The role of MCL-1 phosphorylation for haematopoiesis and lymphoma development:
Gespeichert in:
| 1. Verfasser: | |
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| Format: | Abschlussarbeit Buch |
| Sprache: | English |
| Veröffentlicht: |
2013
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| Schlagworte: | |
| Online-Zugang: | Inhaltsverzeichnis |
| Beschreibung: | X, 178 S. Ill., graph. Darst. 30 cm |
Internformat
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| 100 | 1 | |a Lindner, Silke Elisabeth |e Verfasser |0 (DE-588)103624010X |4 aut | |
| 245 | 1 | 0 | |a The role of MCL-1 phosphorylation for haematopoiesis and lymphoma development |c vorgelegt von Silke Elisabeth Lindner |
| 264 | 1 | |c 2013 | |
| 300 | |a X, 178 S. |b Ill., graph. Darst. |c 30 cm | ||
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| adam_text | IMAGE 1
TABLE OF CONTENTS
A B S T R A C T ; I
Z U S A M M E N F A S S U N G III
T A B L E O F C O N T E N T S V
1 I N T R O D U C T I O N A N D L I T E R A T U R E R E V I E W 1
1.1 CHARACTERISTICS O F APOPTOSIS A N D OTHER FORMS O F CELL DEATH 1
1.1.1 APOPTOSIS 1
1.1.2 NECROSIS AND NECROPTOSIS 3
1.1.3 AUTOPHAGY 3
1 . 2 THE TWO APOPTOSIS SIGNALLING PATHWAYS 4
1.2.1 THE INTRINSIC OR BCL-2-REGULATED MITOCHONDRIAL PATHWAY 4
1.2.1.1 CASPASE-DEPENDENT CELL DEATH 4
1.2.1.2 CASPASE-LNDEPENDENT CELL DEATH 6
1 . 2 . 2 THE EXTRINSIC OR DEATH RECEPTOR PATHWAY 6
1 . 3 THE B C L - 2 PROTEIN FAMILY 8
1.3.1 THE ANTI-APOPTOTIC B C L - 2 PROTEINS 1 0
1.3.2 THE PRO-APOPTOTIC PROTEINS 1 1
1 . 3 . 3 THE B H 3 ONLY PROTEINS 1 2
1 . 4 HOW B C L - 2 FAMILY PROTEINS REGULATE THE MITOCHONDRIAL PATHWAY 1
4
1.4.1 THE INDIRECT ACTIVATOR MODEL 1 5
1.4.2 THE DIRECT ACTIVATOR-DEREPRESSOR MODEL 1 5
1 . 5 MYELOID CELL LEUKEMIA-1 ( M C L - 1 ) 1 6
1.5.1 DISCOVERY AND CHARACTERISATION 1 6
1.5.2 TISSUE DISTRIBUTION AND SUBCELLULAR LOCALISATION 1 7
V
HTTP://D-NB.INFO/1036240290
IMAGE 2
TABLE OF CONTENTS
1.5.3 REGULATION OF M C L - 1 1 8
1.5.3.1 TRANSCRIPTIONAL MECHANISMS 1 9
1.5.3.2 POSTTRANSCRIPTIONAL MODIFICATIONS 2 0
1 . 5 . 3 . 3 POSTTRANSLATIONAL MODIFICATIONS ( P T M ) 2 0
1.5.3.3.1 PHOSPHORYLATION 2 1
1.5.3.3.2 UBIQUITINATION 2 1
1 . 5 . 3 . 3 . 3 CASPASE CLEAVAGE 2 2
1 . 5 . 4 THE ROLE O F M C L - 1 IN HAEMATOPOIESIS 2 3
1 . 5 . 5 THE ROLE OF M C L - 1 IN CANCER 2 5
1 . 6 TRANSCRIPTION FACTOR M Y C AND ITS INTERPLAY WITH THE B C L - 2
FAMILY MEMBERS . 2 7
1.6.1 THE TRANSCRIPTION FACTOR M Y C 2 7
1.6.2 M Y C DEREGULATION AND APOPTOSIS 2 8
1 . 7 THE PI3 KINASE/AKT PATHWAY IS A MAJOR REGULATOR OF G S K - 3 3 0
1.7.1 THE P I 3 K / A K T PATHWAY 3 0
1.7.2 FUNCTION AND REGULATION O F G S K - 3 3 2
1 . 7 . 3 THE ROLE O F G S K - 3 IN APOPTOSIS 3 3
1 . 8 AIM OF THIS STUDY 3 5
2 M A T E R I A L S A N D M E T H O D S 3 7
