바이러스성 단백질 HBx에 의한 DNA 손상 신호 활성화와 그에 의한 외부 DNA 손상 자극에 대한 둔감화
Modulation of the DNA Damage Checkpoint by Hepatitis B virus X protien: A Possible Role in Hepatocarcinogenesis
- 주제(키워드) Hepatitis B Virus X (HBx) , Cell cycle checkpoint , G2 delay , DNA damage , g-H2AX.
- 발행기관 아주대학교
- 지도교수 조혜성
- 발행년도 2006
- 학위수여년월 2006. 8
- 학위명 박사
- 학과 및 전공 일반대학원 분자과학기술학과
- 본문언어 영어
초록/요약
Purpose: Eukaryotic cells have evolved a complex defense mechanism to maintain genomic integrity and it is often destroyed in cancer cells. During the chronic infection of the hepatitis B virus (HBV), HBx viral protein plays a multifunctional role in the development of hepatocellular carcinoma (HCC). Here, we attempted to investigate whether HBx viral protein alters genomic integrity and the DNA damage-induced checkpoint pathway during the pathogenesis of HCC. Methods: To analyze cell cycle profiles and mitotic index, we carried out FACS analysis and aceto-orcein staining, respectively. CyclinB/Cdk1 and cyclinA/Cdk2 kinase activities were determined by immune complex kinase assay. Immunofluorescence staining for gamma-H2AX was used as a marker of double strand break. Activations of ATM and Chk2 were analyzed by western blot analysis using phospho-specific antibodies. Results: Cell cycle profiles revealed a delayed G2/M transition in both HBx-stably expressing ChangX cells and HBx-transiently transfected cells, in which the delayed increases of cyclinA/Cdk2 and cyclinB/Cdk1 kinase activities were accompanied. Utilizing CENP-F staining, we further demonstrated that HBx prevents cells from entering into the prophase. Importantly, we found that one of the DNA double strand breakage markers, gamma-H2AX foci were significantly increased in HBx expressing cells, which was accompanied with the activation of Chk2, a downstream kinase of DNA damage signaling. Consequently, the negative phosphorylation of Cdk1 was increased and cell cycle progression was delayed at G2 phase. Inhibition of ataxia telangiectasia mutated (ATM) and ATM and Rad3 related (ATR) proteins, the upstream kinase of DNA damage checkpoint abrogated the HBx induced G2 delay. Intriguingly, low dose of irradiation (1 Gy) in Chang cells activated the DNA damage checkpoint pathway, evidenced by recruitment of 53BP1 to the DNA foci, increase of the phospho-Chk2 and consequent cell cycle delay at G2 phase. In contrast, the same dose of irradiation in HBx-expressing cells failed to transduce the DNA damage signaling and did not induce any delay in cell cycle progression. Conclusion: Taken together, we concluded that the activation of the DNA damage-induced signaling pathway by HBx viral protein weakens the ability of cells to respond extrinsic DNA damage response, which may provide a chance for increasing DNA damage accumulation.
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ABSTRACT ••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••• i
TABLE OF CONTENTS ••••••••••••••••••••••••••••••••••••••••••iii
LIST OF FIGURES ••••••••••••••••••••••••••••••••••••••••••••••••••••v
I. INTRODUCTION ••••••••••••••••••••••••••••••••••••••••••••••••••••1
A. HBx as a multifunctional regulator in the development of
hepatocellular carcinoma ••••••••••••••••••••••••••••••••••••••••••••1
B. Cell cycle checkpoint •••••••••••••••••••••••••••••••••2
C. Effects of HBx on cell cycle deregulation ••••••••••••••••••••••••••••••••••••••••••••••••3
D. DNA damage checkpoint ••••••••••••••••••••••••••4
E. HBx and DNA damage •••••••••••••••••••••••••••••••8
F. Purpose of this study •••••••••••••••••••••••••••••••9
II. MATERIALS AND METHODS ••••••••••••••••••••••••••••••••10
A. Cell culture and synchronization •••••••••••••••••••10
B. Reagents and antibodies ••••••••••••••••••••••••••11
C. Plasmid and transfection •••••••••••••••••••••••••11
D. DNA FACS analysis and mitotic index counting •••••••••••••12
E. Western blot analysis and Cdk1/cyclin B Kinase Assay •••••••••••••••••••13
F. Ionizing radiation •••••••••••••••••••••••••••••••••••••••14
G. Determination of cellular ROS levels •••••••••••••••14
H. Immuno-fluorescence staining and DNA damaged foci counting ••••••••••••15
I. Statistics •••••••••••••••••••••••15
III. RESULTS •••••••••••••••••••••••••••16
A. G2/M transition was delayed by HBx•••••••••••••••16
B. Early but not late G2 phase was delayed•••••••20
C. HBx induces -H2AX foci formation •••••••••••••••24
D. DNA damage signaling cascade is activated by HBx and its inhibition
rescue the cells from HBx induced G2 delay ••••••••••••••28
E. 1-100 of HBx is important for DNA damage checkpoint activation•••••••••36
F. HBx increases cellular ROS levels which correlated with activation of
DNA damage•••••••••••••••••••••••••••••••39
G. HBx weakens extrinsic DNA damage signaling ••••••••43
IV. DISCUSSION •••••••••••••••••••••••••••••••••••••••••••••••••••51
V. CONCLUSION •••••••••••••••••••••••••••••••••••••••••••••••••••55
VI. REFERENCE ••••••••••••••••••••••••••••••••••••••••••••••••••57
국문요약 •••••••••••••••••••••••••••••••••••••••••••••••••••••••••••67
