umwelt der Technischen Universität München zur Erlangung
des akademischen Grades eines
Doktors der Naturwissenschaften
genehmigten Dissertation.





Vorsitzender: Univ.-Prof. Dr. S. Scherer
Prüfer der Dissertation:
1. apl.Prof. Dr. J. Graw
2. Uni.-Prof. A. Schnieke, Ph.D
3. Priv.-Doz. Dr. N. S. Pellegata

Die Dissertation wurde am 28.02.2011 bei der Technischen Universität München eingereicht
und durch die Fakultät Wissenschaftszentrum Weihenstephan für Ernährung, Landnutzung und
Umwelt am 29.06.2011 angenommen. Erklärung


Ich erkläre hiermit an Eides statt, dass ich die vorliegende Arbeit selbständig ohne
unzulässige fremde Hilfe angefertigt habe.

Die verwendeten Literaturquellen sind im Literaturverzeuchnis vollständig zitiert.



München, den



Mi Su Lee Acknowledgements

I would like to thank all of my colleagues in the Pathology department at the Helmholtz
Zentrum Muenchen for their support. Special thanks go to Dr. Natalia. S. Pellegata and Prof.
Dr. J. Graw for their support, advice and patience not just as my supervisors, but as mentors. I
would also like to express my gratitude to my graduate committee: Prof. Dr. A. Schnieke and
Prof. Dr. S. Scherer for their valuable criticism and suggestions during the course of my
research. I thank all members of Dr. Pellegatas’ lab for their help and advices. I would also
like to thank E. Samson for helping me to take care of my rats.

Finally, I would like to dedicate this dissertation to my family, whose love and support made
all these possible. Table of contents


ABBREVIATIONS .................................................................................................................. 1
SUMMARY ............................................................................................................................... 3
ZUSAMMENFASSUNG ......................................................................................................... 5
1. INTRODUCTION ................................................................................................................ 7
1.1. Multiple endocrine neoplasia (MEN) .............................................................................. 7
1.1.1. MEN syndrome ........................................................................................................ 7
1.1.2. MEN1-like syndrome (MEN4) ................................................................................ 8
1.1.3. MEN-like syndrome in the rat 11
1.2. Cyclin-dependent kinase inhibitor ................................................................................ 12
1.3. Pituitary ......................................................................................................................... 15
1.3.1. Anatomy of the pituitary gland 15
1.3.2. Pituitary development ............................................................................................ 17
1.3.3. Pituitary adenoma ................................................................................................... 18
1.3.4. Current treatment of pituitary adenomas ................................................................ 19
1.3.5. PI3K/AKT/mTOR pathway as a therapeutic target ............................................... 21
1.3.6. Animal models of pituitary adenoma ..................................................................... 23

2. AIMS ................................................................................................................................... 25
3. MATERIALS ...................................................................................................................... 26
3.1. Equipments .................................................................................................................... 26
3.2. Consumable materials ................................................................................................... 28
3.3. Chemicals and reagents ................................................................................................. 29
3.4. Buffers and solutions ..................................................................................................... 31
3.5. Commercially available kits .......................................................................................... 33
3.6. Constructs ....... 33
3.7. Software .................................................................................................................

4. METHODS ......................................................................................................................... 34
4.1. Mutagenesis ................................................................................................................... 34
4.1.1. Primer design.......................................................................................................... 34
4.1.2. PCR reaction .......................................................................................................... 35
4.1.3. Digestion by DpnI .................................................................................................. 35
4.1.4. Transformation & DNA extraction ........................................................................ 36
4.2. Dye-terminator sequencing ........................................................................................... 36
4.3. Animals ......................................................................................................................... 38
4.4. Histology ................................................................................................................
4.5. Cell culture .................................................................................................................... 40
4.6. Cell viability .................................................................................................................. 41
4.7. RNA interference .......................................................................................................... 42
4.8. Transient transfection .................................................................................................... 42
4.9. Protein extraction and western blotting ......................................................................... 43
4.10. RNA isolation ... 44
4.11. Reverse transcription 44 4.12. Quantitative (q) RT-PCR ............................................................................................ 45
4.13. Immunofluorescence ................................................................................................... 47
4.14. Glutathione-S-transferase (GST) pull down assay ...................................................... 47
4.15. Apoptosis assay ........................................................................................................... 47
4.16. Clonogenic assay and growth curve ............................................................................ 48
4.17. Statistical analysis ....................................................................................................... 49

5. RESULTS ............................................................................................................................ 50
Kip15.1. Germline Cdkn1b (p27 ) mutation in the MENX rat syndrome ......................... 50
5.1.1. The MENX mutation in Cdkn1b affects p27 stability ............................................ 50
5.1.2. Proteasome-mediated degradation of p27fs177 in vitro ........................................ 53
5.1.3. Instability of p27fs177 in primary rat cells ............................................................ 58

5.2. Characterization of MENX-associated pituitary tumors60
5.2.1. Macroscopic and histological analysis ................................................................... 60
5.2.2. Immunophenotype of the pituitary tumors ............................................................. 62
5.2.3. Proliferative index of rat pituitary tumors .............................................................. 64

5.3. MENX as a preclinical model to evaluate compounds with therapeutic activity
against neuroendocrine tumors ......................................................................................... 65
5.3.1. Establishing a protocol to obtain single pituitary primary cells from MENX ....... 65
5.3.2. Receptor profiling of pituitary primary cells from MENX .................................... 66
5.3.3. Effect of somatostatin-related drugs on the viability of rat primary pituitary cells 68
5.3.4. Correlation of response to SSA with somatostatin receptor expression ................ 72
5.3.5. Overactivation of the PI3K/mTOR/AKT pathway in rat pituitary tumor cells ...... 74
5.3.6. Effect of RAD0