p73-regulated pathways in Rhabdomyosarcoma:
Gespeichert in:
| 1. Verfasser: | |
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| Format: | Abschlussarbeit Buch |
| Sprache: | English |
| Veröffentlicht: |
2013
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| Schlagworte: | |
| Online-Zugang: | Inhaltsverzeichnis |
| Beschreibung: | III, 109 S. Ill., graph. Darst. 21 cm |
Internformat
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TABLE OF CONTENTS
1 SUMMARY/ZUSAMMENFASSUNG 1
1.1 SUMMARY 1
1.2 ZUSAMMENFASSUNG 2
2 INTRODUCTION 5
2.1 THE P53 FAMILY 5
2.1.1 STRUCTURE OF (HE P53 FAMSY PROTEINS 5
2.1.2 FUNCTIONS OF P53, P63 AND P73 6
2.1.2.1 THE P53 FAMILY IN CANCER 6
2.1.2.2 P63 AND P73 IN DEVELOPMENT 9
2.2 RHABDOMYOSARCOMAS 9
2.2.1 HUMAN RHABDOMYOSARCOMAS: EPIDEMIOLOGY, CLASSIFICATION, DIAGNOSIS
AND TREATMENT 9
2.2.2 MOLECULAR PATHOLOGY OF RHABDOMYOSARCOMAS 10
2.2.2.1 FUSION PROTEINS 10
2.2.2.2 CHROMOSOMAL ABERRATIONS IN FUSION-NEGATIVE RHABDOMYOSARCOMAS 11
2.2.2.3 P53 PATHWAY ALTERATIONS 11
2.2.2.4 REGULATION OF THE RB PATHWAY BY P53 FAMILY MEMBERS 13
2.3 P53 AND P73 IN THE RATIONAL TREATMENT OF CANCER 15
2.3.1 STRATEGIES TO REACTIVATE P53 IN MALIGNANT TUMORS 15
2.3.2 TARGETING P73 IN CANCER 17
2.3.3 THE SMALL MOLECULE RETRA: P73 ACTIVATION BY MUTANT P53 INHIBITION
18
2.4 SPECIFIC AIMS 20
3 MATERIALS AND METHODS 21
3.1 MATERIALS 21
3.1.1 EQUIPMENT, CONSUMABLES AND CHEMICALS 21
3.1.2 BUFFERS AND SOLUTIONS 21
3.1.3 COMMERCIAL KITS, STANDARDS AND READY-TO-USE SOLUTIONS 23
3.1.4 OLIGONUCLEOTIDES 24
3.1.5 SIRNAS 26
3.1.6 ANTIBODIES 26
3.1.7 ENZYMES 27
3.1.8 RECOMBINANT ADENOVIRUSES AND RETROVIRAL EXPRESSION VECTORS 27
3.1.9 BACTERIAL STRAINS 28
3.1.10 MAMMALIAN CELL LINES 28
3.1.11 MEDIA AND REAGENTS FOR TISSUE CULTURE 29
3.1.12 ANTIBIOTICS AND SMALL MOLECULE DRUGS 29
3.1.13 COMPUTER PROGRAMS AND DATABASES 30
3.2 METHODS 32
3.2.1 BIOLOGICAL SAFETY 32
I
HTTP://D-NB.INFO/1045692441
INHALL
3.2.2 CELL BIOLOGICAL METHODS 32
3.2.2.1 CULTURING AND PASSAGING OF CELLS 32
3.2.2.2 FREEZING AND THAWING OF CELLS 32
3.2.2.3 RETRA TREATMENT OF CELLS 33
3.2.2.4 TRANSFECTION OF CELLS WITH SIRNAS 33
3.2.2.5 PRODUCTION OF RECOMBINANT RETROVIRUS AND TRANSDUCTION OF CELLS
33
3.2.2.6 INFECTION OF CELLS WITH ADENOVIRUSES 34
3.2.2.7 FLOW CYTOMETRY 34
3.2.2.8 CELL VIABILITY ASSAY 36
3.2.2.9 ANALYSIS OF CELL MIGRATION 36
3.2.3 MOLECULAR BIOLOGICAL METHODS 37
3.2.3.1 PREPARATION OF GENOMIC DNA 37
3.2.3.2 CLEANUP OF DNA 38
3.2.3.3 POLYMERASE CHAIN REACTION (PCR) 38
3.2.3.4 SEPARATION OF NUCLEIC ACID FRAGMENTS BY AGAROSE GEL
ELECTROPHORESIS 39
3.2.3.5 TOPO CLONING OF PCR PRODUCTS 39
3.2.3.6 TRANSFORMATION OF ELECTROCOMPETENT BACTERIA 39
3.2.3.7 SMALL SCALE PREPARATION OF PLASMID DNA (MINI-PREP) 40
3.2.3.8 RESTRICTION DIGEST OF DNA 40
3.2.3.9 SEQUENCING OF DNA 41
3.2.3.10 RNA ISOLATION 41
3.2.3.11 REVERSE TRANSCRIPTION 41
3.2.3.12 QUANTITATIVE PCR (QPCR) 42
3.2.3.13 GENE EXPRESSION PROFILING BY MICROARRAY 44
3.2.4 BIOCHEMICAL METHODS 44
3.2.4.1 PREPARATION OF TOTAL PROTEIN EXTRACTS FROM CELLS 44
3.2.4.2 DETERMINATION OF PROTEIN CONCENTRATION 45
3.2.4.3 SDS-POLYACRYLAMIDE GEL ELECTROPHORESIS 45
3.2.4.4 WESTERN BLOT 45
3.2.4.5 CHROMATIN IMMUNOPRECIPITATION (CHIP) 46
3.2.5 COMPUTATIONAL AND STATISTICAL METHODS 48
3.2.5.1 ANALYSIS OF QPCR DATA 48
3.2.5.2 STATISTICAL ANALYSES 49
3.2.5.3 GENEPATTERN 50
3.2.5.4 GENE SET ENRICHMENT ANALYSIS 50
