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Regulatory Factors of Cellular Senescence in Human Diploid Fibroblast : SA-p-Erk1/2, PP1/2A, PKCα

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dc.contributor.author김, 홍석-
dc.description.abstractProliferation of normal diploid cells is arrested irreversibly after a certain number of divisions, called replicative senescence, which is paragon of the general process of cellular senescence. Once senescent, cells continue to function metabolically, but will not respond to mitogens. Decreased growth rate, limited cell division, flat and large cell shapes, and increase of cell cycle regulator proteins are well-known characteristics of cells entered into senescence. Extracellular signal-regulated kinase 1/2 (Erk1/2) is one of the mitogenactivated protein kinase (MAPK). Erk1/2 signaling promotes cell proliferation and cell surnival. Ironically, p-Erk1/2 level is up-regulated in senescent cells. In the present study, the mechanism of senescence-associcated cytoplasmic induction of p-Erk1/2 (SA-p-Erk1/2) proteins in human diploid fibroblasts was investigated. Erk1/2 proteins were efficiently dephosphorylated in vitro by protein phosphatases 1 and 2A (PP1/2A). Specific activity of PP1/2A activity significantly decreased during cellular senescence, whereas their protein expression levels did not. SA-p-Erk1/2 was most likely due to the oxidation of PP1/2A, which resulted from continuous exposure of the cells to vast amounts of reactive oxygen species (ROS) generated during cellular senescence. Treatment of senescent human diploid fibroblast (HDF) cells with 12-O-tetradecanoylphorbol-13-acetate (TPA), a well known protein kinase C (PKC) activator, induced morphological change and thymidine incorporation. PKC is a serine/threonine kinase and regulates signal transduction pathways involved in gene expression, cell proliferation, and differentiation. We also investigated activation and down-regulation of PKCa by TPA treatment, which reverses senescent HDF cells to young cell like shapes. Within 30min after TPA treatment, binding of PKCa and p- Erk1/2 was significantly increased, and both of them translocated to nucleus with progression of the cell cycle. When PKCa was down-regulated with siRNA treatment, the generation of ROS in mid-old cells was decreased accompanied with a typical change of the senescent HDF to young-cell like morphology. PKCa down-regulation induced the decrease of p53, p21^(WAF1), and SA-p-Erk1/2 in mid-old cells. In addition, PKCa down regulated midold cells regained its proliferation capacity. Therefore, these results suggest that PKCa works not only for maintenance of senescence phenotypes, but also for regulation of proliferation ability in cellular senescence.-
dc.description.abstract정상 세포가 일정한 횟수의 세포증식 후 비가역적으로 세포분열이 정지하게 되는 현상을 replicative senescence(세포노화)라고 정의한다. 노화된 세포의 대사는 활발하나 mitogen에 반응하지 않는다. 세포성장 감소, 제한된 세포분열, 넓고 퍼진 세포 모양, 그리고 증가된 세포분열주기조절 단백질의 발현 등이 노화 세포의 특징으로 잘 알려져 있다. Erk1/2는 MAPK의 하나로 세포 증식과 세포의 생존을 촉진한다. 아이러니 하게도 활성화된 Erk1/2의 형태인 p-Erk1/2가 노화된 세포에서 증가되어 있었다. 본 연구에서는 노화와 관련하여 증가되어 있는 p-Erk1/2 (SA-p-Erk1/2)의 기작과 이의 역전 방법에 대하여 연구하였다. Erk1/2는 protein phosphatase 1 and 2A (PP1/2A)에 의해서 탈 인산화가 되었으며 노화가 진행되면서 PP1/2A의 활성 정도는 유의하게 감소하였으나 단백질 발현정도에는 차이가 없었다. SA-p-Erk1/2는 노화된 세포에서 활성산소에 의해 PP1/2A의 활성이 감소하였기 때문으로 판명되었다. 노화된 세포에 protein kinase C (PKC) activator로 잘 알려진 12-O-tetradecanoylphorbol-13-acetate (TPA)를 처리하였을 때 세포의 모양 변화와 thymidine incorporation이 증가되었다. PKC는 유전자 발현, 세포증식과 분화에 관련된 세포내 신호 전달을 조절하는 것으로 알려져 있으며 본 연구에서는 노화현상의 유지와 역전에 PKCα의 역할에 대해서 조사하였다. TPA를 세포에 처리 후 30분에 PKCα와 p-Erk1/2의 상호작용이 증가하였으며 핵으로 이동하였고 세포분열 주기의 진행이 관찰되었다. 노화된 세포에서 siRNA를 처리하여 PKCα의 발현을 감소시켰을 때, 노화된 세포의 특징인 활성산소의 감소와 세포모양의 변화가 유발되었다. 또한 PKCα의 발현 감소로 p53, p21WAF1, SA-p-Erk1/2의 정도가 감소하였으며 노화된 세포의 가장 큰 특징인 저하된 세포증식 능력이 회복되는 현상을 관찰할 수 있었다. 따라서 PKCα가 세포 노화 현상의 역전에 관여할 뿐만 아니라 노화 현상을 유지하는데 중요한 역할을 하는 것으로 사료된다.-
dc.description.tableofcontents"―ABSTRACT― = i



A. Cellular Senescence = 1

B. Extracellular Signal-regulated Protein Kinase 1/2 (Erk1/2) = 2

C. Protein Phosphatase 1 and 2A (PP1/2A) = 4

D. Protein Kinase C (PKC) = 5

E. Purposes of This Study = 7


A. Materials = 8

B. HDF Cell Culture = 8

C. Assay of Senescence Associated β-galactosidase = 9

D. Subcellular Fractionation = 10

E. Immunoprecopitation = 11

F. Immunoblot Analysis = 12

G. PKCa Activity Assay = 12

H. Immunocytochemistry = 13

I. Measurement of ROS by FACS Analysis = 13

J. Protein Phosphatases 1 and 2A Assay = 14

K. Restoration of Protein Phosphatases 1 and 2A Activity by Thiol specific Reagents = 15

L. [3H]-thymidine Incorporation Assay = 16

M. Statistics = 16


A. P-Erk1/2 Proteins were Markedly Induced in Senescent Cells = 17

B. Various Phosphatase Can Dephosphorylate p-Erk1/2 = 17

C. Activity of PP1/2A were Significantly Reduced during Cellular Senescence. = 24

D. ROS was the Mechanism of Reduction of PP1/2A Activities during Cellular Senescence. = 29

E. TPA Treatment Induces Reversal of Senescence Phenotype = 34

F. TPA Activates and Induces Translocation of PKCa = 35

G. Activated PKCa Interacts with p-Erk1/2 = 41

H. Increased ROS Level in Old Cells Activates PKCa = 41

I. Senescence Phenotype is Able to be Reversed by PKCa Down-regulation = 42

J. PKCa Down-regulated Old cells Regained Proliferation Ability = 48




―국문요약― = 80"
dc.titleRegulatory Factors of Cellular Senescence in Human Diploid Fibroblast : SA-p-Erk1/2, PP1/2A, PKCα-
dc.title.alternative인간섬유아세포에서 세포노화조절인자에 관한 연구-
dc.subject.keywordProtein phosphatase 1/2A-
dc.subject.keyword세포 노화-
dc.contributor.department대학원 의학과-
dc.contributor.affiliatedAuthor김, 홍석-
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