2.1 CHEMICALS, REAGENTS AND EQUIPMENT 3 7
2.1.1 CHEMICALS AND REAGENTS 3 7
2 . 1 . 2 CONSUMABLES AND EQUIPMENT 4 0
2 . 1 . 3 KITS 4 2
2 . 1 . 4 BACTERIA 4 3
2 . 1 . 5 PLASMIDS 4 3
2 . 1 . 6 PRIMERS 4 4
2 . 1 . 7 CELL LINES 4 5
2 . 1 . 8 ANTIBODIES 4 5
2.1.8.1 ANTIBODIES FOR WESTERN BLOTTING 4 5
2 . 1 . 8 . 2 ANTIBODIES FOR F A C S ANALYSIS 4 6
2 . 1 . 9 CYTOKINES 4 7
2 . 1 . 1 0 MICE 4 8
2.1.10.1 C 5 7 B L / 6 4 8
2 . 1 . 1 0 . 2 B6.SJL-PFPRC- PEP3-/ BOYJ 4 8
2 . 1 . 1 0 . 3 B6.CG-TG(LGHMYC)22BRI/J 4 8
2 . 1 . 1 1 COMPUTER SOFTWARE AND HOMEPAGES USED 4 8
2 . 2 MOLECULAR BIOLOGY TECHNIQUES 4 9
2.2.1 REVERSE TRANSCRIPTION POLYMERASE CHAIN REACTION ( R T - P C R ) 4
9
2 . 2 . 2 AGAROSE GEL ELECTROPHORESIS 5 0
2 . 2 . 3 ISOLATION AND PURIFICATION O F D N A FROM AGAROSE GELS 5 1
2 . 2 . 4 DIGESTION O F D N A WITH RESTRICTION ENZYMES 5 1
2 . 2 . 5 DEPHOSPHORYLATION O F LINEARIZED D N A VECTOR 5 2
2 . 2 . 6 LIGATION OF D N A FRAGMENTS 5 3
2 . 2 . 7 TRANSFORMATION OF COMPETENT CELLS 5 3
2 . 2 . 8 MINIPREPS O F PLASMID D N A (MINIPREP) 5 4
VI
IMAGE 3
TABLE OF CONTENTS
2 . 2 . 9 ISOLATION O F PLASMID D N A FROM BACTERIA (MINIPREP) 5 4
2.2.9.1 CONTROL DIGEST 5 5
2 . 2 . 9 . 2 SEQUENCING 5 5
2 . 2 . 1 0 D N A QUANTIFICATION (OD) 5 5
2.2.11 LARGE SCALE PREPARATION O F D N A (MAXIPREP) 5 6
2 . 2 . 1 2 QUICK CHANGE SIDE DIRECTED MUTAGENESIS 5 6
2 . 2 . 1 3 R N A ISOLATION WITH TRIZOL 5 7
2 . 3 CELL BIOLOGICAL TECHNIQUES 5 8
2.3.1 THAWING OF CELLS 5 8
2 . 3 . 2 FREEZING O F CELLS 5 8
2 . 3 . 3 TESTING CELLS FOR MYCOPLASMA CONTAMINATION 5 9
2 . 3 . 4 COUNTING CELLS 5 9
2.3.4.1 NEUBAUER COUNTING CHAMBER (HAEMOCYTOMETER) 5 9
2 . 3 . 4 . 2 Z1 COULTER CELL COUNTER 5 9
2 . 3 : 5 CULTIVATION O F ADHERENT CELLS 6 0
2 . 3 . 6 CULTIVATION OF SUSPENSION CELLS 6 1
2 . 3 . 7 TRANSFECTION OF MAMMALIAN CELLS 6 1
2.3.7.1 TRANSFECTION USING SUPERFECT* 6 1
2 . 3 . 7 . 2 TRANSFECTION USING ATTRACTENE 6 2
2 . 3 . 8 RETROVIRAL EXPRESSION SYSTEM 6 2
2.3.8.1 RETROVIRUS PRODUCTION 6 3
2 . 3 . 8 . 2 VIRAL INFECTION OF ADHERENT CELLS 6 3
. 2 . 3 . 8 . 3 VIRAL INFECTION OF SUSPENSION CELLS 6 3
2 . 3 . 9 CELL SORTING 6 4
2 . 3 . 1 0 FLOW CYTOMETRY ANALYSIS 6 4
2.3.10.1 F A C S ANALYSIS OF ADHERENT CELLS 6 5
2 . 3 . 1 0 . 2 F A C S ANALYSIS OF SUSPENSION CELLS 6 5
2.3.11 CYTOKINE DEPRIVATION OF I L - 3 DEPENDENT CELLS 6 6
2.3.11.1 F A C S ANALYSIS OF PRIMARY CELLS 6 6
2 . 3 . 1 1 . 2 CELL CYCLE ANALYSIS 6 6
2 . 3 . 1 2 PROLIFERATION ANALYSIS 6 7
2 . 4 BIOCHEMICAL METHODS 6 8
2.4.1 TOTAL EXTRACT PROTEIN ISOLATION 6 8
2 . 4 . 2 BRADFORD ASSAY 6 8
2 . 4 . 3 REDUCING SODIUM DODECYL SULFATE POLYACRYLAMIDE GEL
ELECTROPHORESIS ( S D S - P A G E ) . 6 9
2.4.3.1 PREPARATION OF PROTEIN SAMPLES FOR S D S - P A G E 6 9
2 . 4 . 3 . 2 PREPARATION OF SDS-GELS 6 9
