Role of phosphoinositide-3-kinase (PI3K), Akt signaling in apoptosis regulation of neuroectodermal tumors [Elektronische Ressource] / Daniela Opel

Daniela Opel

Sujets

• Gesundheit

Informations

• Publié par: universitat_ulm
• Publié le: 01 janvier 2008
• Nombre de lectures: 40
• Langue: English
• Poids de l'ouvrage: 12 Mo

Extrait

University Children’s Hospital
Prof. Dr. K.-M. Debatin




Role of phosphoinositide-3-kinase (PI3K)/Akt
signaling in apoptosis regulation of
neuroectodermal tumors



Dissertation zur Erlangung des Doktorgrades der Humanbiologie der Medizinischen
Fakultät der Universität Ulm





Daniela Opel
geb. in Herzberg/Elster



2008


























Amtierender Dekan: Prof. Dr. Klaus-Michael Debatin

1. Berichterstatter: Prof. Dr. Simone Fulda
2. Berichterstatter: Prof. Dr. Thomas Seufferlein
Tag der Promotion: 09. Mai 2008






























Meinem Mann und meinem Sohn

Table of contents i
Table of contents................................................................................................................... i
Abbreviations............................................................................................................................................. iii
1 Introduction ...................................................................................................................... 1
1.1 Cancer.......................................................................................................................... 1
1.2 Neuroectodermal tumors ............................................................................................. 3
1.2.1 Glioblastoma – a brain tumor.......................................................................................................... 3
1.2.2 Neuroblastoma – a neuroblastic tumor ........................................................................................... 5
1.3 Apoptosis..................................................................................................................... 8
1.3.1 Apoptosis signaling......................................................................................................................... 9
1.3.2 Apoptosis regulation ..................................................................................................................... 10
1.3.3 Apoptosis resistance mechanisms................................................................................................. 12
1.4 Survival signaling.......................................................................................................14
1.4.1 PI3K/Akt signaling................ 14
1.4.2 mTOR signaling............................................................................................................................ 18
1.4.3 Raf/MEK/ERK signaling.............................................................................................................. 19
1.4.4 Inhibitors targeting survival pathways for cancer therapy ............................................................ 20
1.5 Aim of the study .........................................................................................................23
2 Materials and Methods ...................................................................................................24
2.1 Materials .....................................................................................................................24
2.1.1 Cell culture reagents................. 24
2.1.2 Protein electrophoresis and Western blot analysis........................................................................ 24
2.1.3 RT-PCR and related material........................................................................................................ 25
2.1.4 siRNA duplex oligoribonucleotides.............................................................................................. 25
2.1.5 Protein and caspase inhibitors and related material ...................................................................... 25
2.1.6 Other material ............................................................................................................................... 26
2.1.7 Plasticware.................................................................................................................................... 26
2.1.8 Hardware................................................................................................................. 26
2.1.9 Antibodies............................................................................................................... 27
2.1.10 Glioblastoma cell lines................................................................................................................ 29
2.1.11 Neuroblastoma cell lines............................................................................................................. 29
2.1.12 Establishment of primary cultured glioblastoma cells ................................................................ 29
2.2 Methods ......................................................................................................................30
2.2.1 Cell culture.................................................................................................................................... 30
2.2.2 Induction and determination of apoptosis ..................................................................................... 30
2.2.3 Determination of cell viability ...................................................................................................... 31
2.2.4 Determination of proliferation ...................................................................................................... 31
2.2.5 Colony forming assay ................................................................................................................... 32
2.2.6 Knockdown of PI3K and mTOR by RNA interference ................................................................ 32
2.2.7 Protein extraction.......................................................................................................................... 32
2.2.8 Western blot analysis .................................................................................................................... 33
2.2.9 RT-PCR ........................................................................................................................................ 33
2.2.10 Caspase activity assay................................................................................................................. 34
2.2.11 Caspase inhibition....................................................................................................................... 34
2.2.12 Determination of mitochondrial membrane potential ................................................................. 34
2.2.13 Determination of cytochrome c release by flow cytometry ........................................................ 35
2.2.14 Determination of cytochrome c release by immunofluorescence microscopy............................ 35
2.2.15 Cell surface staining.................................................................................................................... 35
2.2.16 Human Tissue microarray........................................................................................................... 36
2.2.17 Tumor lysates.............................................................................................................................. 38
2.2.18 Statistical analysis.................................................................................................... 39
2.2.19 Densitometric analysis.............. 39
3 Results Part 1: Importance of PI3K signaling in glioblastoma...................................40
3.1 Constitutive activity of PI3K signaling in human glioblastoma cell lines .................40
3.2 Inhibition of PI3K sensitizes glioblastoma cells for death receptor-induced apoptosis
....................................................................................................................................42
3.3 Inhibition of PI3K sensitizes glioblastoma cells for drug-induced apoptosis ............47 Table of contents ii
3.4 No sensitization of glioblastoma cells for TRAIL-or Doxorubicin-induced apoptosis
by inhibition of mTOR or MEK.................................................................................51
3.5 Knockdown of PI3K rather than mTOR sensitizes glioblastoma cells for TRAIL- or
Doxorubicin-induced apoptosis.58
3.6 Inhibition of PI3K sensitizes glioblastoma cells for TRAIL- or Doxorubicin-induced
caspase activation .......................................................................................................60
3.7 Inhibition of PI3K sensitizes A172 glioblastoma cells for TRAIL- or Doxorubicin-
induced mitochondrial perturbations..........................................................................66
3.8 Modulation of apoptosis regulatory molecules by inhibition of PI3K .......................69
3.9 PI3K inhibition and TRAIL or Doxorubicin collaborate to reduce cell viability or
proliferation................................................................................................................71
3.10 Inhibition of PI3K cooperates with TRAIL and Doxorubicin