Molecular functional analysis of the tumor suppressor gene PDCD4 [Elektronische Ressource] / vorgelegt von Rajeshwari Marikkannu

Molecular functional analysis of the tumor suppressor gene PDCD4 [Elektronische Ressource] / vorgelegt von Rajeshwari Marikkannu

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MOLECULAR FUNCTIONAL ANALYSIS OF THE TUMOR SUPPRESSOR GENE PDCD4 Westfälische Wilhelms-Universität and International NRW Graduate School of Chemistry Muenster, Deutschland Rajeshwari Marikkannu From Eriyodu, Tamil Nadu, India October-2006 Biochemie MOLECULAR FUNCTIONAL ANALYSIS OF THE TUMOR SUPPRESSOR GENE PDCD4 Inaugural-Dissertation zur Erlangung des Doktorgrades der Naturwissenschaften in der NRW Graduate School of Chemistry im Fachbereich Chemie und Pharmazie der Mathematisch-Naturwissenschaftlichen Fakultät der Westfälischen Wilhelms-Universität Münster vorgelegt von Rajeshwari Marikkannu aus Eriyodu, Tamil Nadu, Indien Oktober-2006 Dekan : Prof. Dr. Bernhard Wünsch Erster Gutachter : Prof. Dr. Karl-Heinz Klempnauer Zweiter Gutachter : Prof. Dr. Hans-Joachim Galla Tag der mündlichen Prüfung : Tag der Promotion : To the nature..... the undeniable nature.......... the four seasons........................ the rain in the four seasons..

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MOLECULAR FUNCTIONAL ANALYSIS OF THE
TUMOR SUPPRESSOR GENE PDCD4







Westfälische Wilhelms-Universität and
International NRW Graduate School of Chemistry
Muenster, Deutschland



Rajeshwari Marikkannu
From
Eriyodu, Tamil Nadu, India


October-2006


Biochemie



MOLECULAR FUNCTIONAL ANALYSIS OF THE TUMOR
SUPPRESSOR GENE PDCD4




Inaugural-Dissertation
zur Erlangung des Doktorgrades
der Naturwissenschaften
in der NRW Graduate School of Chemistry
im Fachbereich Chemie und Pharmazie
der Mathematisch-Naturwissenschaftlichen Fakultät
der Westfälischen Wilhelms-Universität Münster



vorgelegt von
Rajeshwari Marikkannu

aus
Eriyodu, Tamil Nadu, Indien

Oktober-2006





























Dekan : Prof. Dr. Bernhard Wünsch

Erster Gutachter : Prof. Dr. Karl-Heinz Klempnauer
Zweiter Gutachter : Prof. Dr. Hans-Joachim Galla

Tag der mündlichen Prüfung :
Tag der Promotion :




To
the nature.....
the undeniable nature..........
the four seasons........................
the rain in the four seasons.............
the transition between the four seasons...........
the human race, who continuously battle against and constantly falling and raising again.........











There was a bad blot in my day.........
There was a fine blot in my day as well.........









The Metamorphosis.............................
M.C Eischer, Dutch,1898-1972

TABLE OF CONTENTS I
TABLE OF CONTENTS
ABBREVIATIONS ---------------------------------------------------------------------IV
1 SUMMARY ---------------------------------------------------------------------------- 1
2 INTRODUCTION 3
2.1 Cancer------------------------------------------------------------------------------ 3
2.1.1 Cancer Statistics--------------------------------------------------------------------3
2.1.2 Cancer Cell Physiology -------------------------------------------------------------3
2.1.3 Cancer Cell Genetics ---------------------------------------------------------------7
2.1.4 Tumor Suppressor Genes ----------------------------------------------------------8
2.1.5 Mechanisms of Inactivation of the Tumor Suppressor Genes --------------------8
2.1.6 Cancer Therapeutics9
2.2 Pdcd4 – A Novel Putative Tumor Suppressor -------------------------------- 11
2.2.1 The Cloning of Pdcd4 11
2.2.2 The Sequence Motifs of Pdcd4--------------------------------------------------- 11
2.2.3 The Structure of Pdcd4----------------------------------------------------------- 11
2.2.4 The Subcellular Localization of Pdcd4 ------------------------------------------- 12
2.2.5 The Regulation and Expression of Pdcd4---------------------------------------- 12
2.2.6 The Cellular Functions of Pdcd4 ------------------------------------------------- 14
2.2.7 The Molecular Mechanisms of the Functions of Pdcd4-------------------------- 15
2.3 Objectives and Rationale of the Study --------------------------------------- 17
2.3.1 Pdcd4 Models --------------------------------------------------------------------- 17
2.3.2 Pdcd4 Downregulation System -------------------------------------------------- 17
2.3.3 Upregulation System------------------------------------------------------ 19
3 MATERIALS AND METHODS ------------------------------------------------------ 20
3.1 Materials------------------------------------------------------------------------- 20
3.1.1 Antibodies 20
3.1.2 Cell Culture Products------------------------------------------------------------- 20
3.1.3 Chemicals and Reagents --------------------------------------------------------- 21
3.1.4 Devices and Instruments -------------------------------------------------------- 22
3.1.5 Enzymes -------------------------------------------------------------------------- 23
3.1.6 Genotype of Escherichia coli K12 strains---------------------------------------- 24
3.1.7 Kits-------------------------------------------------------------------------------- 24
3.1.8 Plasmids and Constructs 24
3.1.9 Primers---------------------------------------------------------------------------- 27
3.1.10 Standard Buffers and Solutions ------------------------------------------------ 27
3.2 The Molecular Biology Techniques -------------------------------------------- 33
3.2.1 Media and Agar plates ----------------------------------------------------------- 33
3.2.2 Preparation of Competent E.coli cells ------------------------------------------- 33
3.2.3 Transformation of Competent Bacteria and Blue/White Screening ------------ 33
3.2.4 Plasmid DNA Isolation------------------------------------------------------------ 34
3.2.5 Quantification of DNA 35
3.2.6 Modification of DNA by Enzymes 35
3.2.7 Agarose Gel Electrophoresis ----------------------------------------------------- 36
3.2.8 Extraction of DNA Fragments from Agarose gels ------------------------------- 36
3.2.9 Ligation --------------------------------------------------------------------------- 36
3.2.10 Polymerase Chain Reaction ---------------------------------------------------- 37

