La lecture en ligne est gratuite
Le téléchargement nécessite un accès à la bibliothèque YouScribe
Tout savoir sur nos offres
Télécharger Lire

Functions of human DNA topoisomerases in cel proliferation and effects of anticancer drungs and natural compounds on the type II enzymes [Elektronische Ressource] / vorgelegt von Faiza Kalfalah

159 pages
Functions of human DNA topoisomerases in cellproliferation and effects of anti-cancer drugs andnatural compounds on the type II enzymesInaugural-Dissertationzur Erlangung des Doktorgrades der Mathematisch-Naturwissenschaftlichen Fakultätder Heinrich-Heine-Universität Düsseldorfvorgelegt vonFaiza Kalfalahaus Tripoli, LibyenDüsseldorf, Dezember 2009aus dem Institut für Klinische Chemie und Laboratoriumsdiagnostikder Heinrich-Heine Universität DüsseldorfGedruckt mit der Genehmigung derMathematisch-Naturwissenschaftlichen Fakultät derHeinrich-Heine-Universität DüsseldorfReferent: Prof. Dr. Fritz BoegeKoreferent: Prof. Dr. Peter ProkschTag der mündlichen Prüfung:ContentsContentsSummary ____________________________________________ VIIZusammenfassung ______________________________________ IX1. Introduction __________________________________________ 11.1 DNA topoisomerases_________________________________________ 31.1.1 Human nuclear topoisomerase I_______________________________________ 41.1.1.1 Human topoisomerase I domain structure______________________________ 51.1.1.2 Topoisomerase I catalytic cycle _____________________________________ 61.1.2 Human topoisomerases II ____________________________________________ 61.1.2.1 Topoisomerase II isoforms _________________________________________ 71.1.2.2 Topoisomerase II domain structure___________________________________ 81.2 Topoisomerase II catalytic cycle _______________________________ 91.
Voir plus Voir moins

Functions of human DNA topoisomerases in cell
proliferation and effects of anti-cancer drugs and
natural compounds on the type II enzymes
Inaugural-Dissertation
zur Erlangung des Doktorgrades
der Mathematisch-Naturwissenschaftlichen Fakultät
der Heinrich-Heine-Universität Düsseldorf
vorgelegt von
Faiza Kalfalah
aus Tripoli, Libyen
Düsseldorf, Dezember 2009aus dem Institut für Klinische Chemie und Laboratoriumsdiagnostik
der Heinrich-Heine Universität Düsseldorf
Gedruckt mit der Genehmigung der
Mathematisch-Naturwissenschaftlichen Fakultät der
Heinrich-Heine-Universität Düsseldorf
Referent: Prof. Dr. Fritz Boege
Koreferent: Prof. Dr. Peter Proksch
Tag der mündlichen Prüfung:Contents
Contents
Summary ____________________________________________ VII
Zusammenfassung ______________________________________ IX
1. Introduction __________________________________________ 1
1.1 DNA topoisomerases_________________________________________ 3
1.1.1 Human nuclear topoisomerase I_______________________________________ 4
1.1.1.1 Human topoisomerase I domain structure______________________________ 5
1.1.1.2 Topoisomerase I catalytic cycle _____________________________________ 6
1.1.2 Human topoisomerases II ____________________________________________ 6
1.1.2.1 Topoisomerase II isoforms _________________________________________ 7
1.1.2.2 Topoisomerase II domain structure___________________________________ 8
1.2 Topoisomerase II catalytic cycle _______________________________ 9
1.3 Subnuclear distribution of human topoisomerases _______________ 11
1.3.1 Topoisomerase I___________________________________________________ 11
1.3.2 Topoisomerase II __________________________________________________ 11
1.4 Biological functions of human topoisomerases I and II ___________ 12
1.4.1 Role of topoisomerases in replication _________________________________ 12
1.4.2 Role of topoisomerases in transcription _______________________________ 15
1.5 Topoisomerase inhibitors____________________________________ 16
1.5.1 Topoisomerase I poins as cancer drugs ________________________________ 18
1.5.2 Inhibitors of topoisomerase II _______________________________________ 19
1.5.2.1 Topoisomerase II poisons in cancer therapy ___________________________ 19
1.5.2.2 Topoisomerase II catalytic inhibitors ________________________________ 22
1.5.3 Natural and xenobiotic topoisomerase poisons__________________________ 23
1.5.3.1 Topoisomerase II poisoning by bioflavonoids _________________________ 24
1.5.3.2 Alkaloids ______________________________________________________ 25
1.5.3.3 Quinones ______________________________________________________ 26
1.5.3.4 Topoisomerase inhibition by DNA base modifications __________________ 26
1.5.4 Topoisomerase II-initiated chromosome translocations and leukemia ______ 27
IContents
