Regulation of death receptor-induced apoptosis by c-FLIP in pancreatic carcinoma [Elektronische Ressource] / vorgelegt von Christian Haag

Sujets

• Biologie

Informations

• Publié par: universitat_ulm
• Publié le: 01 janvier 2010
• Nombre de lectures: 27
• Langue: English
• Poids de l'ouvrage: 2 Mo

Extrait

Regulation of death receptor-induced apoptosis
by c-FLIP in pancreatic carcinoma
Dissertation zur Erlangung des Doktorgrades Dr. rer. nat. der Fakultät
für Biologie der Universität Ulm
Vorgelegt von
Christian Haag
aus Heidelberg
2010Regulation of death receptor-induced apoptosis
by c-FLIP in pancreatic carcinoma
Dissertation zur Erlangung des Doktorgrades Dr. rer. nat. der Fakultät
für Biologie der Universität Ulm
Vorgelegt von
Christian Haag
aus Heidelberg
2010Current Dean: Prof. Dr. Axel Groß
First supervisior: Prof. Dr. Thomas Wirth
Second supervisor: Prof. Dr. Klaus-Michael Debatin
Day doctorate awarded: 08.06.2011N
Table of content
Table of content
Table of content………………………………………………………………………... I
Abbreviations……………………………………………………… IV
A Abstract…………………………………………………………………………... 1
1 Introduction………………………………………………… 2
1.1 Cancer cell biology…..……………………………………… 2
1.2 Apoptosis………………………………………………………………………….. 7
1.3 The DISC protein c-FLIP…………………………………... 11
1.4 Biology of pancreatic cancer……………………………… 15
1.5 Aim of the study………………………………………………………………….. 18
192 Materials and Methods……………………………………
2.1 Eukaryotic cell culture……………………………………… 19
202.2 Cell viability assay (MTT Assay)………………………………………………..
202.3 Cell count………………………………………….
212.4 Protein preparation………………………….
212.5 Determination of protein concentration………………………………………...
212.6 SDS-Polyacrylamide-Gelelectrophoresis (SDS-PAGE)……………………...
2.7 Western blot analysis.................................................................................... 22
232.8 Overexpression of c-FLIP ………………………………………………………L
242.9 Cloning of RNA interference vectors……………………...
Genetic downregulation of c-flip mRNA by RNA interference………………. 252.10
262.11 Quantitative Real-time PCR……………………………………………………...
272.12 Immunoprecipitation of ubiquitylated c-FLIP protein………………………….
2.13 Immunoprecipitation of the Death Inducing Signaling Complex (DISC)…… 27
2.14 Determination of apoptosis……………………………………………………… 28
2.15 Receptor staining…………………………… 29
2.16 Nuclear fractionation……….……………………………….. 29
2.17 Electrophoretic mobility shift assay (EMSA)………………………………….. 30
3 Results…………………………………………………………………….……… 31
3.1 Expression of c-FLIP isoforms in pancreatic carcinoma cells……………….. 31
3.2 Effect of TRAIL-stimulation in TRAIL-resistant pancreatic carcinoma
cells………………………………………………………………………………… 32
3.3 Downregulation of c-FLIP proteins by CHX sensitizes pancreatic carcinoma
cells for death receptor-induced apoptosis……………………….. 32
3.4 Simultaneous knockdown of c-FLIP and c-FLIP sensitizes pancreaticL S
carcinoma cells for death receptor-induced apoptosis……………………….. 35
3.5 Induction of NF- B activity in ASPC1 cells with simultaneous knockdown of
IN
Table of content
c-FLIP and c-FLIP protein expression………………………………………… 39L S
3.6 Selective knockdown of c-FLIP and c-FLIP protein expression in ASPC1L S
cells sensitizes for death receptor-induced apoptosis…..……………………. 39
3.7 Selective knockdown of c-FLIP protein expression sensitizes PancTu1 cellsL
for death receptor-induced apoptosis..………………………………………… 41
3.8 Downregulation of c-FLIP proteins by 5-FU occurs on mRNA level and
sensitizes pancreatic carcinoma cells for death receptor-induced
apoptosis…………………………………………………………………………... 43
3.9 Molecular events involved in the synergistic interaction of 5-FU and TRAIL. 48
3.10 Overexpression of c-FLIP rescues 5-FU-pre-treated cells from TRAIL-L
induced apoptosis…………………………………………………………………... 51
3.11 Cisplatin-mediated downregulation of c-FLIP sensitizes pancreatic
carcinoma cells towards TRAIL-induced apoptosis……...... 53
3.12 Gemcitabine treatment sensitizes pancreatic carcinoma cells towards
TRAIL-induced apoptosis..………………………………………………………… 55
3.13 Primary cultured pancreatic carcinoma cells can be sensitized for TRAIL-
induced apoptosis by 5-FU or Cisplatin…………………...... 57
4 Discussion………………………………………………………………………..... 58
4.1 c-FLIP proteins are highly expressed in pancreatic carcinoma cells…………. 58
