Oxidative stress 에 의한 Nuclear shape 의 변화
Nuclear shape alteration by oxidative stress
- 주제(키워드) ROS , H2O2 , Mitosis , Abnormal nuclear shape , Cdk1 , PP2A
- 발행기관 아주대학교
- 지도교수 이재호
- 발행년도 2018
- 학위수여년월 2018. 8
- 학위명 박사
- 학과 및 전공 일반대학원 의생명과학과
- 실제URI http://www.dcollection.net/handler/ajou/000000027777
- 본문언어 영어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
Many types of cancer exhibit abnormal nuclear shapes and increased reactive oxygen species (ROS) level compared to normal cells. However, whether ROS induce nuclear deformation has not been fully addressed. Here, hydrogen peroxide (H2O2) treatment induced concentration-dependent alterations in nuclear shape that were abolished by pretreatment with the antioxidant N-acetyl-L-cysteine (NAC) or overexpression of catalase. Interestingly, treatment with H2O2 during mitosis induced nuclear shape alterations significantly more compared with treatment to asynchronous cells, suggesting that nuclear shape alteration by H2O2 was mainly due to its effect on nuclear envelope disassembly and/or reassembly processes. Because protein phosphatase 2A (PP2A) activity is reported to be involved in nuclear envelope reassembly during mitosis, the possibility of PP2A involved in nuclear shape alteration has been investigated. Indeed, H2O2 reduced the activity of PP2A and okadaic acid, a PP1 and PP2A inhibitor, induced nuclear shape alteration. Moreover, overexpression of PP2A, but not PP1 or PP4, partially overcame H2O2-induced alterations in nuclear shape, indicating that a decrease in PP2A activity induced by H2O2 is specifically involved in the observed nuclear shape alterations. I also observed that the treatment of mitotic cells with H2O2 formed lamin aggregates during early mitosis, and it correlated with the abnormal nucleus formation. Since phosphorylation by Cdk1 is important in lamin disassembly during mitosis, I wondered whether Cdk1 was involved in the formation of lamin aggregates by H2O2. Treatment of mitotic cells with RO3306, an inhibitor of Cdk1, also induced lamin aggregates and an abnormal nucleus. Moreover, in vitro kinase assay and immunoprecipitation study demonstrated that H2O2 reduced the activity of Cdk1 via decreasing its interaction with cyclin B. In addition, the reduction of Cdk1 activity induced prematurely lamin reassembly, which seems to contribute to the formation of abnormal nuclei. Collectively, during early mitosis, inhibition of the Cdk1 activity by H2O2 leads to lamin aggregates probably due to a decrease in the phosphorylation of lamin. Remaining lamin aggregates seem to affect the lamin reassembly process and eventually contribute to the formation of abnormal nucleus. In addition, during mitotic exit, H2O2 inhibits the activity of PP2A, which plays an important role in nuclear envelope reassembly, resulting in the formation of the abnormal nucleus. Therefore, I propose that the inhibition of Cdk1 and PP2A activity by H2O2 during mitotic entry and exit, respectively, is the underlying mechanisms involved in the change of nuclear shape.
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TABLE OF CONTENTS
ABSTRACT. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . i
TABLE OF CONTENTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . iii
LIST OF FIGURES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vi
LIST OF TABLES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . viii
ABBREVIATIONS . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix
I. INTRODUCTION . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
II. MATERIALS AND METHODS. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
A. Antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
B. Cell culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
C. Synchronization and drug treatment. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
D. H2O2 treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
E. Determination of intracellular ROS level . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
F. Measurements of intracellular level of H2O2 in living cells . . . . . . . . . . . . . . . . . . . . . 18
G. Plasmids and transfection experiment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
H. Transmission electron microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
I. Immunocytochemistry (ICC). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
J. Immunoblotting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .19
K. Protein Phosphatase 2A (PP2A) activity assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .20
L. Time-lapse microscopic analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .20
M. In vitro Cdk1 kinase assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .21
N. Immunoprecipitation (IP) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .21
O. Phos-tag analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .22
P. S-glutathionylation assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .22
Q. Statistical analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
III. RESULTS
PART Ⅰ. Inhibition of PP2A activity by H2O2 during mitosis disrupts nuclear envelope reassembly and alters nuclear shape.
A. Treatment of mitotic cells with H2O2 induces abnormal nucleation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
B. Formation of abnormal nuclei following H2O2 treatment is prevented by NAC or catalase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
C. Quantitative changes in lamin B1, structural changes in the cytoskeleton and ER, and DNA damage are not major contributors to H2O2-induced nuclear shape changes . . . . . . . . . . . . . . . . . . . . 36
D. H2O2 inhibits PP2A activity during mitosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
E. Ectopic expression of PP2A rescues H2O2-induced aberrant nuclear shape changes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44
F. Inhibition of PP2A activity by H2O2 causes mislocalization of core proteins during mitotic exit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .47
PART Ⅱ. Oxidative stress during early mitosis induces formation of aggregated lamin through decreasing Cdk1 activity, leading to abnormal nuclear shape.
A. Lamin aggregates are formed by H2O2 treatment during mitosis . . . . . . . . . . . . . . 53
B. The lamin aggregates formed during mitosis correlates to nuclear shape hanges . . 58
C. The activity of Cdk1 is reduced by H2O2 treatment during early mitosis . . . . . . . . . 62
D. The inhibition of Cdk1 by H2O2 during early mitosis reduces the phosphorylation of the substrates, especially lamin protein . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65
E. Ectopic expression of Cdk1 rescues H2O2-induced lamin aggregates formation and aberrant nuclear shape changes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . , . . . . . . . . . . . . . . . . . . . . . . . . . 70
F. Cdc25 is not involved in the inhibition of the activity of Cdk1 by H2O2 . . . . . . . . . .73
G. Glutathionylation is not related to inhibition of Cdk1 activity by H2O2 . . . . . . . . . .76
H. Analysis of cysteine modification of Cdk1 119 residue using mass spectrometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79
I. Reduction in the activity of Cdk1 by H2O2 during mitosis leads to nuclear envelope reassembly earlier than normal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81
J. Progerin expression induces nuclear shape alteration in interphase cells and lamin aggregates in mitotic cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .84
IV. DISCUSSION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87
REFERENCES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100
국문요약 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109
