The role of the apoptosis and splicing associated protein Acinus during apoptotic nuclear changes [Elektronische Ressource] / vorgelegt von Alvin Paul Joselin

Alvin Joselin

Sujets

• Biologie

Informations

• Publié par: heinrich-heine-universitat_dusseldorf
• Publié le: 01 janvier 2006
• Nombre de lectures: 14
• Langue: English
• Poids de l'ouvrage: 12 Mo

Extrait

The role of the apoptosis and splicing associated protein
Acinus during apoptotic nuclear changes




Inaugural-Dissertation
zur Erlangung des Doktorgrades der
Mathematisch-Naturwissenschaftlichen Fakultät
der Heinrich-Heine-Universität Düsseldorf






vorgelegt von
ALVIN PAUL JOSELIN
aus
Nagercoil, Indien
Düsseldorf, 2006



Aus dem Institut für Molekulare Medizin
der Heinrich-Heine Universität Düsseldorf









Gedruckt mit der Genehmigung der
Mathematisch-Naturwissenschaftlichen Fakultät der
Heinrich-Heine-Universität Düsseldorf


Referent: Prof. Dr. Klaus Schulze-Osthoff
Koreferent: Prof. Dr. Heinz Mehlhorn


Tag der mündlichen Prüfung:
09 Nov 2006 Acknowledgements
I. ACKNOWLEDGEMENTS
This work was performed at the Institut für Molekulare Medizin, Heinrich-
Heine-Universität Düsseldorf.
I would like to thank Prof. Dr. Klaus Schulze-Osthoff for his support,
encouragement and advise concerning the project.
I would also like to thank Dr. Christian Schwerk, my supervisor and mentor,
for his invaluable discussions, criticisms and timely motivation, which helped me
on to thinking independently and acquiring the skills that I needed to perform
experimentation successfully.
I thank Prof. Dr. Heinz Mehlhorn for making it possible for me to present this
thesis.
I am grateful to Fr. Dr. med. Grete Berns and Dr. Matthias Drechsler for their
help with performing pulsed-field gel electrophoresis. Thanks also to Dr. Axel
Schwerk for his help with the statistical analyses.
My gratitude and appreciation to all my colleagues at the Institut für
Molekulare Medizin, especially Ute, Frank, Vilma and Stephan, for their support
and assistance, not to mention the innumerable phone calls they made on my
behalf.
Thanks to my mom and dad for giving me the opportunity to study. And
finally, I’m thankful to my wife for her presence, patience and for proofreading
and correcting the many passive voices in my dissertation.