4.2 TRAIL-stimulation does not lead to increased proliferation in TRAIL-resistant
pancreatic carcinoma cells…………………………………….…………………... 59
4.3 Downregulation of c-FLIP protein levels by Cycloheximide sensitizes
pancreatic carcinoma cells for death receptor-induced apoptosis in a
caspase-dependent manner………………………………………………………. 60
4.4 Knockdown of c-FLIP and c-FLIP protein expression is sufficient toL S
sensitize pancreatic cancer cells for death receptor-induced apoptosis, but
not for chemotherapeutic drug-induced apoptosis…………………………....... 61
4.5 63c-FLIP knockdown increases NF- B activity…………………………………......
4.6 Both c-FLIP or c-FLIP inhibit death receptor-mediated apoptosis in aL S
caspase-dependent manner, with c-FLIP being a more potent inhibitor…….. 64S
4.7 Already small changes in the stoichiometry of c-FLIP protein levels at the
DISC induce apoptosis……………………………………………………………. 65
4.8 5-FU interferes with c-flip mRNA expression and sensitizes pancreatic
carcinoma cells for death receptor-induced apoptosis..………………………... 66
4.9 5-FU treatment enhances DISC formation and caspase activation…………… 67
4.10 Downregulation of c-FLIP is a key event in 5-FU-mediated sensitization forL
TRAIL-induced apoptosis………………………………………………………….. 68
II Table of content
4.11 Sensitization for TRAIL-induced apoptosis by Cisplatin is mainly mediated
by downregulation of c-FLIP………….…………………………………………. 69
4.12 Gemcitabine treatment sensitizes pancreatic cancer cells towards TRAIL
independent of c-FLIP downregulation………………………………………… 70
4.13 Primary cultured pancreatic carcinoma cells can be sensitized towards
TRAIL-induced apoptosis by 5-FU or Cisplatin..……………………………… 71
5 Literature………………………………………………………….………………… 73
6 Appendix……………………………………………………………….…………… 88
6.1 Table of Figures…………………………………………………………………...... 88
6.2 Vector maps……………………………………. 90
926.3 All c-FLIP knockdown sequences……………………………
6.4 5-FU metabolism………………………………………………………………….. 93
7 Curriculum Vitae……………………………... 94
8 Publications and Poster Presentations……………………………………….. 96
9 Statutory Declaration……………………………………………………………... 97
10 Acknowledgements………………………………………….. 98
IIIE
Abbrevations
Abbreviations
°C degree Celsius
5-FU 5-Fluorouracil
APS Ammonium Persulfate
AIF Apoptosis Inducing Factor
Amp Ampicillin
Apaf-1 Apoptosome-associated factor 1
Bak Bcl-2 antagonist/killer
Bax Bcl-2 associated X protein
bp base pair
BSA Bovine serum albumine
CARD Caspase recruitment domain
Caspase Cysteine-dependent aspartate-specific protease
CHX Cycloheximide
DD Death Domain
dATP Deoxyadenosine triphosphate
DED Death Effector Domain
DIABLO Direct IAP-binding protein with low pI
DISC Death Inducing Signaling Complex
DNA Desoxyribonucleic acid
ECL Enhanced chemoluminescence
EDTA Ethylene diamine tetraacetate
FACS Fluorescence-activated cell sorting
FCS Fetal Calf Serum
FADD Fas-Associated protein with Death Domain
FLIP FADD-Like Interleukin-1 Converting Enzyme Inhibitory Protein
2g 9,81 m/s
h hour(s)
IAP Inhibitor of Apoptosis Protein
IP immunoprecipitation
kb kilobases
kDa kilo Dalton
LB Lysogeny Broth
M Molar
mA Milliampere
min Minute(s)
ml Millilitre(s)
IV Abbrevations
mRNA messenger RNA
µg Microgram
ng Nanogram
nm Nanometer
PanIN Pancreatic Intraepithelial Neoplasia
PBS Phosphate Buffered Saline
PBS/T Phosphate Buffered Saline with Tween
PCR Polymerase Chain Reaction
PDAC Pancreatic ductal adenocarcinoma
SDS-PAGE Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis
RIP Receptor interacting protein
RT-PCR Reverse Transcriptase Polymerase Chain Reaction
RNAi RNA interference
S Svedberg factor
SEM Standard Error of Mean
shRNA Short hairpin RNA
Smac Second mitochondria-derived activator of caspases
TBE TRIS/Borate/EDTA
TNF Tumor Necrosis Factor
TRAIL TNF-related Apoptosis Inducing Ligand
V Volt
v/v volume per volume
w/v weight per volume
WB Western blot
XIAP X-linked Inhibitor of Apoptosis Protein
V Abstract
A. Abstract
Pancreatic adenocarcinoma is one of the leading causes of cancer death and despite
aggressive surgical or medical management, the mean life expectancy is only 3 - 6 months
for patients with metastatic disease. Combination of surgery and current chemo- or
radiotherapy on long-term survival is minimal, since escape from apoptosis (programmed cell
death) is a characteristic feature of pancreatic cancer cells. This calls for new strategi