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Differentially expressed proteins in prostate cancer and functional characterization of proteins with altered expression [Elektronische Ressource] / vorgelegt von Ramesh Ummanni

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136 pages
Differentially expressed proteins in prostate cancer and functional characterization of proteins with altered expression I n a u g u r a l d i s s e r t a t i o n zur Erlangung des akademischen Grades doctor rerum naturalium (Dr. rer. nat.) an der Mathematisch-Naturwissenschaftlichen Fakultät der Ernst-Moritz-Arndt-Universität Greifswald vorgelegt von Ramesh Ummanni geboren am 15.05.1978 Hyderabad, India Dekan: ............................................................................................................. 1. Gutachter: ............................................................................................................. 2. Gutachter: ............................................................................................................. Tag der Promotion: ............................................................................................................. This thesis is dedicated to my parents and family members Results of this work are part of the following publications and manuscripts: Zimmermann U, Ummanni R, Junker H, Venz S, Teller S, Giebel J, Walther R. Establishing a protein signature from prostate tissue biopsies. Urologe A. 2007 Sep;46(9):1089-91. Ummanni R, Junker H, Zimmermann U, Venz S, Teller S, Giebel J, Scharf C, Woenckhaus C, Dombrowski F, Walther R.
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Differentially expressed proteins in prostate cancer and
functional characterization of proteins with altered expression





I n a u g u r a l d i s s e r t a t i o n
zur
Erlangung des akademischen Grades
doctor rerum naturalium (Dr. rer. nat.)
an der Mathematisch-Naturwissenschaftlichen Fakultät
der
Ernst-Moritz-Arndt-Universität Greifswald




vorgelegt von
Ramesh Ummanni
geboren am 15.05.1978
Hyderabad, India































Dekan: .............................................................................................................

1. Gutachter: .............................................................................................................

2. Gutachter: .............................................................................................................

Tag der Promotion: .............................................................................................................






This thesis is dedicated to my parents and family members
Results of this work are part of the following publications and
manuscripts:


Zimmermann U, Ummanni R, Junker H, Venz S, Teller S, Giebel J, Walther R.
Establishing a protein signature from prostate tissue biopsies. Urologe A. 2007
Sep;46(9):1089-91.

Ummanni R, Junker H, Zimmermann U, Venz S, Teller S, Giebel J, Scharf C, Woenckhaus
C, Dombrowski F, Walther R. Prohibitin identified by proteomic analysis of prostate
biopsies distinguishes hyperplasia and cancer. Cancer Letters, 2008, 266: 171-185


Ummanni R, Teller S, Junker H, Zimmermann U, Giebel J, Walther R. Altered expression
of TPD52 regulates apoptosis and migration of prostate cancer cells. Manuscript in revison
(FEBS Journal)

Ummanni R, Lehnigk U, Zimmermann U, Woenckhaus C, Walther R, Giebel J.
Immunohistochemical expression of caspases 1 and 9, uncleaved caspases 3 and 6, cleaved
caspases 3 and 6 as well as bcl-2 in benign prostate epithelium (BPE) and prostate cancer
(PCa). Manuscript submitted









