The HER3 receptor [Elektronische Ressource] : role as an intervention target in ovarian cancer / Martin Bezler

Bezler

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• Gesundheit

Informations

• Publié par: technische_universitat_munchen
• Publié le: 01 janvier 2010
• Nombre de lectures: 33
• Langue: Deutsch
• Poids de l'ouvrage: 6 Mo

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TECHNISCHE UNIVERSITÄT MÜNCHEN 
   Lehrstuhl für Genetik 
 
 
 

The HER3 Receptor: Role as an Intervention
Target in Ovarian Cancer

Martin Bezler



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. K. H. Schneitz
Prüfer der Dissertation: 1. Univ.-Prof. Dr. A. Gierl
2. Hon.-Prof. Dr. Dr. h.c. A. Ullrich
(Eberhard-Karls-Universität Tübingen)


Die Dissertation wurde am 29.04.2010 bei der Technischen Universität München
eingereicht und durch die Fakultät Wissenschaftszentrum Weihenstephan für
Ernährung, Landnutzung und Umwelt am 12.07.2010 angenommen.
 




 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
  Dedicated to my parents

1.   Introduction ......................................................................................... 7 
1.1  The epidermal growth factor receptor (EGFR) family .................................... 7 
1.1.1   Epidermal growth factor receptor (EGFR, ErbB1) ...................................... 9 
1.1.2   HER2 (ErbB2, neu) ...................................................................................... 9 
1.1.3   HER3 (ErbB3) ............................................................................................ 10 
1.1.4   HER4 (ErbB4) 12 
1.2  Neuregulin isoforms ........................................................................................... 13 
1.3.  AKT/PKB kinase and the PI3K-AKT pathway .............................................. 15 
1.4  Apoptosis (programmed cell death) ................................................................. 17 
1.5  Chemotherapeutic drugs ................................................................................... 19 
1.5.1   Chemotherapeutic resistance ...................................................................... 21 
1.6  Targeted therapies ............................................................................................. 23 
1.6.1   Small-molecule therapeutics 24 
1.6.2   Therapeutic monoclonal antibodies ........................................................... 25 
1.7  Ovarian cancer ................................................................................................... 27 
1.8  Single Nucleotide Polymorphisms (SNP) ......................................................... 28 
2.   Aims of the study ............................................................................... 30 
3.   Material and Methods ...................................................................... 31 
3.1  Materials ............................................................................................................. 31 
3.1.1  Laboratory Chemicals ................................................................................ 31 
3.1.2  Enzymes ..................................................................................................... 32 
3.1.3  “Kits“ and other materials .......................................................................... 32 
3.1.4.  Chemotherapeutic drugs ............................................................................. 32 
3.1.5  Small molecule inhibitors ........................................................................... 33 
3.1.6  Growth factors and ligands ........................................................................ 33 
3.2  Cell culture media .............................................................................................. 33 
3.3  Stock solutions and commonly used buffers ................................................... 34 
3.4  Eukaryotic cell lines ........................................................................................... 36 
3.5  Antibodies ........................................................................................................... 37 
3.5.1  Primary antibodies ...................................................................................... 37 
3.5.2  Secondary antibodies .................................................................................. 38 
3.5.3  Therapeutic monoclonal antibodies ........................................................... 39 
3.6.  Oligonucleotides ................................................................................................. 39 
3.6.1  siRNAs ....................................................................................................... 39 
3.6.2  RT-PCR-Primers ........................................................................................ 40 
3.7  Methods of mammalian cell culture ................................................................. 41 
3.7.1  General cell culture techniques .................................................................. 41 
3.7.2  Cell culture in SILAC media ...................................................................... 41 
3.7.3  RNA interference ....................................................................................... 41 
3.7.4  Treatment of cells for western blot analysis ............................................... 42 
3.8  Methods of Biochemistry and Cell Biology ..................................................... 42 
3.8.1  Lysis of cells with Triton X-100 lysis buffer ............................................. 42 
3.8.2  Determination of total protein concentration in cell lysates ...................... 42 
3.8.3  Immunoprecipitation of proteins ................................................................ 42 
3.8.4  Cell lysis with NP-40 and anti-HER3 immunoprecipitation ...................... 43 
3.8.5  SDS-polyacrylamide-gelelectrophoresis (SDS-PAGE) ............................. 43 
3.8.6  Western blotting ......................................................................................... 43 
3.8.7  In-gel protein digestion for MS-based study .............................................. 44 
3.8.8  RNA isolation and RT-PCR analysis ......................................................... 44 
3.8.9  Cell proliferation assay ............................................................................... 45 
3.8.10  Flow cytometry (PI-assay) ......................................................................... 45 
3.8.11  Flow cytometry (doxorubicin accumulation) ............................................. 45 
3.8.12  Caspase 3/7-Glo assay ................................................................................ 45 
3.8.13  Statistical analysis ...................................................................................... 46 
3.8.14  MS analysis on the LTQ-Orbitrap .............................................................. 46 
3.8.15  Peptide identification and quantification .................................................... 46 
4.   Results ................................................................................................ 48 
4.1  HER3/ErbB3 in ovarian cancer cells and its role in doxorubicin (chemo)
sensitivity ............................................................................................................ 48 
4.1.1.  Doxorubicin induces phosphorylation of AKT in four out of nine
ovarian cancer cell lines ............................................................................. 48 
4.1.2.   Inhibition of PI3K activity blocks the doxorubicin induced increase in
AKT phosphorylation and induces apoptosis ............................................. 49 
4.1.3.  Expression analysis of EGFR family members in ovarian cancer cell
lines ............................................................................................................ 52 
4.1.4  Doxorubicin induces phosphorylation of HER3 and activated HER3
interacts with the PI3K regulatory subunit p85 .......................................... 53 
4.1.4.   Downregulation of HER3 increases doxorubicin mediated apoptosis ....... 54 
4.1.5.  Lapatinib or Erlotinib effectively blocks the doxorubicin mediated
increase in HER3 and AKT phosphorylation and enhances apoptosis ...... 56 
4.1.6   Doxorubicin up-regulates the expression of different NRG1/Hrg
isoforms ............................