1.6 Scope of the dissertation_____________________________________ 29
2. Materials____________________________________________ 31
2.1 Vectors and cDNAs_________________________________________ 31
2.1.1 Expression of bicistronic vectors _____________________________________ 31
2.1.2 Expression of tricistronic vectors_____________________________________ 32
2.1.3 cDNA____________________________________________________________ 32
2.1.4 DNA oligonucleotides ______________________________________________ 33
2.2 Bacterial strains and growth media ___________________________ 33
2.2.1 E. coli strains _____________________________________________________ 33
2.2.2 Bacterial growth media _____________________________________________ 33
2.3 Cell culture ________________________________________________________ 34
2.3.1 Cell lines _________________________________________________________ 34
2.3.2 Supplements and Antibiotics ________________________________________ 34
2.3.3 Media ___________________________________________________________ 35
2.4 Buffers and Stock Solutions__________________________________ 35
2.5 Enzymes __________________________________________________ 36
2.6 Chemicals_________________________________________________ 36
2.7 Topoisomerase toxins and inhibitors __________________________ 37
2.8 Antibodies ________________________________________________ 38
2.8.1 Primary antibodies ________________________________________________ 38
2.8.2 Secondary antibodies_______________________________________________ 38
2.9 Consumed items ___________________________________________ 38
2.10 Kits _____________________________________________________ 39
2.11 Instruments ______________________________________________ 39
3. Methods ____________________________________________ 41
3.1 Cloning___________________________________________________ 41
3.1.1 Construction of bicistronic vectors ___________________________________ 41
3.1.1.1 Generation of the fluorescent chimera of Cdc6_________________________ 41
3.1.1.2 Generation of the fluorescent chimera of PCNA _______________________ 42
3.1.2 Construction of tricistronic vector____________________________________ 42
3.1.2.1 Generation of yellow colored Topo with blue colored Cdc6 ______________ 43
IIContents
3.1.2.2 Generation of yellow colored Topo with blue colored PCNA _____________ 43
3.1.3 Standard PCR ____________________________________________________ 43
3.1.4 Purification of PCR products ________________________________________ 44
3.1.5 Gel electrophoresis and recovery of DNA from agarose gels ______________ 44
3.1.6 Restriction digestion _______________________________________________ 44
3.1.6.1 Analytical restriction digestion _____________________________________ 44
3.1.6.2 Preparative restriction digestion ____________________________________ 45
3.1.6.3 Partial restriction digestion ________________________________________ 45
3.1.7 Ligation__________________________________________________________ 45
3.1.8 Transformation and isolation of plasmid DNA__________________________ 45
3.1.8.1 Generation of competent E. coli cells ________________________________ 45
3.1.8.2 Transformation of E. coli _________________________________________ 46
3.1.8.3 Plasmid preparation at a small scale (Minipreps) _______________________ 46
3.1.8.4 Plasmid preparation at a large scale (Maxipreps) _______________________ 46
3.1.8.5 Sequencing of plasmids___________________________________________ 47
3.2 Cell culture _______________________________________________ 47
3.2.1 Maintenance of mammalian cells_____________________________________ 47
3.2.2 Freezing and thawing of cells ________________________________________ 47
3.2.3 Transfection and selection of HT-1080 cells ____________________________ 47
3.2.4 Cell cycle analysis _________________________________________________ 48
3.2.5 Cell Synchronization _______________________________________________ 48
3.2.5.1 Synchronization of cells by double thymidine block ____________________ 48
3.2.5.2 Synchronization of cells by Mevinolin _______________________________ 48
3.2.5.3 Synchronization of cells by serum starvation __________________________ 49
3.2.5.4 Synchronization of cells by Nocodazole______________________________ 49
3.3 Microscopy _______________________________________________ 49
3.3.1 Fluorescence microscopy____________________________________________ 49
3.3.2 Confocal microscopy _______________________________________________ 49
3.3.3 Photobleaching____________________________________________________ 49
3.3.4 Immunocytochemistry______________________________________________ 50
