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비정상적인 Met활성화에 의한 중심체의 수적 증가와 유전체의 불안정성
| DC Field | Value | Language |
|---|---|---|
| dc.contributor.advisor | 이재호 | - |
| dc.contributor.author | 남현자 | - |
| dc.date.accessioned | 2018-11-08T07:39:30Z | - |
| dc.date.available | 2018-11-08T07:39:30Z | - |
| dc.date.issued | 2008-02 | - |
| dc.identifier.other | 6165 | - |
| dc.identifier.uri | https://dspace.ajou.ac.kr/handle/2018.oak/6585 | - |
| dc.description | 학위논문(박사)----아주대학교 일반대학원 :분자과학기술학과,2008. 2 | - |
| dc.description.tableofcontents | 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 | - |
| dc.language.iso | eng | - |
| dc.publisher | The Graduate School, Ajou University | - |
| dc.rights | 아주대학교 논문은 저작권에 의해 보호받습니다. | - |
| dc.title | 비정상적인 Met활성화에 의한 중심체의 수적 증가와 유전체의 불안정성 | - |
| dc.type | Thesis | - |
| dc.contributor.affiliation | 아주대학교 일반대학원 | - |
| dc.contributor.department | 일반대학원 분자과학기술학과 | - |
| dc.date.awarded | 2008. 2 | - |
| dc.description.degree | Master | - |
| dc.identifier.localId | 566931 | - |
| dc.identifier.url | http://dcoll.ajou.ac.kr:9080/dcollection/jsp/common/DcLoOrgPer.jsp?sItemId=000000006165 | - |
| dc.subject.keyword | Met | - |
| dc.subject.keyword | Centrosome Hyperamplification | - |
| dc.subject.keyword | Chromosomal Instability | - |
| dc.subject.keyword | the PI13K-Akt | - |
| dc.description.alternativeAbstract | 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. | - |
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