Table of contents
Abbreviations
1 Introduction 1
1.1 Anatomy and physiology of Prostate …………………………………………… 1
1.2 Epidemiology and Etiology Prostate cancer…………………………………….. 1
1.3 Hormonal control of prostate……………………………………………………. 4
1.4 Prostate cancer ………………………………………………………………….. 5
1.4.1 Prostate cancer progression …………………………………………….. 5
1.4.2 Prostatic intraepithelial neoplasia ………………………………………. 5
1.4.3 Classification of Pca ……………………………………………………. 6
1.4.4 Diagnosis ……………………………………………………………….. 6
1.4.5 Biomarkers for early diagnosis of PCa …………………………………. 7
1.4.6 Treatment ……………………………………………………………….. 7
1.5 Proteomics ……………………………………………………………………… 8
1.5.1 Highlights of proteomics technology …………………………………… 8
1.5.2 Classification of proteomics technology………………………………… 9
1.5.3 Two-dimensional electrophoresis ………...…………………………….. 10
1.5.4 Mass spectrometry ……………………………………………………… 12
1.6 Prostate cancer proteomics ……………………………………………………… 14
1.7 Caspases in prostate cancer ……………………………………………………... 16
1.8 Aims of the study ……………………………………………………………….. 18
2 Materials and Methods 19
2.1 Materials ………………………………………………………………………… 19
2.1.1 Instruments………………………………………………………………. 19
2.1.2 Chemicals ……………………………………………………………….. 20
2.1.3 Kits and solutions ready to use …………………………………………. 22
2.1.4 Enzymes and inhibitors ………………………………………………… 23
2.1.5 Antibodies ……………………………………………………………… 24
2.1.6 Cell lines and materials used in cell culture …………………………… 24
2.1.7 Microbial cultures and culture medium used .......................................... 25
2.1.8 Primers …………………………………………………………………. 26
2.2 Plasmids ............................................................................................................... 28
2.3 Methods ………………………………………………………………………… 34
2.3.1 Clinical sample …………………………………………………………. 34
2.3.2 Cell culture ……………………………………………………………... 34
2.3.3 Preparation of protein extracts/RNA from cell lines …………………… 35
2.3.4 Protein extracts/RNA preparation from biopsies ………………………. 36
2.3.5 Two-dimensional gel electrophoresis (2-DE) ………………………….. 36
2.3.6 Imaging and analysis …………………………………………………… 37
2.3.7 Mass spectrometry ……………………………………………………… 37
2.3.8 Sodium Dodecyl Sulfate-PolyaAcrylamide Gel Electrophoresis ………. 38
2.3.9 Western blotting ………………………………………………………… 39
2.3.10 Histopathological evaluation ……………………………………………. 41
2.3.11 Immunohistochemistry ………………………………………………….. 41
2.3.12 Amplification of target genes by PCR ………………………………….. 42
2.3.13 Restriction digestion of DNA …………………………………………… 42
2.3.14 Annealing and phosphorylation of oligo nucleotides …………………… 43
2.3.15 DNA ligation ……………………………………………………………. 43
2.3.16 Preperation of competent cells ………………………………………….. 44
2.3.17 Bacterial transformation ………………………………………………… 44
2.3.18 Screening for positive clones ……………………………………………. 45
2.3.19 Preparation of glycerol stocks …………………………………………... 45
2.3.20 Transfection of cells …………………………………………………….. 45
2.3.21 Semi quantitative or quantitative real time PCR ....................................... 46
2.3.22 Agarose gel electrophoresis ……………………………………………... 47
2.3.23 Gel extraction of DNA ………………………………………………….. 48
2.3.24 Isolation of plasmid DNA from bacteria ……………………………….. 48
2.3.25 Formaulas for calcultion of moleculr weight and concentration ……….. 49
2.3.26 Downregulation of TPD52 ……………………………………………… 49
2.3.27 MTT assay for cell proliferation ………………………………………… 50
2.3.28 Cell migration assay …………………………………………………….. 51
2.3.29 Propidium iodide uptake (PI) for cell death …………………………….. 52
2.3.30 Caspase 3 and Caspase 9 activity determination ………………………... 52
2.3.31 Measurement of mitochondrial membrane transmembrane
potential (∆ψ ) ………………………………………………………….. 53 m
2.3.32 GST fusion protein expression and GST pull down assay ……………… 53
2.3.33 Co-immunoprecipitation of Prx1 ……………………………………….. 54
2.3.34 Immunofluoroscence ……………………………………………………. 55
2.3.35 (His )-TPD52 expression and purification ……………………………… 55 6
3 Results 57
3.1 Proteomic analysis of prostate needle biopsies …………………………………. 57
3.2 Prohibitn is overexpressed in prostate cancer ………………………………….. 64
3.3 TPD52 is overexpressed in prostate cancer …………………………………….. 68
3.4 Functional characterization of TPD52 in LNCaP cells …………………………. 71
3.4.1 Cloning and expression of EGFP-TPD52 ………………………………. 71
3.4.2 Downregulation of TPD52 ……………………………………………… 71
3.4.3 Dysregulation of TPD52 causes changes in the proliferation rate of
LNCaP cells ……………………………………………………………. 73
3.4.5 Silencing of TPD52 by shRNA leads to apoptosis in LNCaP cells ……. 74
3.4.6 Influence of TPD52 overexpression on LNCaP cell migration ………… 76
3.4.7 TPD52 interacts with the peroxiredoxin1 (Prx1) ………………………. 78
3.4.8 Localization of TPD52 …………………………………………………. 80
3.4.9 Overexpression and purification of (His )-TPD52 ……………………... 81 6
3.5 Expression of active and inactive caspases in prostate cancer …………. 82
3.5.1 Histology and Grading of tumours ……………………………………… 82
3.5.2 Imunohistochemistry for expression of caspases and statistical analysis. 83
4 Discussion 88
4.1 Proteomic analysis of Prostate biopsies ………………………………………… 88
4.2 Prohibitin can distinguish hyperplasia and cancer ………………………………. 89
4.3 TPD52 is over expressed in prostate cancer ……………………………………. 90
4.4 Functional Characterization of TPD52 in LNCaP cells ………………………… 91
4.4.1 Downregulation of TPD52 induces apoptosis ………………………….. 91
4.4.2 Dysregulation TPD52 alters LNCaP cell proliferation …………………. 92
4.4.3 Cell migration and activation of AKT/PKB pathway …………………... 92
4.4.4 TPD52 interacts with Prx 1 in LNCaP cells …………………………….. 95
4.4.5 Localization TPD52 ……………………………………………………... 96
4.5 Caspases in prostate cancer ……………………………………………………... 97
4.5.1 Active and inactive caspase-3 expression in PCa and BPE …………….. 97
4.5.2 Active and inactive caspase-6 expression in PCa and BPE …………….. 98
4.5.3 Immunostaining for caspase-1, -9 and Bcl-2 …………………………… 98
Summary 100
5 References 103
Acknowledgements 122
Erklärung 124
Curriculum vitae 125
Abbreviations