i Index
II. INDICES
II.1. TABLE OF CONTENTS
I. ACKNOWLEDGEMENTS I
II. INDICES II
II.1. TABLE OF CONTENTS II
II.2. ABBREVIATIONS VI
II.3. FIGURE INDEX IX
II.4. TABLE INDEX XI
III. INTRODUCTION 1
III.1. PROGRAMMED CELL DEATH: AN OVERVIEW 1
III.2. MOLECULAR MECHANISMS DURING APOPTOSIS 2
III.2.A. ACTIVATION OF CASPASES 3
III.2.B. NUCLEAR CHANGES 7
III.2.B.1. DNA fragmentation 7
III.2.B.2. Apoptotic chromatin condensation 14
III.3. APOPTOSIS AND SPLICING ASSOCIATED PROTEIN (ASAP) COMPLEX 16
III.4. ALTERNATIVE SPLICING AND APOPTOSIS 17
III.5. RNA INTERFERENCE 18
III.5.A. MECHANISM OF GENE SILENCING 19
III.5.B. VECTOR BASED, INDUCIBLE EXPRESSION OF SIRNAS 22
IV. AIM OF THE STUDY 26
V. MATERIALS AND METHODS 27
V.1. GENERAL MATERIALS 27
V.1.A. CHEMICALS AND REAGENTS 27
V.1.B. ENZYMES AND PROTEINS 28
V.1.C. KITS 29
V.1.D. CONSUMABLES 29
V.1.E. EQUIPMENT 29
V.1.F. SOFTWARE 30
V.1.G. CELL LINES 30
ii Index
V.1.H. CELL CULTURE REAGENTS 31
V.1.I. OTHER REAGENTS 31
V.1.J. EXPRESSION VECTORS 31
V.1.K. OLIGONUCLEOTIDES 32
V.1.L. ANTIBODIES 32
V.2. METHODS 33
V.2.A. MAINTENANCE OF CELL LINES AND BACTERIAL CULTURES 33
V.2.A.1. Culturing bacteria and preparation of stocks 33
V.2.A.2. Growth and maintenance of insect cells 33
V.2.A.3. Growth and maintenance of mammalian cell lines 33
V.2.A.4. Preparation of liquid nitrogen stocks 34
V.2.B. CLONING AND ANALYSIS 34
V.2.B.1. Restriction digestion 34
V.2.B.2. Agarose gel electrophoresis 34
V.2.B.3. Purification of DNA fragments form agarose gels 34
V.2.B.4. Ligation and transformation 35
V.2.B.5. Purification of plasmid DNA from bacteria 35
V.2.B.5.a. Small-scale plasmid isolation (Mini-prep) 35
V.2.B.5.b. Large-scale plasmid isolation (Maxi-prep) 36
V.2.B.6. Quantification of DNA 36
V.2.C. TRANSFECTION OF MAMMLIAN CELL LINES AND GENERATION OF STABLES 37
V.2.D. RNA INTERFERENCE 37
V.2.D.1. shRNA design 37
V.2.D.2. Plasmid generation and transfection 37
V.2.D.3. Induction of Acinus knockdown 38
V.2.E. RNA METHODS 38
V.2.E.1. Purification of cellular RNA 38
V.2.E.2. Reverse transcriptase PCR (RT-PCR) 38
V.2.F. SURVIVAL ASSAYS 38
V.2.F.1. Measurement of cell numbers 38
V.2.F.2. Crystal violet staining and quantification 39
V.2.F.3. Senescence-associated β-galactosidase assay 39
V.2.F.4. LDH assay 39
V.2.G. EXPRESSION AND PURIFICATION OF RECOMBINANT PROTEINS 39
V.2.G.1. Infection of SF9 cells 39
V.2.G.2. Purification of recombinant FLAG-Acinus 40
V.2.H. PROTEIN STUDIES 40
V.2.H.1. Preparation of cell extracts 40
iii Index
V.2.H.2. Protein gel electrophoresis (SDS-PAGE) 41
V.2.H.3. Western blotting 41
V.2.H.4. Immunoprecipitations 42
V.2.I. INDUCTION OF APOPTOSIS 43
V.2.J. CASPASE ASSAYS 43
V.2.K. FACS ANALYSIS 43
V.2.L. DNA LADDERING ASSAYS 44
V.2.M. PULSED-FIELD GEL ELECTROPHORESIS (PFGE) 44
V.2.N. IN VITRO CHROMATIN CONDENSATION ASSAYS 45
VI. RESULTS 46
VI.1. KNOCKDOWN OF ACINUS 46
VI.1.A. SHRNA DESIGN 46
VI.1.B. EVALUATION OF THE SILENCING POTENCY OF THE VARIOUS SHRNA 47
VI.1.C. STABLE AND INDUCIBLE KNOCKDOWN OF ACINUS 48
VI.1.D. ACINUS HAS A SLOW PROTEIN TURNOVER RATE 50
VI.1.E. KNOCKDOWN OF ACINUS IS REVERSIBLE 50
VI.2. EFFECT OF ACINUS KNOCKDOWN ON ASAP COMPLEX 52
VI.2.A. SAP18 AND RNPS1 LOCALIZE TO THE NUCLEUS 52
