비정상적인 Met활성화에 의한 중심체의 수적 증가와 유전체의 불안정성
Aberrant Activation of Met Induces Centrosome Hyperamplification and Chromosomal Instability via the PI3K-Akt Pathway
- 주제(키워드) HGF/Met signaling , chromosomal instability , centrosome amplification , PI3K , 염색체 불안정성 , 중심체 이상증가
- 발행기관 아주대학교 대학원
- 지도교수 이재호
- 발행년도 2008
- 학위수여년월 2008. 2
- 학위명 박사
- 학과 및 전공 일반대학원 분자과학기술학과
- 본문언어 영어
초록/요약
Purpose: Genetic instability is considered as one of the main pathways in human carcinogenesis. Since aberrant HGF/c-Met signaling causes human cancers, we investigated whether activation of aberrant c-Met signaling induced genetic instability and what the underlying mechanisms were. Methods: Aneuploidy was assessed by Giemsa staining. The number of centrosome was determined by staining with ?-tubulin antibody. To analyze causes of centrosome amplification, we performed immunostaing for Cep-170 protein as a mature centrosome marker. Abnormality of chromosome segregation was measured by counting lagging and/or bridge chromosomes. Activation of Akt, Erk, JNK and p38 was analyzed by western blot analysis using corresponding phospho-specific antibodies. To investigate signaling pathways involved in centrosome amplification, we used signaling inhibitors, siRNA and dominant-negative mutants. To activate mitotic checkpoint, we used microtubule stabilizing drug, Taxol. Aurora-A and Polo-like kinase-1 (Plk-1) kinase activities were determined by immune complex kinase assay. Results: Both stable and transient expression of M1268T, a constitutively active form of Met, increased the cell population with aneuploidy. In addition, supernumerary centrosomes and multinucleated cells were increased, indicating that aberrant HGF/c-Met signaling indeed caused genomic instability. Among the possible down-stream signaling molecules, phospho-Erk and phospho-Akt levels were found to be elevated in M1268T expressing cells compared to MOCK-transfected cells. LY294002, a PI3K inhibitor, but not U0126, an MEK inhibitor, could abolish both centrosome hyperamplification and multinucleated cell formation. Moreover, Akt gene knockdown by Akt siRNA as well as expression of dominant-negative mutant forms of Akt or PTEN significantly inhibited both phenotypes. Interestingly, ectopic expression of wild-type Akt promoted supernumerary centrosomes indicating that activation of PI3K-Akt axis is both necessary and sufficient. Furthermore, we observed that the increase in aneuploidy by M1268T was found in p53-/- HCT116 cells, but not in p53+/+ HCT116 cells, which strongly suggests that M1268T induces chromosomal instability depending on p53 status. We further checked the CIN (chromosomal instability)-inducing effect of M1268T in mouse embryonic fibroblasts from p53-/- background. As expected, M1268T expression also resulted in the increase of aneuploidy and polyploidy as well as multi- and micronuclei formation, thus expanding our observation to non-transformed cells. Conclusion: Taken together, the data demonstrated that the aberrant Met signaling induces centrosome hyperamplification and the resultant increase of chromosomal instability via PI3K pathway depending on p53 status.
more목차
ABSTRACT = i
TABLE OF CONTENTS = ⅲ
LIST OF FIGURES = ⅵ
I. INTRODUCTION = 1
A. HGF/Met signaling and cancer = 1
B. Genomic instability and cancer = 3
C. Centrosome structure and centrosome duplication = 4
D. Centrosome amplification and chromosomal instability = 5
E. Purpose of this study = 6
II. MATERIALS AND METHODS = 12
A. Cell culture = 12
B. Antibodies and chemicals = 12
C. Cell synchronization and flow cytometry = 13
D. Plasmid and transfection = 14
E. Knockdown experiment = 14
F. Immuno-fluoresence staining = 15
G. Western blot analysis = 16
H. In vitro kinase assay = 17
I. RT-PCR = 17
J. Giemsa staining = 18
K. Transfection by microporation = 19
L. Statistics = 19
III. RESULTS = 20
A. Induction of aneuploidy by M1268T expression = 20
B. Increase of cells with multinuclei and micronuclei in M1268T expressing cells = 24
C.Constitutive activation of HGF/c-Met signaling results in hyperamplification of centrosome resulting in multipolar mitotic spindle formation = 27
D. Deregulation of centrosome duplication causes supernumerary centrosomes by M1268T expression = 31
E. Supernumerary centrosomes by M1268T is not correlated with CDK2/cyclinE activity = 35
F. Phosphorylation of Erk and Akt in M1268T expressing cells = 37
G. Activation of the PI3K-Akt pathway is necessary for centrosome hyperamplification = 39
H. Activation of the PI3K-Akt pathway is sufficient for centrosome hyperamplifcation = 46
I. Aneuploidy induction by M1268T is p53-dependent = 49
J. Increase of cells with multi-nuclei and micronuclei by M1268T expression in Mouse Embryonic Fibroblast (MEF) p53^(-/-) cells = 53
K. Increase of cells with aneuploidy and polyploidy by M1268T expression in Mouse Embryonic Fibroblast (MEF) p53^(-/-) cells = 56
IV. DISCUSSION = 59
V. CONCLUSION = 69
VI. REFERENCE = 71
국문요약 = 81
