Cellular and molecular mechanisms of glioma growth control [Elektronische Ressource] / von Sridhar Reddy Chirasani

Sridhar Reddy Chirasani

Sujets

• Biologie

Informations

• Publié par: humboldt-universitat_zu_berlin
• Publié le: 01 janvier 2009
• Nombre de lectures: 33
• Langue: English
• Poids de l'ouvrage: 3 Mo

Extrait

Cellular and molecular mechanisms of glioma growth control
Dissertation
Zur Erlangung des akademischen Grades
doctor rerum naturalium
(Dr. rer. nat.)
im Fach Biologie
eingereicht an der
Mathematisch-Naturwissenschaftlichen Fakultät I
der Humboldt-Universität zu Berlin
von
M.Tech.Biotechnology. Sridhar Reddy Chirasani
geboren am 01.08.1978 in Chodipalem, Indien
Präsident der Humboldt-Universität zu Berlin
Prof. Dr. Dr. h.c. Christoph Markschies
Dekan der Mathematisch-Naturwissenschaftlichen Fakultät I
Prof. Dr. Lutz-Helmut Schön


Gutachter :
1. Prof.Dr.Richard Lucius
2. Prof.Dr.Helmut Kettenmann
3. Prof.Dr.Bozena Kaminska
Tag der mündlichen Prüfung: 28-08-2009






Abbreviations

Abbreviations ..........................................................................................................5
Abstract....................................................................................................................7
1. Introduction.....................................................................................................9
1.1. Brain tumors and their classification.........................................................9
1.2. Epidemiology of gliomas ........................................................................10
1.3. Diagnosis and treatment of gliomas........................................................11
1.4. Pathophysiology of gliomas....................................................................12
1.5. Cell of origin of gliomas .........................................................................13
1.6. Glioma Stem cells (GSCs) ......................................................................14
1.7. Glioma stem cells may represent as novel therapeutic targets................15
1.8. Targeting glioma stem cells via bone morphogenic proteins..................16
2. Molecular mechanisms of glioma growth control......................................19
2.1. ETS transcription factors and tumors......................................................19
2.2. Transferrin receptor (TfR).......................................................................22
2.3. r and its expression in brain23
2.4. rs and tumor cells .....................................................24
2.5. Transcriptional regulation of TfR ...........................................................25
2.6. TfR over expression and reactive oxygen species generation ................26
3. Aim of the study ............................................................................................28
4. Materials and Methods.................................................................................29
4.1. Materials..................................................................................................29
4.1.1. Devices29
4.1.2. Plastic ware and other material .......................................................30
4.1.3. Chemicals........................................................................................30
4.1.4. Enzymes ..........................................................................................31
4.1.5. Kits32
4.1.6. Antibodies .......................................................................................32
4.1.7. Oligonucleotides (PCR primers).....................................................34
4.1.8. Plasmids35
4.1.9. Media and buffer.............................................................................35
4.1.10. Software37
4.2. Methods...................................................................................................37
4.2.1. In vivo inoculation of glioma or glioma stem cells into the mouse 37
4.2.2. Survival study .................................................................................38
4.2.3. Tumor size measurement ................................................................38
4.2.4. Paraformaldehyde fixation..............................................................39
4.2.5. Immunohistochemistry of brain sections (floating sections) ..........39
4.2.6. Cell lines and plasmid constructs....................................................39
4.2.7. Cell culture of glioma cells .............................................................40
4.2.8. Cell culture of neural precursor cells ..............................................40
4.2.9. Isolation and Cell culture of Glioma stem cells..............................41 Abbreviations
4.2.10. Hippocampal cell culture and immunocytochemistry.....................41
4.2.11. Hematoxylin and Eosin staining .....................................................42
4.2.12. BrdU proliferation assay .................................................................42
4.2.13. Immunolabelling .............................................................................42
4.2.14. Microscopy......................................................................................43
4.2.15. Transfection methods......................................................................43
4.2.16. G418 sensitivity test........................................................................44
4.2.17. Cell Cycle Analysis using FACS analyser......................................45
4.2.18. Iron Imaging....................................................................................45
4.2.19. ROS measurement...........................................................................46
4.2.20. Reporter assays ...............................................................................46
4.2.21. Western blot47
4.2.22. Oxyblot and detection of oxidised proteins ....................................48
4.2.23. Gelatin zymography........................................................................49
4.2.24. Chromatin immunoprecipitation assay ...........................................49
4.2.25. Identification of mRNA transcripts.................................................50
5. Results-Part I: Mechanism of anti-tumorigenic effect of endogenous
neural precursor cells against glioma..................................................................53
5.1. Neural precursor cell-derived BMP7 inhibit the tumorigenic potential of
glioma stem cells.................................................................................................53
5.2. Neural precursor cells (NPCs) express BMP7 in tumor (glioma) margin ..
.................................................................................................................53
5.3. BMP7 expression in the glioma margin is predominantly contributed by
migrating NPC ....................................................................................................54
5.4. Neural precursors in subventricular zone (SVZ) expressess BMPs........54
5.5. Neurosphere cultures abundantly express BMP7 mRNA.......................55
5.6. PSA-NCAM positive NPC neurosphere cultures express and release
BMP7 protein......................................................................................................56
5.7. Glioma stemc cells express receptors for BMPs.....................................57
5.8. BMP-release from neurospheres downregulates CD133 expression in
Glioma Stem Cells ..............................................................................................58
5.9. Glioma Stem Cells respond to NPC-derived BMP7...............................60
5.10. NPC-derived BMP7 downstream signalling in glioma stem cells......61
5.11. BMP7 represses sphere formation of glioma stem cells .....................62
5.12. NPC derived BMP7 elicits a pro-differentiation effect on glioma stem
cells .............................................................................................................63
5.13. BMP7 supressess the proliferation of glioma stem cells and induce cell
death64
5.14. NPC-derived BMP7 inhibits tumorigenicity of glioma stem cells and
prolongs survival in a mouse model....................................................................64 Abbreviations
6. Results part II: Cell autonomous molecular mechanism of ets
transcription factor induced pro-tumorigenic signalling in glioma .................67
6.1. Proliferating glioma cells overexpress the Transferrin receptor.............67
6.2. Transferrin receptor overexpression in glioma is regulated via an ets
transcription factor binding site in the TfR promoter .........................................68
6.3. Trancription factor ets1 directly binds to the promoter region of the TfR
gene .................................................................................................................69
6.4. Alteration in expression of transferrin receptors in glioma changed their
morphology .........................................................................................................69
6.5. Over expression of transferrin receptors in glioma is independent of
culture conditions................................................................................................70
6.6. TfR expression in glioma in vitro is not altered by hypoxia and Hif1....71
6.7. TfR expression in glioma in vitro is largely independent of the cellular
iron concentration, but depends on the oxidant levels .......