4 RESULTS 53
4.1 P73-DEPENDENT PATHWAYS IN TUMOR DEVELOPMENT S3
4.1.1 KNOCKDOWN OF
P73
DOWNREGULATES P53 AND TAP73 TARGET GENES 54
4.1.2 GENE SETS RELATED TO DEVELOPMENT AND IMMUNITY ARE UPREGULATED IN
P73-DEPIETED
CELLS 55
4.1.3 KNOCKDOWN OF P73 INDUCES A MUSCLE DIFFERENTIATION PROGRAM 57
II
INHALT
4.1.4 KNOCKDOWN OF P73 INDUCES EXPRESSION OF CXCR4 59
4.1.5 KNOCKDOWN OF P73 ACTIVATES THE TGF-(3 SIGNALING PATHWAY 61
4.1.6 KNOCKDOWN OF P73 INHIBITS CELL MIGRATION 63
4.1.7 ANP73 PROMOTES CELL MIGRATION 65
4.2 ROLE OF P73 IN THE MODE OF ACTION OF THE SMALL MOLECULE RETRA 66
4.2.1 P53 MUTATIONAL STATUS DOES NOT PREDICT SENSITIVITY TO RETRA 67
4.2.2 THE SENSITIVITY OF RHABDOMYOSARCOMA CELL LINES TO RETRA DOES NOT
CORRELATE WITH
P73 EXPRESSION 68
4.2.3 RETRA CAUSES A G2/M CELL CYCLE ARREST AND APOPTOSIS 69
4.2.4 RETRA TREATMENT INDUCES P21 AND PUMA 71
4.2.5 RETRA REPRESSES MUTANT P53 AND P73 IN RH1 CELLS, BUT INDUCES P63
72
4.2.6 RETRA INCREASES BINDING OF P63 BUT NOT OF P73 TO THE P21 PROMOTER
75
5
DISCUSSION
77
5.1 THE P73 KNOCKDOWN GENE SIGNATURE IN RHABDOMYOSARCOMA CELLS 77
5.1.1 CLASSICAL TUMORSUPPRESSOR FUNCTIONS AND OTHER ESTABLISHED ROLES OF
P73 78
5.1.2 P73 AND MYOGENIC DIFFERENTIATION 79
5.1.3 EMERGING ROLES FOR P73 IN METABOLISM 60
5.1.4 REGULATION OF CELL MIGRATION BY P73 82
5.1.4.1 ROLE OF CXCR4 IN P73-DEPENDENT MIGRATION 82
5.1.4.2 P73 AND TGF-0: IMPLICATIONS FOR CELL MIGRATION AND STEM CELL
MAINTENANCE 83
5.1.4.3 REGULATION OF CELL MIGRATION BY DIFFERENT P73 ISOFORMS 84
5.2 MUTANT P53 AND
P73 IN THE MODE OF ACTION OF THE SMALL MOLECULE RETRA
....86
5.2.1 SPECIFICITY OF RETRA FOR MUTANT P53-EXPRESSING CELLS 86
5.2.2 DEPENDENCY OF RETRA EFFECTS ON P73 88
5.3 FUTURE DIRECTIONS 90
6
REFERENCES 92
7 APPENDIX 103
7.1 ABBREVIATIONS 103
7.2 LEBENSLAUF [ENTFERNT] 106
7.3 VERZEICHNIS DER AKADEMISCHEN LEHRER 107
7.4 DANKSAGUNG 108
7.5 EHRENWORTLICHE ERKLARUNG [ENTFERNT] 109
III
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| author | Mack, Elisabeth Karin Maria |
| author_facet | Mack, Elisabeth Karin Maria |
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| dewey-ones | 610 - Medicine and health |
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| spelling | Mack, Elisabeth Karin Maria Verfasser aut p73-regulated pathways in Rhabdomyosarcoma vorgelegt von Elisabeth Karin Maria Mack 2013 III, 109 S. Ill., graph. Darst. 21 cm txt rdacontent n rdamedia nc rdacarrier Marburg, 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=027479470&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Mack, Elisabeth Karin Maria p73-regulated pathways in Rhabdomyosarcoma |
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| title | p73-regulated pathways in Rhabdomyosarcoma |
| title_auth | p73-regulated pathways in Rhabdomyosarcoma |
| title_exact_search | p73-regulated pathways in Rhabdomyosarcoma |
| title_full | p73-regulated pathways in Rhabdomyosarcoma vorgelegt von Elisabeth Karin Maria Mack |
| title_fullStr | p73-regulated pathways in Rhabdomyosarcoma vorgelegt von Elisabeth Karin Maria Mack |
| title_full_unstemmed | p73-regulated pathways in Rhabdomyosarcoma vorgelegt von Elisabeth Karin Maria Mack |
| title_short | p73-regulated pathways in Rhabdomyosarcoma |
| title_sort | p73 regulated pathways in rhabdomyosarcoma |
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