B형 간염 바이러스 X 단백질(HBx)에 의한 유사분열 체크포인트의 기능 이상에 대한 조절 기전
Mitotic Checkpoint Control by Hepatitis B Virus X (HBx) Oncoprotein
- 주제(키워드) HBx , Mitotic checkpoint
- 발행기관 아주대학교
- 지도교수 조혜성
- 발행년도 2010
- 학위수여년월 2010. 2
- 학위명 박사
- 학과 및 전공 일반대학원 분자과학기술학과
- 실제URI http://www.dcollection.net/handler/ajou/000000010549
- 본문언어 영어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
Purpose: Hepatitis B virus X (HBx), encoded by HBV genome, deeply involves in the development of HBV-mediated liver cancer, of which occurrence rate is highly correlated with chromosomal instability. Here, we attempted to investigate whether viral HBx protein contributes to increase chromosomal instability through mitotic checkpoint dysfunction in hepatocarcinogenesis. Methods: Interaction of BubR1 with HBx and CDC20 in vivo was determined by immunoprecipitation. To activate mitotic checkpoint, we used microtubule destabilizing drug, nocodazole. To analyze BubR1 localization in HBxAP/Rsf-1 depleted cells, we performed co-immunostaining for BubR1 along with ACA as a kinetochore marker. The level of HBxAP/Rsf-1 expression was analysed by western blot analysis. To determine the localization of HBxAPα/Rsf-1, we carried out chromosome spreading experiment and cellular fractionation analysis. Abnormality of chromosome segregation was measured by counting formation of lagging chromosomes and chromosomal bridge. Results: HBx mainly binds hBubR1 during mitosis and its interaction appeared to be dependent on BubR1 phosphorylation at mitotic phase. This HBx/hBubR1interaction was strong in cells and however, we found notably weak HBx/hBubR1 interaction in vitro, suggesting that a strong HBx/hBubR1 interaction in vivo require other protein(s). We found that depletion of HBx-interacting protein (HBxIP) by siRNA did not distort the HBx/hBubR1 interaction but rather enhanced it. In contrast, RNA interference against HBxAP (HBx-associated protein), also known as Rsf-1 (a subunit of chromatin remodeling factor complex), almost completely abolished the HBx/hBubR1 interaction in vivo. Reconstitution of HBxAP/Rsf-1 into the HBxAP depleted cells restored the HBx/hBubR1 interaction, indicating that HBxAP/Rsf-1 mediates the HBx/hBubR1 interaction in vivo. Cell fractionation experiment further demonstrated that these interactions occur during mitosis in the chromatin fraction. A series of deletion mutant analysis revealed that two Kunitz domains of HBx and the Cdc20 binding domain of hBubR1 are essential for these interactions. Consequently, the truncated mutant deleting Kunitz domain of HBx failed to bind hBubR1 and did not cause mitotic aberration. Furthermore, Binding of HBx to hBubR1 by HBxAP/Rsf-1 attenuated the association between BubR1 and CDC20 and increased the rate of chromosome instability. Conclusion: Together, our data show that the HBx impairs BubR1 function and induces chromosomal instability through HBxAP/Rsf-1. This provides a novel mechanism for a viral pathogen to induce genomic instability through mitotic checkpoint dysfunction in hepatocarcinogenesis.
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ABSTRACT ---------------------------------------------------------------------------------------ⅰ
TABLE OF CONTENTS -----------------------------------------------------------------------ⅲ
LIST OF FIGURES ------------------------------------------------------------------------------ⅴ
Ⅰ. INTRODUCTION
A. Chromosomal instability (CIN) and mitotic checkpoint ----------------------------- 1
B. Hepatocellular carcinoma (HCC) and chromosomal instability (CIN) ------------ 5
C. Role of HBx in the development of hepatocellular carcinoma -------------------- 6
D. HBx-associating protein (HBxAP)/chromatin remodeling factor (Rsf-1) -------- 7
E. Purpose of the study --------------------------------------------------------------------- 10
Ⅱ. MATERIALS AND METHODS
A. Cell culture and synchronization ------------------------------------------------------ 11
B. Reagent and antibodies ----------------------------------------------------------------- 12
C. siRNA, plasmid and transfection ------------------------------------------------------ 12
D. Immunoblotting and immunoprecipitation ------------------------------------------- 13
E. Cellular fractionation analyses --------------------------------------------------------- 14
F. Immunofluorescence staining ----------------------------------------------------------- 15
G. Chromosome spreading ----------------------------------------------------------------- 15
H. Statistical analyses ---------------------------------------------------------------------- 16
Ⅲ. RESULTS
A. HBx interacts with BubR1 during mitosis ------------------------------------------ 17
B. HBxAP/Rsf-1 but not HBxIP is required for the HBx-BubR1 interaction ----- 21
C. Cell cycle-dependent expression of HBxAP/Rsf-1 protein ------------------------ 26
D. HBxAP/Rsf-1 localizes to centromere in prometaphase --------------------------- 30
E. HBx interacts with BubR1 in chromatin region during mitosis ------------------ 33
F. HBxAP/Rsf-1 depletion does not affect the localization of BubR1 to kinetochore
----------------------------------------------------------------------------------------------- 35
G. Kuniz domains of HBx bind to the cdc20 binding domain of the BubR1 ------- 38
H. HBx and BubR1 interact with the full length of HBxAP/Rsf-1 ------------------- 43
I. Binding of HBx to hBubR1 by HBxAP/Rsf-1 attenuates the association between
BubR1 and CDC20 and increases the rate of chromosome instability ------------ 45
Ⅳ. DISCUSSION ------------------------------------------------------------------------------- 49
Ⅴ. CONCLUSION ------------------------------------------------------------------------------ 52
Ⅵ. REFERENCES ------------------------------------------------------------------------------ 53
Ⅶ. 국문요약 ----------------------------------------------------------------------------------- 61