2 . 4 . 3 . 3 S D S - P A G E 7 0
2 . 4 . 3 . 4 TRANSFER 7 1
2 . 4 . 3 . 5 PONCEAU S STAINING 7 1
2 . 4 . 3 . 6 BLOCKING THE MEMBRANE 7 2
2 . 4 . 3 . 7 DETECTION OF PROTEINS B Y ANTIBODIES 7 2
2 . 5 IN VIVO STUDIES 7 3
2.5.1 GENOTYPING O F MICE 7 3
2 . 5 . 2 PHENOTYPING O F MICE 7 4
2 . 5 . 3 5-FLUOROURACIL (5-FU) MOBILIZATION OF H S C AND PROGENITOR
CELLS 7 5
2 . 5 . 4 ISOLATION AND CULTIVATION OF PRIMARY BONE MARROW CELLS 7 5
VII
IMAGE 4
TABLE OF CONTENTS
2.5.5 VIRAL INFECTION O F PRIMARY BONE MARROW CELLS 7 6
2.5.6 BONE MARROW ENGRAFTMENT 7 7
2 . 5 . 7 SURVIVAL BLEEDING OF MICE 7 8
2.5.7.1 RETRO-ORBITAL BLEEDING 7 8
2 . 5 . 7 . 2 MANDIBULAR VEIN BLEEDING 7 8
2 . 5 . 8 A D V I A 2 1 2 0 / 1 2 0 7 9
2 . 5 . 9 LYSIS OF ERYTHROCYTES 7 9
2 . 5 . 1 0 DISSECTING MICE 8 0
2.5.11 STAINING O F PRIMARY CELLS FOR FLOW CYTOMETRY ANALYSIS 8 0
2 . 5 . 1 2 EX VIVO EXPERIMENTS O F PRIMARY CELLS 8 1
2 . 5 . 1 3 HISTOLOGY 8 1
3 R E S U L T S : 8 3
3.1 VERIFICATION O F THE EXPERIMENTAL TOOLS FOR IN VIVO STUDIES 8 3
3.1.1 WILD-TYPE M C L - 1 IS PHOSPHORYLATED ON SERINE 1 5 9 B Y G S K -
3 8 3
3 . 1 . 2 THE SERINE 1 5 9 MUTATION O F M C L - 1 PROTECTS CELLS AGAINST
CYTOKINE DEPRIVATION
INDUCED APOPTOSIS 8 5
3 . 1 . 3 CELL CYCLE PROGRESSION IS INDEPENDENT OF SERINE 1 5 9
PHOSPHORYLATION ON M C L - 1 8 7
3 . 2 OPTIMIZING BONE MARROW INFECTIONS 9 1
3 . 3 THE ROLE OF M C L - 1 PHOSPHORYLATION FOR HAEMATOPOIESIS 9 3
3.3.1 RETROVIRAL INFECTION O F PRIMARY BONE MARROW 9 4
3 . 3 . 2 IMPACT OF SERINE 1 5 9 PHOSPHORYLATION-DEFICIENT M C L - 1
MUTANT ON LEUKOCYTE
NUMBERS 9 5
3 . 3 . 3 EQUAL EXPRESSION LEVELS OF M C L - 1 M AND M C L - 1 S 1 5 9 A
IN BONE MARROW ALLOW A
CORRELATION BETWEEN SURVIVAL AND STABILITY 9 8
3 . 3 . 4 INCREASED CELLULARITY REVEALS A SURVIVAL ADVANTAGE O F M C L -
1 S 1 S 9 A 1 0 0
3 . 3 . 5 M C L - 1 S 1 S 9 A PLAYS A CRUCIAL ROLE FOR THE SURVIVAL O F
H S C S BUT NOT FOR MEYLOPOIESISI 0 2
3 . 3 . 6 THE LOSS O F S 1 5 9 PHOSPHORYLATION O F M C L - 1 DOES NOT
IMPACT ON THE DISTRIBUTION O F
B-CELL SUBPOPULATIONS BUT ON THEIR SURVIVAL 1 0 4
3 . 3 . 7 PHOSPHORYLATION-DEFICIENT M C L - 1 DOES NOT PLAY A ROLE FOR
T-CELL DEVELOPMENT AND
SURVIVAL 1 0 9
3 . 4 M C L - 1 S 1 5 9 A IN DIRECT COMPETITION WITH M C L - 1 ^ IN
ACHIMERISM MODEL 1 1 2
3.4.1 GENERATION OF MIXED M C L - 1 ^ AND M C L - 1 S 1 5 9 A BONE
MARROW CHIMERIC MICE 1 1 3
3 . 4 . 2 LY-5.2 CELLS DISPLAYED A N ENGRAFTMENT ADVANTAGE INDEPENDENT O
F THE EXPRESSED
GENOTYPE 1 1 4
3 . 4 . 3 M C L - 1 S 1 S 9 PHOSPHORYLATION DOES NOT PLAY A CRUCIAL ROLE
FOR LYMPHOPOIESIS 1 1 6
3 . 4 . 4 T-CELL DEVELOPMENT IS INDEPENDENT O F S 1 5 9 PHOSPHORYLATION
O F M C L - 1 1 1 8
3 . 4 . 5 THE ABSENCE O F S 1 5 9 PHOSPHORYLATION DOES NOT AFFECT
MYELOPOIESIS 1 1 9
3 . 5 THE ROLE O F M C L - 1 S 1 5 9 A PHOSPHORYLATION FOR LYMPHOMA