VI.2.B. ACINUS IS NOT REQUIRED FOR THE MAINTENANCE OF SAP18-RNPS1 INTERACTION 52
VI.3. GROWTH SUPPRESSIVE EFFECT OF ACINUS KNOCKDOWN 54
VI.3.A. KNOCKDOWN OF ACINUS RESULTS IN A SLOW GROWTH PHENOTYPE 54
VI.3.B. GROWTH SUPPRESSION IS NOT A RESULT OF SENESCENCE 57
VI.4. APOPTOSIS IN ACINUS KNOCKDOWN CELLS 58
VI.4.A. EARLIER CASPASE ACTIVATION IN ACINUS KNOCKDOWN CELLS 58
VI.4.A.1. Western blot analysis of caspase-3 58
VI.4.A.2. Cleavage of PARP 59
VI.4.A.3. Cleavage of ICAD 60
VI.4.A.4. Caspase activity assay 61
VI.5. APOPTOTIC CHROMATIN CONDENSATION IN ACINUS KNOCKDOWN CELLS 62
VI.5.A. IN VIVO CHROMATIN CONDENSATION ASSAY 62
VI.5.B. IN VITRO CELL-FREE APOPTOTIC CHROMATIN CONDENSATION ASSAY 67
VI.5.C. IN VITRO CHROMATIN CONDENSATION ASSAYS 69
VI.5.C.1. Caspase cleavage of recombinant Acinus 69
VI.5.C.2. In vitro chromatin condensation assays with recombinant Acinus 70
VI.6. APOPTOTIC DNA FRAGMENTATION IN ACINUS KNOCKDOWN CELLS 72
iv Index
VI.6.A. FLOW CYTOMETRIC ANALYSIS OF APOPTOTIC HYPODIPLOID NUCLEI 72
VI.6.B. LACTATE DEHYDROGENASE RELEASE ASSAY 74
VI.6.C. OLIGONUCLEOSOMAL DNA LADDERING ASSAY 75
VI.6.D. HIGH MOLECULAR WEIGHT DNA FRAGMENTATION 77
VI.6.D.1. Pulse-field gel electrophoresis 77
VI.6.D.2. Histone H2A.X phosphorylation 78
VI.6.D.3. H2A.X phosphorylation and LMW in MCF7 cells 79
VII. DISCUSSION 81
VII.1. A STABLE, INDUCIBLE AND REVERSIBLE KNOCKDOWN OF ACINUS 82
VII.2. CONSEQUENCE OF ACINUS KNOCKDOWN 83
VII.2.A. A NUCLEAR RNPS1-SAP18 INTERACTION PERSISTS IN THE ABSENCE OF ACINUS 83
VII.2.B. KNOCKDOWN OF ACINUS RESULTS IN GROWTH SUPPRESSION 83
VII.2.C. EARLIER CASPASE ACTIVATION 84
VII.3. CHROMATIN CONDENSATION IS UNAFFECTED BY THE ABSENCE OF ACINUS 85
VII.4. DNA FRAGMENTATION IN ACINUS KNOCKDOWN CELLS 87
VII.4.A. OLIGONUCLEOSOMAL DNA FRAGMENTATION IS IMPAIRED 88
VII.4.B. HMW FRAGMENTATION IS UNAFFECTED 90
VII.4.C. IMPLICATIONS OF ACINUS IN NUCLEAR APOPTOSIS 91
VIII. SUMMARY 94
IX. BIBLIOGRAPHY 96
v Abbreviations
II.2. ABBREVIATIONS
ΔΨ mitochondrial membrane potential m
A/T adenine thymine
aa amino acid
AIF apoptosis inducing factor
Amp ampicillin
Apaf-1 apoptotic protease activating factor-1
ASAP apoptosis and splicing associated protein
ASF/SF2 alternative splicing factor/ splicing factor 2
ATP adenosine triphosphate
Bak Bcl-2 homologous antagonist/killer
Bax Bcl-2 associated X-protein
Bcl-2 B cell leukemia-2
BSA bovine serum albumin
CAD caspase-activated DNase
CARD caspase recruitment domain
CED cell death-defective
CIDE cell-inducing DFF45-like effector
CPAN caspase activated nuclease
C-terminal carboxy terminal
dATP deoxy adenosine triphosphate
DFF DNA fragmentation factor
DNA deoxyribonucleic acid
dNTP deoxynucleotide triphosphate
DR death receptor
dsDNA double-stranded DNA
dsRNA double-stranded RNA
DTT dithiothreitol
ECL enhanced chemo luminescence
EDTA ethylenediamine tetraacetic acid
EJC exon junction complex
vi Abbreviations
ER endoplasmic reticulum
FACS fluorescence-activated cell sorting
FADD Fas associated protein with death domain
HDAC histone deacetylases
HEPES N- (2-hydroxyethyl) piperazine-N`-(2-ethanesulfonicacid)
HMGB high mobility group protein
HMW high molecular weight
HSP heat shock protein
ICAD