TABLE OF CONTENTS II
3.2.11 T/A-cloning of PCR products with TOPO TA System--------------------------- 38
3.2.12 DNA Sequencing ---------------------------------------------------------------- 38
3.2.13 Sequencing Gel ----------------------------------------------------------------- 39
3.2.14 Isolation of Genomic DNA from the Eucaryotic cells -------------------------- 39
3.2.15 Isolation of PolyA RNA from the Eucaryotic cells ------------------------------ 40
3.2.16 Genomic DNA-Agarose gel Electrophoresis and Southern Blotting----------- 41
3.2.17 RNA-Agarose gel Electrophoresis and Northern Blotting---------------------- 41
32 33.2.18 Radioactive Labeling of DNA with α P-dCTP and with H-dUTP -------------- 42
3.2.19 Hybridization and Washing ----------------------------------------------------- 42
3.2.20 Cell Cycle Analysis by FACS Method ------------------------------------------- 43
3.3 The Cell Culture Techniques --------------------------------------------------- 44
3.3.1 Cell lines and Medium------------------------------------------------------------ 44
3.3.2 Passage and Cultivation of cells ------------------------------------------------- 44
3.3.3 Cell Counting --------------------------------------------------------------------- 45
3.3.4 Transfection and Harvesting of Adherent and Suspension Cell lines ---------- 45
3.4 The Protein Biochemical Techniques ----------------------------------------- 47
3.4.1 Reporter-gene Assays 47
3.4.2 2-Dimensional-Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis - 48
3.4.3 Coomassie Brilliant Blue Staining------------------------------------------------ 51
3.4.4 Western Blotting and Immunodetection----------------------------------------- 51
353.4.5 S-Methionine Labeling---------------------------------------------------------- 51
3.4.6 Immunoprecipitation ------------------------------------------------------------- 52
4 RESULTS --------------------------------------------------------------------------- 53
4.1 Downregulation of Pdcd4 – A Knock-Down System------------------------- 53
4.1.1 Targeted Silencing of the Human Pdcd4 gene ---------------------------------- 53
4.1.2 The effect of Hpdcd4 on Cap-Dependent Translation--------------------------- 56
4.1.3 d4 on IRES-Dependentation 57
4.1.4 The search for Novel Molecular Targets of Hpdcd4 – Transcription Factors--- 61
4.1.5 The effect of Hpdcd4 on Transcription Factor C/EBP β -------------------------- 64
4.1.6 The search for Novel Molecular Targets of Hpdcd4 – A Proteomic Approach-- 67
4.1.7 The Effect of Hpdcd4 on the Biochemical Modification of CK-8 ---------------- 72
4.1.8 Expression of Pdcd4 Targets at the RNA Level---------------------------------- 74
4.1.9 The effect of Hpdcd4 on mRNA Stability ---------------------------------------- 76
4.1.10 The effect of Hpdcd4 on Nonsense-Mediated mRNA Decay------------------- 78
4.2 Downregulation of Pdcd4 – Knock-Out-System ----------------------------- 80
4.2.1 Targeted Disruption of the Chicken Pdcd4 gene -------------------------------- 80
4.2.2 The effect of the Disruption of Cpdcd4 on the Growth Characteristics -------- 83
4.2.3 Cpdcd4 on Translation--------------------------------------------- 83
4.2.3 The effect of Cpdcd4 on Translation 84
4.2.4 2D-SDS-PAGE Analysis of Cpdcd4 Knock-out Clones--------------------------- 87
4.3 Upregulation of Pdcd4 - Overexpression System 89
4.3.1 Overexpression of the Human Pdcd4 gene-------------------------------------- 89
4.3.2 2D-SDS-PAGE Analysis of Cpdcd4 Overexpression Clones--------------------- 91
5 DISCUSSION----------------------------------------------------------------------- 93
5.1 Analysis of Pdcd4 in the HeLa Cells------------------------------------------- 93
5.1 Analysis of Pd 94
5.1.1 Downregulation of Human Pdcd4 by siRNA Technology ------------------------ 94
5.1.2 Hpdcd4 inhibits Translation of Secondary Structured RNAs-------------------- 95