DEVELOPMENT 1 2 0
3.5.1 INFECTION O F EM-MYC TRANSGENIC BONE MARROW 1 2 1
3 . 5 . 2 PHOSPHORYLATION-DEFICIENT M C L - 1 COOPERATES WITH EP-MYC T O
INDUCE LYMPHOMA 1 2 2
3 . 5 . 3 PHOSPHORYLATION-DEFICIENT-MCL-1 ACCELERATES LYMPHOMA 1 2 5
3 . 5 . 4 MICE WITH M C L - 1 / E M - M Y C TRANSPLANTED B M SHOW
SYMPTOMS O F BURKITT LYMPHOMA... 1 2 6
4 D I S C U S S I O N 1 2 9
VIII
IMAGE 5
TABLE OF CONTENTS
4.1 THE ROLE OF M C L - 1 IN CELL PROLIFERATION AND APOPTOSIS IN VITRO 1
2 9
4 . 2 PHOSPHORYLATION O F M C L - 1 A T SERINE 1 5 9 PLAYS A ROLE IN
HAEMATOPOIESIS 1 3 1
4 . 3 M C L - 1 S 1 5 9 A IN DIRECT COMPETITION WITH M C L - 1 ^ IN A
CHIMERISM MODEL 1 3 8
4 . 4 PHOSPHORYLATION-DEFICIENT M C L - 1 ACCELERATES LYMPHOMA IN THE
EM-MYC
MOUSE 1 4 0
5 G R A P H I C A L S U M M A R Y 1 4 7
6 B I B L I O G R A P H Y 1 4 9
7 A P P E N D I X : 1 7 1
7.1 ABBREVIATIONS 1 7 1
7 . 2 PUBLICATION LIST & PRIZES 1 7 5
7 . 3 CURRICULUM VITAE 1 7 6
7 . 4 CONFERENCES 1 7 8
IX
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| spelling | Lindner, Silke Elisabeth Verfasser (DE-588)103624010X aut The role of MCL-1 phosphorylation for haematopoiesis and lymphoma development vorgelegt von Silke Elisabeth Lindner 2013 X, 178 S. Ill., graph. Darst. 30 cm txt rdacontent n rdamedia nc rdacarrier Freiburg (Breisgau), Univ., Diss., 2013 (DE-588)4113937-9 Hochschulschrift gnd-content DNB Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=026234367&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Lindner, Silke Elisabeth The role of MCL-1 phosphorylation for haematopoiesis and lymphoma development |
| subject_GND | (DE-588)4113937-9 |
| title | The role of MCL-1 phosphorylation for haematopoiesis and lymphoma development |
| title_auth | The role of MCL-1 phosphorylation for haematopoiesis and lymphoma development |
| title_exact_search | The role of MCL-1 phosphorylation for haematopoiesis and lymphoma development |
| title_full | The role of MCL-1 phosphorylation for haematopoiesis and lymphoma development vorgelegt von Silke Elisabeth Lindner |
| title_fullStr | The role of MCL-1 phosphorylation for haematopoiesis and lymphoma development vorgelegt von Silke Elisabeth Lindner |
| title_full_unstemmed | The role of MCL-1 phosphorylation for haematopoiesis and lymphoma development vorgelegt von Silke Elisabeth Lindner |
| title_short | The role of MCL-1 phosphorylation for haematopoiesis and lymphoma development |
| title_sort | the role of mcl 1 phosphorylation for haematopoiesis and lymphoma development |
| topic_facet | Hochschulschrift |
| url | http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=026234367&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
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