1,722,663 research outputs found
Park Sun Yi
No full text학위논문(박사)--아주대학교 일반대학원 :분자과학기술학과,2007. 8TABLE OF CONTENTS
ABSTRACT ⅰ
LIST OF FIGURES ⅸ
PARTⅠ
Ⅰ. INTRODUCTION
A. Cell cycle checkpoints 1
B. Mitotic checkpoint 3
C. hBubR1 in cell cycle regulation 5
D. Regulation of G2-M transition 6
E. Purpose of the study 9
Ⅱ. MATERIALS AND METHODS 10
A. Reagents and antibodies 10
B. Cell culture and synchronization 10
C. Small-interfering RNA (siRNA) transfection 11
D. In vitro kinase assay 11
E. Immunofluorescence microscopy 12
F. Immunoblot analysis 13
G. Flow cytometry 13
H. Mitotic index counting 14
I. Statistics 14
Ⅲ. RESULTS 15
A. Knockdown of hBubR1 expression by siRNA 17
B. hBubR1 knockdown induces premature mitotic slippage under the condition of nocodazole treatment. 17
C. The hBubR1 knockdown induces early mitotic entry 17
D. hBubR1 depletion induces early accumulation of mitotic cells under time-lapse microscopy 19
E. hBubR1 depletion lowers intracellular cyclin B levels 23
F. hBubR1 overexpression restores cyclin B protein levels 27
G. hBubR1 depletion rapidly activates cyclin B/Cdk1 kinase activities 27
H. hBubR1 depletion increases the phosphorylated form of histone H3 30
I.Cyclin B was rapidly targeted to centrosomes in hBubR1 siRNA cells. 32
Ⅳ. DISCUSSION 36
Ⅴ. CONCLUSION 39
PART Ⅱ
Ⅰ. INTRODUCTION 41
F. Antephase checkpoint(mitotic stress checkpoint) 41
G. Chfr as a mitotic stress checkpoint 42
H. Regulation of protein degradation 45
I. Purpose of the study 46
Ⅱ. MATERIALS AND METHODS 47
J. Reagents and antibodies 47
K. Cell Culture and Synchronization 47
L. Immunofluorescence 48
M. Immunoblotting 49
N. Flow cytometry 49
O. Mitotic index counting 50
P. Statistics 50
Ⅲ. RESULTS 51
J. Nocodazole treatment delays mitotic entry and induces antephase in HeLa cells 51
K. Cell cycle profiles of Chfr transfected cells 51
L. Chfr is degraded at G2-M transition 55
M. Cell cycle-dependent degradation of Chfr 58
N. Gradual degradation of Chfr during mitotic progression 58
O. Chfr interacts with Cdc20 61
Ⅳ. DISCUSSION 63
Ⅴ. CONCLUSION 65
REFERENCE 66
국문요약 76
LIST OF FIGURES
Fig. 1. Diagram of cell cycle checkpoints. 2
Fig. 2. Localization of mitotic checkpoint proteins during mitosis 4
Fig. 3. Regulation of the cyclin B/Cdk1 activity during G2/M transition 8
Fig. 4. Knockdown of hBubR1 expression by siRNA 16
Fig. 5. Phenotype of hBubR1 siRNA cells after nocodazole treatment 18
Fig. 6. Cell cycle profiles of hBubR1 depleted cells 20
Fig. 7. Early mitotic entry by hBubR1 depletion 21
Fig. 8. Live-cell time-lapse image and analysis 24
Fig. 9. hBubR1 depletion lowers intracellular cyclin B level. 26
Fig. 10. Restoration of cyclin B protein by hBubR1 overexpression in SKBR3 cells lacking hBubR1 protein 28
Fig. 11. Premature activation of cyclin B/Cdk1 kinase activities by hBubR1 depleted cells 29
Fig. 12. Increase of phosphorylation of histone H3 in hBubR1 depleted cells 31
Fig. 13. Subcellular localization of endogenous cyclin B1 during G2-M transition. 33
Fig. 14. Rapid targeting of cyclin B to centrosomes by hBubR1 depletion 34
Fig 15. Schematic presentation of antephase checkpoint 44
Fig. 16. Nocodazole treatment delays mitotic entry and induces antephase in Chang cells 52
Fig. 17. Cell cycle profiles of Chfr transfected cells 54
Fig. 18. Chfr is degraded at G2-M transition 56
Fig. 19. Immunofluorescence analysis of Chfr expression during G2-M progression 57
Fig. 20. Cell cycle-dependent degradation of Chfr 59
Fig. 21. Gradual degradation of Chfr during mitotic progression 60
Fig. 22. Chfr interacts with Cdc20 62MasterPURPOSE: Cell cycle progression is tightly regulated by several kinds of checkpoint molecules that control proper timing for DNA replication and initiation of chromosomal segregation during mitosis. BubR1 is known as one of the key components of the mitotic checkpoint that ensures the fidelity of chromosome segregation during mitosis. Since BubR1 is expressed through the cell cycle in mammalian cells, other regulatory function of hBubR1 in cell cycle might be present unraveled. In addition, Chfr is known as a mitotic stress checkpoint gene that is broadly inactivated in a wide variety of human cancers. Chfr delays cells from entering mitosis under the condition of microtubule poisoning. So far, the mechanism by which Chfr regulates mitotic entry remains largely unknown. Here, I attempted to explore cellular functions of hBubR1 during transition from G2 phase to mitotic phase. In parallel, I examined expression profiles of Chfr protein in cell cycle progression and analyzed different expression patterns of Chfr during G2 and M progression in the presence of microtubule poisons.
