Aldehyde dehydrogenase 1 A1 is expressed by cancer stem cells and mediates chemoresistance in human glioblastoma:
Gespeichert in:
| Beteilige Person: | |
|---|---|
| Format: | Hochschulschrift/Dissertation Buch |
| Sprache: | Englisch |
| Veröffentlicht: |
2012
|
| Schlagwörter: | |
| Links: | http://mediatum.ub.tum.de/node?id=1113009 https://nbn-resolving.org/urn:nbn:de:bvb:91-diss-20121210-1113009-0-3 http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=025861019&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
| Umfang: | 127 Bl. Ill., graph. Darst. |
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| 100 | 1 | |a Schäfer, Andrea |e Verfasser |4 aut | |
| 245 | 1 | 0 | |a Aldehyde dehydrogenase 1 A1 is expressed by cancer stem cells and mediates chemoresistance in human glioblastoma |c Andrea Schäfer |
| 264 | 1 | |c 2012 | |
| 300 | |a 127 Bl. |b Ill., graph. Darst. | ||
| 336 | |b txt |2 rdacontent | ||
| 337 | |b n |2 rdamedia | ||
| 338 | |b nc |2 rdacarrier | ||
| 502 | |a München, Techn. Univ., Diss., 2012 | ||
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| 650 | 0 | 7 | |a Aldehyddehydrogenasen |0 (DE-588)4324574-2 |2 gnd |9 rswk-swf |
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Datensatz im Suchindex
| DE-BY-TUM_call_number | 0001 DM 30705 1001 Diss.4 4478 |
|---|---|
| DE-BY-TUM_katkey | 1915975 |
| DE-BY-TUM_location | Mag |
| DE-BY-TUM_media_number | TEMP3219655 040050976057 |
| _version_ | 1821934747163557888 |
| adam_text | IMAGE 1
TABLE O F CONTENTS
TABLE OF CONTENTS
A. SUMMARY 1
B. INTRODUCTION 3
1. MALIGNANT GLIOMAS 3
2. GLIOBLASTOMA 3
2.1 EPIDEMIOLOGY AND ETIOLOGY 3
2.2 HISTOLOGY AND MOLECULAR PATHOLOGY 4
2.2.1 PRIMARY AND SECONDARY GBM 4
2.2.2 IDH1/2 MUTATIONS 6
2.2.3 EGFR ALTERATIONS 7
2.2.4 PTEN MUTATIONS 9
2.3 MOLECULAR SUBCLASSES OF GLIOBLASTOMA 10
3. THE CELL OF ORIGIN: CLONAL EVOLUTION VERSUS CANCER STEM CELL THEORY
11
4. BRAIN TUMOR STEM CELLS OR BRAIN TUMOR CELLS WITH STEM CELL
CHARACTERISTICS 12
5. TREATMENT OF GLIOBLASTOMA 14
6. MECHANISMS O F THERAPY RESISTANCE 15
7. ALDEHYDE DEHYDROGENASE 1A1 16
8. OBJECTIVE 18
C. MATERIALS AND METHODS 20
1. TECHNICAL DEVICES 20
2. CHEMICALS, REAGENTS AND CYTOSTATICS 21
3. SOFTWARE 23
4. CELL CULTURE 23
4.1 CONSUMABLES AND ADDITIVES 23
4.2 BUFFERS AND MEDIA 24
4.3 CULTIVATION AND CRYOPRESERVATION OF GBM CELL LINES 25
4.4 PRIMARY CELL CULTURE 26
4.5 CELL AUTHENTICATION AND MYCOPLASMA-TEST 26
4.6 NEUROSPHERE FORMATION AND DIFFERENTIATION 27
4.7 IMMUNOFLUORESCENCE : 28
4.8 ALDH1A1 KNOCKDOWN WITH SHRNA 28
4.9 CELLULAR VIABILITY ASSAYS 29
HTTP://D-NB.INFO/1034436953
IMAGE 2
TABLE O F CONTENTS
4.10 COLONY FORMATION ASSAY 29
4.11 B-GALACTOSIDASE STAINING TO ANALYZE SENESCENCE O F GBM CELLS 30
5. WESTERN BLOT ANALYSIS 30
5.1 BUFFERS AND SOLUTIONS 30
5.2 ANTIBODIES 31
5.3 PROTEIN ISOLATION 32
5.4 SDS-PAGE AND WESTERN BLOTTING 32
6. ANALYSIS ON MRNA LEVEL: RT-PCR AND QPCR 34
6.1 BUFFERS AND SOLUTIONS 34
6.2 RNA ISOLATION AND REVERSE TRANSCRIPTION 34
6.3 OLIGONUCLEOTIDES FOR RT-PCR AND QPCR 35