2-DE 2-dimentional gel electrophoresis
aa Amino acid
APS Ammonium persulphate
at al. and others
AMACR Alpha-methylacyl-CoA-racemase
AR Androgen receptor
bp Base pairs
BPE Benign prostate epithelium
BPH Benign prostate hyperplasia
BSA Bovine serum albumin
CDS Coding sequence
CHCA α-Cyano-4-hydroxycinnamic acid
CHAPS 3-[(3-Cholamidopropyl)dimethylammonio]-propanesulfonate
COFRADIC Combined FRActional DIagonal Chromatography
dd H O Deionized distilled water 2
DEPC Diethyl pyrocarbonate
DIOC Dihexyloxacarbocyanine iodide
DNA Deoxyribonucleic acid
dNTP Deoxyribonucleic acid triphosphate
DDT Dithiothreitol
DHT Dihydroxy testosterone
DRE Digital rectal examination
E coli Escherichia coli
EDTA Ethylendiamintetraacetic acid
EGTA Ethylenebis(oxyethylenenitrilo) tetraacetic acid
ESI Electrospray ionization
FFPE Formalin-fixed paraffin-embedded
FPC Familial prostate cancer
FCS Fetal calf serum
For forward
GFP Green Fluorescent Protein
GST Glutathione -S-Transferase
h Hour
HDC Hereditary prostate cancer
HEPES Hydroxyethyl piperazinyl ethanesulfonic acid
HUGO Human Genome Organization
HRP Horseradish peroxidase
IgG Immunoglobulin G
IGF-1 Insulin-like growth factor-1
IP Immunoprecipitation
IPTG Isopropyl β-thiolgalactoside
IPG Immobilzed pH gradient
IEF Isoelectric focusing
kDa kilodalton
LB Luria Bertani medium
LCM Laser capture microdissection
MALDI Matrix Assisted Laser Desorption Ionization
MCS Multiple cloning site
min Minute
mRNA messenger ribonucleic acid
MS Mass spectrometry
MW Molecular weight
NMR Nuclear magnetic resonance
OD Optical density
PAGE polyacrylamide gel electrophoresis
PF-2D Two-dimensional liquid-phase proteome profiling
PCR Polymerase chain reaction
PCa Prostate cancer
PI Propidium iodide
PIN Prostatic intra epithelial neoplasia
PHB Prohibitin
PMSF Phenyl methyl sulphonylfluoride

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