METHODS: Small-interfering RNA (siRNA)-mediated knockdown of hBubR1 was applied to mammalian Chang and HeLa cells. Expression patterns of Chfr protein in cell cycle progression were analyzed after transfection of the flag-tagged Chfr expression vector into HeLa cells. Cells were synchronized by the double thymidine block method and cell cycle profiles and mitotic index were determined by flow cytometric analysis, time-lapse microscopy, aceto-orcein staining or cell cycle-specific CENP-F localization. Expression profiles and subcellular distribution of proteins during G2 to M transition were analyzed by immunoblotting and immunofluorescence staining, respectively. The cyclin B/Cdk1 kinase activities were determined by in vitro kinase assay and interaction of Chfr protein with Cdc20 protein in vivo was determined by co-immunoprecipitation assay.
RESULTS: Lowering hBubR1 protein levels by siRNA in HeLa and Chang cells shortened the G2 phase and promoted early mitotic entry under time-lapse microscopy as well as by the aceto-orcein staining. Intriguingly, the cyclin B levels of hBubR1-depleted cells were reduced. However, the cyclin B/Cdk1 kinase activity reached its peak earlier then control cells, which was accompanied with early increase of phospho-histone H3 levels in these cells. Immunofluorescence staining revealed that cyclin B in hBubR1 depleted cells co-localized with centrosomal γ-tubulin during early G2 phase, indicating that cyclin B in these cells prematurely move to centrosomes. Early centrosomal cyclin B localization was accompanied with early breakdown of nuclear envelope. In parallel, I found that overexpression of Chfr gene in HeLa cells did not significantly alter cell cycle progression profiles during G2-M progression by flow cytometric analysis. Intracellular cyclin A and B as well as Cdc20 levels were accumulated during G2-M progression in both control cells and Chfr-transfected cells. Interestingly, gradual decreases of Chfr proteins levels were observed during G2-M progression. Immunofluorescence staining against flag-Chfr protein confirmed gradual decrease of nuclear Chfr protein during G2-M progression. Next, HeLa cells expressing Chfr gene were treated with nocodazole up to 24 hrs and changes in Chfr protein expression were assessed in floating cells entered to mitotic phase and adherent cells trapped at G2 phase after nocodazole poisoning. In mitotic cells, a dramatic decrease of Chfr protein levels was observed in a time-dependent manner. In contrast, Chfr levels remained constant in cells of G2 phase. Finally, Chfr protein was found to interact with Cdc20, in vivo.
CONCLUSION: These results for the first time demonstrate that hBubR1 can control mitotic entry by regulating timing of centrosomal localization of cyclin B that appears to lead to early activation of cyclinB/Cdk1 kinase complex. Thus, hBubR1 may contribute to the suppression of premature targeting of cyclin B to the centrosomes during early G2 phase. In parallel, I demonstrated degradation of Chfr protein during G2-M progression as well as its persistence in the presence of nocodazole. The data suggest that degradation of intracellular Chfr levels may be a key regulatory step for the mitotic stress checkpoint activity
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