6.4 ANALYSIS OF VARIOUS ALDH ISOFORMS ON MRNA LEVEL 36
6.5 OPTIMIZATION OF ANNEALING TEMPERATURES BY GRADIENT PCR 36
6.6 QUANTIFICATION OF ALDH1A1 KNOCKDOWN BY QPCR 37
7. QUANTIFICATION OF MGMT PROMOTER METHYLATION 38
8. IMMUNOHISTOCHEMICAL ANALYSIS OF ALDH1A1 AND IDH1 R132N EXPRESSION 40
8.1 BUFFERS AND SOLUTIONS 40
8.2 IMMUNOHISTOCHEMISTRY AND PATIENT CHARACTERISTICS 40
9. ANALYSIS OF CELL CYCLE DISTRIBUTION 42
10. ANALYSIS OF NITRIC OXIDE AND SUPEROXIDE FORMATION 44
11. ALDH1A1 EXPRESSION UNDER HYPOXIC CONDITIONS 44
12. IRRADIATION OF GBM CELL LINES 45
13. CLONING OF EGFR CONSTRUCTS AND ALDH1A1 INTO MAMMALIAN EXPRESSION
VECTORS 45
13.1 MEDIA, BUFFERS AND SOLUTIONS 46
13.2 PREPARATION OF PLASMID STOCKS 46
13.3 RESTRICTION DIGEST 47
13.4 DEPHOSPHORYLATION OF PCDNA3 48
13.5 LIGATION OF EXCISED CONSTRUCTS AND LINEARIZED PCDNA3 48
13.6 TRANSFORMATION OF COMPETENT E.COLI 48
13.7 PLASMID PREPARATION TO CHECK FOR POSITIVE CLONES 49
13.8 STABLE TRANSFECTION OF EUKARYOTIC CELLS 50
14. STATISTICAL ANALYSIS 51
IMAGE 3
TABLE O F CONTENTS
D. RESULTS 52
1. ALDH1A1 EXPRESSION IN PRIMARY AND ESTABLISHED GLIOBLASTOMA CELL LINES
52
2. STABLE AND SPECIFIC KNOCKDOWN O F ALDH1A1 IN GLIOBLASTOMA CELL LINES
BY SHRNA TRANSDUCTION.. 54
RESULTS I: ALDH1A1 AND PTEN/PI3K/AKT SIGNALING 56
1. PTEN STATUS O F GLIOBLASTOMA CELL LINES CORRELATES WITH NEUROSPHERE
FORMATION 56
2. AKT ACTIVATION AND HIGH-LEVEL ALDH1A1 EXPRESSION ARE FOUND IN
NEUROSPHERES 57
3. INHIBITION OF ALDH1A1 AND AKT IMPAIRS NEUROSPHERE FORMATION 59
4. INHIBITION OF ALDH1A1 AND AKT UNDER SERUMFREE CONDITIONS INDUCES
APOPTOSIS AND G1 ARREST.. 62
RESULTS II: ALDH1A1 AND CHEMORESISTANCE 64
1. ALDH1A1 EXPRESSION IN MGMT+ AND MGMT- GLIOBLASTOMA CELL LINES
CORRELATES WITH RESISTANCE T O
TEMOZOLOMIDE 64
2. COMBINATION OF TMZ AND ALDH1A1 DEPLETION REDUCES THE CLONOGENIC
POTENTIAL O F ALDH1A1+
GLIOBLASTOMA CELL LINES 65
3. SENSITIVITY TO TMZ IN MGMT+/ALDH1A1+ CELLS IS RESTORED BY INHIBITION
OR KNOCKDOWN OF
ALDH1A1 IN VITRO 68
4. TMZ INDUCES PROLONGED G2/M CELL CYCLE ARREST IN ALDH1A1+/MGMT+ GBM
CELLS AFTER INHIBITION
OR KNOCKDOWN OF ALDH1A1 69
5. CELL CYCLE ARREST FOLLOWING EXPOSURE TO TMZ AND DEAB IS ACCOMPANIED
BY NEURAL DIFFERENTIATION
OF ALDH1A1 POSITIVE GLIOBLASTOMA CELLS 74
6. ALDH1A1 INHIBITION OR KNOCKDOWN LEADS T O PHOSPHORYLATION OF H2AX
AFTER TMZ TREATMENT 75
7. ALDH1A1 PLAYS NO MAJOR ROLE IN MEDIATING RESISTANCE TO RADIOTHERAPY
76
8. ALDH1A1 PLAYS A PIVOTAL ROLE IN THE PROTECTION AGAINST OXIDATIVE
STRESS 77
8.1 ALDH1A1 PROTEIN LEVELS INCREASE UNDER HYPOXIC CONDITIONS 77
8.2 OXIDATIVE STRESS IS INDUCED BY TREATMENT WITH TEMOZOLOMIDE 78
9. HIGH-LEVEL ALDM1A1 EXPRESSION IS ASSOCIATED WITH AN UNFAVORABLE
PROGNOSIS IN GLIOBLASTOMA
PATIENTS 81
10. RELAPSED GLIOBLASTOMAS SHOW A HIGH PERCENTAGE O F ALOH1A1 POSITIVE
CELLS 85
IMAGE 4
TABLE O F CONTENTS
E. DISCUSSION 86
1. ALDH1A1 IS EXPRESSED BY STEM-LIKE GLIOBLASTOMA CELLS 87
2. PTEN/PI3K/AKT PATHWAY AND ALDH1A1 EXPRESSION PLAY A SUBSTANTIAL ROLE
IN NEUROSPHERE
FORMATION 89
3. ALDH1A1 OVEREXPRESSION PREDICTS TEMOZOLOMIDE RESISTANCE IN VITRO 93