TABLE OF CONTENTS III
5.1.3 Hpdcd4 inhibits IRES-Dependent Translation----------------------------------- 95
5.1.4 Novel Molecular Targets of Hpdcd4---------------------------------------------- 98
5.1.5 Expression of c-Myc is lower in the Absence of Hpdcd4------------------------ 98
5.1.6 Phosphorylation of ATF-2 is decreased in the Absence of Hpdcd4------------- 99
5.1.7 Expression of C/EBP β is increased in the absence of Hpdcd4----------------- 100
5.1.8 Biochemical Modification of Cytokeratin-8 in the absence of Hpdcd4 -------- 100
5.1.9 Expression of Cytokeratin-17 is higher in the absence of Hpdcd4------------ 104
5.1.10 Expression of AKR1C2 and C3 are sence of Hpdcd4-------- 106
5.1.11 Biochemical Modification of GluProRS in the absence of Hpdcd4 107
5.1.12 Potential Role of Hpdcd4 in mRNA Stability ---------------------------------- 107
5.1.13 Potential role of Hpdcd4 in Nonsense-Mediated mRNA Decay--------------- 108
5.2. Analysis of Pdcd4 in the DT40 Cells----------------------------------------- 110
5.2.1 Downregulation of Chicken Pdcd4 by Homologous Recombination ----------- 110
5.2.2 Growth Characteristics are unaffected by Cpdcd4----------------------------- 110
5.2.3 Cpdcd4 has no Effect on Total Protein Synthesis ------------------------------ 111
5.2.4 Cpdcd4 on Cap-Independent Translation ----------------------- 111
5.3 Analysis of Pdcd4 in A549 Cells ---------------------------------------------- 112
6 FUTURE PERSPECTIVES --------------------------------------------------------- 113
7 REFERENCES---------------------------------------------------------------------- 114
8 APPENDIX------------------------------------------------------------------------- 125
8.1 Clone Charts-------------------------------------------------------------------- 125
8.2 Sequence ----------------------------------------------------------------------- 133
8.3 Zusammenfassung------------------------------------------------------------- 134
LEBENSLAUF ------------------------------------------------------------------------ 136
BIO-DATA --------------------------------------------------------------------------- 137
ACKNOWLEDGEMENT -------------------------------------------------------------- 138


ABBREVIATIONS IV
ABBREVIATIONS

1D First dimension
2D Second dimension
2D-SDS-PAGE Two dimensional sodium dodecyl sulfate polyacrylamide gel
electrophoresis
A Adenine base and the corresponding nucleotide
aa Amino acid
Ab Antibody
AKR1 Aldo keto reductases family 1
APS Ammonium persulfate
AraC Cytosine arabinoside
ATF Activating transcription factor
ATP Adenosine triphosphate
r AMP Ampicillin-resistant gene
bp Base pairs
BSA Bovine serum albumin
C/EBP CCAAT/enhancer-binding protein
cAMP Cyclic adenosine monophosphate
CBB Coomassie brilliant blue
CK-8 Cytokeratin-8
CK-17 Cytokeratin-17
CMV Cytomegalovirus
Cpdcd4 Chicken programmed cell death 4 protein ggene
C Cytosine base and the corresponding nucleotide
dd Double distilled
DNA Deoxyribonucleic acid
dNTP Desoxynucleotide
DTT 1,4-Dithio-DL-threitol
E.coli Escherichia coli
EDTA Ethylene diamine tetraacetate
eIF Eucaryotic initiation factor
EMS Ethyl methane sulphonate
Fig Figure
GluProRS Glutamyl prolyl bifunctional tRNA synthetase
G Guanine base and the corresponding nucleotide
hr Hour(s)