Mechanism of cell death in Burkitt lymphomas [Elektronische Ressource] : apoptosis or mitotic catastrophe / von Cindrilla Chumduri

Cindrilla Chumduri

Sujets

• Biologie

Informations

• Publié par: humboldt-universitat_zu_berlin
• Publié le: 01 janvier 2009
• Nombre de lectures: 64
• Poids de l'ouvrage: 8 Mo

Extrait

Mechanism of cell death in Burkitt
lymphomas: apoptosis or mitotic catastrophe
Dissertation
zur Erlangung des Akademischen Grades
doctor rerum naturalium
(Dr. rer. nat.)
im Fach Biologie

eingereicht an der
Mathematisch-Naturwissenschaftlichen Fakultät I
Humboldt-Universität zu Berlin

Von
Cindrilla Chumduri (M.Sc. Biotechnology)
geboren am 11.10.1980 in Davangere-KS, Indien
Dekan der Mathematisch-Naturwissenschaftlichen Fakultät I
Prof. Dr. Lutz-Helmut Schön
Gutachter/innen:
1) Prof. Dr. Wolfgang Uckert
2) Prof. Dr. Peter T. Daniel
3) Dr. Ingeborg Tinhofer
Tag der mündlichen Prüfung: 18.December 2009 1






Dedicated
to my family











1

ZUSAMMENFASSUNG ................................................................................................................ 6
ABSTRACT ................................... 8
1 INTRODUCTION ................. 10
1.1 Cell death .................................................................................................................... 10
1.1.1 Apoptosis .............. 11
1.1.1.1 Cellular and molecular basis of apoptosis ..................... 11
1.1.1.2 Caspases ........................................................................................................ 12
1.1.1.3 Apoptosis signalling pathways ....... 13
1.1.1.3.1 Extrinsic apoptosis .................................................................................... 13
1.1.1.3.2 Intrinsic apoptosis ..................... 14
1.1.1.3.3 Cross-talk between the extrinsic and intrinsic pathways.......................... 16
1.1.1.4 Regulation of apoptosis signalling ................................................................. 16
1.1.1.4.1 Regulation of death receptor mediated extrinsic signalling ..................... 16
1.1.1.4.2 Regulation of mitochondrially-mediated intrinsic apoptosis signalling by
Bcl-2 family members ................................................................................................. 17
1.1.1.4.3 Regulation of apoptosis by IAPs 21
1.1.1.4.4 Regulation of apoptosis by p53 21
1.1.2 Non-apoptotic cell death ..................................................................................... 22
1.1.2.1 Necrosis ......................................... 22
1.1.2.2 Autophagy...................................................................... 23
1.1.2.3 Mitotic catastrophe ....................................................................................... 23
1.2 Apoptosis resistance in malignant disease . 25
1.2.1 Apoptosis resistance confers insensitivity to cancer therapy .............................. 25
1.3 The Cell Cycle .............................................................................................................. 26
1.3.1 The cell cycle phases ............................ 26
1.3.2 Regulation of the cell cycle .................. 27
1.3.3 Cyclin dependent kinases (Cdks) and cyclins ....................................................... 27
1.3.4 Cell cycle checkpoints and their role in tumor formation ................................... 29
1.3.4.1 Structural and functional aspects of PLK1 ..................... 30
1.4 Burkitt lymphoma ....................................................................... 33
1.5 Carcinogenesis ............................................ 35
1.5.1 Treatment modalities ........................................................................................... 35 2

1.5.1.1 Chemotherapy ............................................................................................... 35
1.5.1.1.1 Taxol .......................................... 36
1.5.1.1.2 Vincristine and nocodazole ....................................... 36
2 Aim of the study ................................................ 38
3 Results ................................................................................................................................ 39
3.1 Inverse relationship between apoptosis and polyploidy in various Burkitt
lymphoma cells upon microtubule inhibitors treatment ..................................................... 39
3.2 Vincristine treatment does not induce polyploidy implying different cell death
mechanism compared to that of taxol and nocodazole ....................... 46
3.3 Microtubule inhibitors induce endoreduplication during mitosis leading to
enhanced ploidy in apoptosis resistant cell lines ................................................................. 46
3.4 Mitotic catastrophe is not associated with immediate cell death ............................. 51
3.5 Taxol induced loss of viability is irreversible............................... 51
3.6 Taxol induced caspase activation ............................................................................... 52
3.7 Taxol induced cell death is p53 independent ............................. 55
3.8 Lack of Bax and Bak can be the cause of apoptosis resistance to taxol in Burkitt
lymphomas ............................................................................................................................ 56
3.9 Role of pro-apoptotic BH3-only proteins in taxol induced cell death ........................ 57
3.10 Taxol induces downregulation of the anti-apoptotic Bcl-2 family protein Mcl-1 ... 59
3.11 Taxol induced apoptosis is Bax and Bak dependent ............................................... 60
3.12 Individual pro-apoptotic BH3-only proteins have no impact on taxol induced
apoptosis in FDM cells .......................................................................... 63
3.13 Apoptosis induced by the microtubule inhibitors is caspase dependent and
inhibition of caspase activity leads to polyploidy ................................................................. 65
3.14 Caspase-8 plays an important role in taxol induced apoptosis ............................... 70
3.15 Induction of apoptosis upon deregulation of PLK1 function in Burkitt lymphomas72
3.16 PLK1 deregulation leads to G2/M cell cycle arrest, while PLK1 overexpression
promotes cell cycle progression ........................................................................................... 73
3.17 PLK1 deregulation triggers G2 and mitotic arrest ................... 75
3.18 Similar cell cycle characteristics observed in different Burkitt lymphomas: DG75
and CA46 in response to PBD overexpression. ..................................................................... 77 3

3.19 PLK1 deregulation promotes polyploidy in Burkitt lymphomas lacking Bak/Bak
expression ............................................................................................................................. 79
3.20 Deregulation of PLK1 function has an antiproliferative effect followed by necrosis
in Burkitt lymphomas ............................................................................................................ 80
3.21 Deregulation of PLK1 and treatment with microtubule inhibitors failed to show
an increase or inhibition of apoptosis induction .................................................................. 82
3.22 Deregulation of PLK1 function induces apoptosis but lacks synergistic effect with
microtubule inhibitors in HeLa cells ..................................................................................... 83
3.23 PBD induces aberrant spindle formation and chromosomal congressional defects85
3.24 PBD induced apoptosis in HeLa cells is caspase dependent ................................... 86
4 Discussion .......................................................................................................................... 89
5 Materials and methods ... 108
5.1 Materials ................... 108
5.1.1 Cell lines.............................................................................................................. 108
5.1.2 Genetically modified cell lines ........... 109
5.1.3 Cell culture media .............................................................................................. 109
5.1.4 Buffers and solutions .......................... 109
5.1.5 Fine chemicals .................................... 112
5.1.6 Kits used in this study ......................................................................................... 112
5.1.7 Antibodies .......................................................................... 112
5.1.8 Primers ............................................... 113
5.1.9 Plasmids and expression constructs .................................. 113
5.1.10 Enzymes .......................................... 114
5.2 Methods .................................................................................... 115
5.2.1 Cell biology methods .......................... 115
5.2.1.1 Culturing of mammalian cells ...................................................................... 115
5.2.1.2 Freezing and thawing procedure . 115
5.2.1.3 Microtubule inhibitor treatment . 115
5.2.1.4 Transient transfection of BL cells ................................................................ 116
5.2.1.5 Stable transfection and establishment of single cell clones ....................... 116
5.2.1.6 Measurement of cell death .................