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G2/M 세포주기 진행에 있어 hBubR1과 Chfr 단백질의 새로운 기능에 관한 연구
- Alternative Title
- Park Sun Yi
- Author(s)
- 박선이
- Alternative Author(s)
- Park Sun Yi
- Advisor
- 조혜성
- Department
- 일반대학원 분자과학기술학과
- Publisher
- The Graduate School, Ajou University
- Publication Year
- 2007-08
- Language
- eng
- Keyword
- function; Regulatory; cycle progression
- Alternative Abstract
- PURPOSE: 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.
- Fulltext
- Appears in Collections:
- Graduate School of Ajou University > Department of Molecular Science and Technology > 3. Theses(Master)
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