4. SENSITIVITY OF ALDH1A1+/MGMT+ GLIOBLASTOMA CELLS TO TEMOZOLOMIDE IS
RESTORED BY INHIBITION
OR KNOCKDOWN OF ALDH1A1 95
5. HIGH-LEVEL ALDH1A1 EXPRESSION CORRELATES WITH AN UNFAVORABLE
PROGNOSIS IN GBM PATIENTS 97
6. ALDH1A1 IS EXTENSIVELY EXPRESSED IN TUMORS OF PATIENTS WITH RECURRENT
GBM 99
7. MECHANISMS O F ALDH1A1 MEDIATED RESISTANCE TO TEMOZOLOMIDE 100
7.1 DEPLETION OR INHIBITION OF ALDH1A1 IMPAIRS BASE EXCISION REPAIR 101
7.2 TEMOZOLOMIDE TREATMENT CAUSES OXIDATIVE STRESS 102
8. OUTLOOK 103
F. DANKSAGUNG 105
G. REFERENCES 107
H. ABBREVIATIONS 120
I. SUPPLEMENTARY DATA 122
J. LIST OF PUBLICATIONS 126
K. LEBENSLAUF 127
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| spellingShingle | Schäfer, Andrea Aldehyde dehydrogenase 1 A1 is expressed by cancer stem cells and mediates chemoresistance in human glioblastoma Arzneimittelresistenz (DE-588)4143180-7 gnd Aldehyddehydrogenasen (DE-588)4324574-2 gnd Marker (DE-588)4037588-2 gnd Glioblastom (DE-588)4157617-2 gnd |
| subject_GND | (DE-588)4143180-7 (DE-588)4324574-2 (DE-588)4037588-2 (DE-588)4157617-2 (DE-588)4113937-9 |
| title | Aldehyde dehydrogenase 1 A1 is expressed by cancer stem cells and mediates chemoresistance in human glioblastoma |
| title_auth | Aldehyde dehydrogenase 1 A1 is expressed by cancer stem cells and mediates chemoresistance in human glioblastoma |
| title_exact_search | Aldehyde dehydrogenase 1 A1 is expressed by cancer stem cells and mediates chemoresistance in human glioblastoma |
| title_full | Aldehyde dehydrogenase 1 A1 is expressed by cancer stem cells and mediates chemoresistance in human glioblastoma Andrea Schäfer |
| title_fullStr | Aldehyde dehydrogenase 1 A1 is expressed by cancer stem cells and mediates chemoresistance in human glioblastoma Andrea Schäfer |
| title_full_unstemmed | Aldehyde dehydrogenase 1 A1 is expressed by cancer stem cells and mediates chemoresistance in human glioblastoma Andrea Schäfer |
| title_short | Aldehyde dehydrogenase 1 A1 is expressed by cancer stem cells and mediates chemoresistance in human glioblastoma |
| title_sort | aldehyde dehydrogenase 1 a1 is expressed by cancer stem cells and mediates chemoresistance in human glioblastoma |
| topic | Arzneimittelresistenz (DE-588)4143180-7 gnd Aldehyddehydrogenasen (DE-588)4324574-2 gnd Marker (DE-588)4037588-2 gnd Glioblastom (DE-588)4157617-2 gnd |
| topic_facet | Arzneimittelresistenz Aldehyddehydrogenasen Marker Glioblastom Hochschulschrift |
| url | http://mediatum.ub.tum.de/node?id=1113009 https://nbn-resolving.org/urn:nbn:de:bvb:91-diss-20121210-1113009-0-3 http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=025861019&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
| work_keys_str_mv | AT schaferandrea aldehydedehydrogenase1a1isexpressedbycancerstemcellsandmediateschemoresistanceinhumanglioblastoma |
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0001 DM 30705 Lageplan 1001 Diss.4 4478 Lageplan |
|---|---|
| Exemplar 1 | Ausleihbar Am Standort |
| Exemplar 1 | Ausleihbar Am Standort |