3.3.4.1 Fixation of cells by formaldehyde___________________________________ 50
3.3.4.2 Fixation of cells by methanol/ acetone _______________________________ 50
3.3.4.3 DRT Assay ____________________________________________________ 50
3.4 Proteins analysis ___________________________________________ 51
IIIContents
3.4.1 Preparation of whole cells lysates_____________________________________ 51
3.4.2 Chromatine fractionation ___________________________________________ 51
3.4.3 Polyacrylamide gel electrophoresis ___________________________________ 52
3.4.3.1 Gel run________________________________________________________ 52
3.4.3.2 Western blotting ________________________________________________ 52
3.4.4 Catalytic activity of topoisomerase ___________________________________ 52
3.4.4.1 Relaxation assay of topoisomerase __________________________________ 52
3.4.4.2 Cleavage assay _________________________________________________ 53
4. Results______________________________________________ 55
4.1 Human Cdc6 and PCNA as versatile markers for cell cycle stages __ 55
4.1.1 The replication initiation protein Cdc6 ________________________________ 55
4.1.2 The Polymerase anchorning factor PCNA _____________________________ 57
4.1.3 Production of stable cell lines expressing Cdc6 tagged YFP _______________ 58
4.1.4 Normal cell cycle-dependent regulation of over-expressed Cdc6-YFP ______ 60
4.1.5 Degradation and nuclear export of Cdc6 are temporally separated ________ 61
4.1.6 Relocalization of Cdc6 to the nucleus due to treatment of cells with hypotonic
buffer ________________________________________________________________ 63
4.1.7 Crm1-controlled association of Cdc6-YFP with centrosomes and microtubuli65
4.1.8 A model for Cdc6 regulation during the cell cyle ________________________ 66
4.1.9 Dissecting the cell cycle using PCNA and Cdc6 as markers _____________ 69
4.2 In vivo - Disposition of topoisomerases I and II during interphase __ 71
4.2.1 Characterization of cells co-expressing YFP-fused topoisomerases I, IIα,
IIβ with CFP-fused Cdc6 or PCNA _______________________________________ 71
4.2.2 Nuclear distribution and mobility of topoisomerases during interphase stages
_____________________________________________________________________ 73
4.2.2.1 Spatiotemporal distribution of topoisomerase I and II in G- and S phases____ 74
4.2.2.2 Mobility of topoisomerase I, II α and II β in interphase cells______________ 76
4.2.3 Active involvement of topoisomerases in replication factories _____________ 77
4.2.3.1 Effects of topoisomerase poisoning on the architecture of replication foci ___ 78
4.2.3.2 Differential recruitment of topoisomerases to replication foci _____________ 79
4.3 Monitoring Topoisomerases-directed effects of drugs, toxins, and
micro nutrients in living cells____________________________________ 83
4.3.1 In vitro and in vivo models for Topo II targeted subtances. _______________ 84
IVContents
4.3.2 Efficacy and isoform selectivity of clinical Topo II drugs _________________ 87
4.3.3 Validation of experimental Topo II drugs_____________________________ 100
4.3.3.1 Putative Topo IIα – selective poisons _______________________________ 100
4.3.3.2 Putative Topo IIβ _______________________________ 103
4.3.4 In vivo targeting of Topo IIα and IIβ by plant Polyphenols.______________ 106
5. Discussion __________________________________________ 111
5.1 Disposition and function of topoisomerase during interphase _____ 111
5.1.1 Our model is suitable and adequate__________________________________ 111
5.1.2 Specific functions of human topoisomerases in vivo during cell cycle ______ 112
5.2 Topo II directed effects of cancer drugs, environmental toxins and
food ingredients______________________________________________ 114
5.2.1 An attempt at a new classification of Topo II targeted compounds according
to their effect on enzyme mobility in vivo __________________________________ 114
5.2.2 A reappraisal of Topo II targeted cancer drugs ________________________ 117
5.2.3 The hazard of Topo II targeted toxins in food _________________________ 120
6. References__________________________________________ 123
7. List of abbreviations _________________________________ 137
8. Appendix___________________________________________ 139
8.1 Plasmid maps_____________________________________________ 139
8.1.A pMC-2PS-delta HindIII-P _________________________________________ 139
8.1.B pMC-EYFP-P-N _________________________________________________ 140
8.1.C pMC- EYFP-P___________________________________________ 141
9. Acknowledgments ___________________________________ 143
10. Erklärung _________________________________________ 145
VContents
VI