| Project/Area Number |
11670008
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
General anatomy (including Histology/Embryology)
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| Research Institution | NAGOYA UNIVERSITY |
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Principal Investigator |
NISHIO Koji SCHOOL OF MEDICINE, NAGOYA UNIVERSITY, RESEARCH ASSOCIATE, 医学部, 助手 (60252235)
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| Co-Investigator(Kenkyū-buntansha) |
TATSUMI Hitoshi SCHOOL OF MEDICINE, NAGOYA UNIVERSITY, ASSOCIATE PROFESSOR, 医学部, 助教授 (20171720)
TONE Shigenobu KAWASAKI MEDICAL SCHOOL, SCHOOL OF MEDICINE, ASSOCIATE PROFESSOR, 医学部, 助教授 (70211399)
INOUE Akira OSAKA CITY UNIVERSITY, SCHOOL OF MEDICINE, ASSOCIATE PROFESSOR, 医学部, 助教授 (50109857)
HONDA Takashi SCHOOL OF MEDICINE, NAGOYA UNIVERSITY, RESEARCH ASSOCIATE, 医学部, 助手 (20165608)
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| Project Period (FY) |
1999 – 2000
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| Project Status |
Completed (Fiscal Year 2000)
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| Budget Amount *help |
¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 2000: ¥1,400,000 (Direct Cost: ¥1,400,000)
Fiscal Year 1999: ¥2,200,000 (Direct Cost: ¥2,200,000)
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| Keywords | cytoskeleton / vimentin / p53 / apoptosis / anchoring function / nuclear translocation / functional domain / 核移行反応 |
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| Research Abstract |
To reveal the molecular mechanism of anchoring of p53 and S1protein on the vimentin cytoskeleton, We constructed the expression vectors that produce the GFP-chimera of the full length, amino (N)-terminal or carboxyl (C)-terminal truncated p53. The N-terminal 20 or 40 amino acid and C-terminal 33 or 63 amino acid were truncated by PCR.The truncated p53 cDNA were subcloned into pEGFP vectors. Vimentin expressing cells, Cos7 and human fibroblasts or vimentin knockout Vim-/- cells were transfected with the truncated or full-length p53-GFP expression vectors. The wild type p53-GFP localized in the nuclei and occasionally in the cytoplasm. The truncated wild type p53-GFP localized in the nuclei in most cells. The localization of class III mutant p53V143A-GFP is cytoplasmic and dependent on the presence of vimentin. In contrast, the truncated mutant p53V143A-GFP (mp53V143AN41 and mp53V143AC330) significantly localized in the nuclei of Vim +/+ cells as like as wild type p53. These results indicates that both of the N-terminal and C-terminal domains of p53 involve with the anchoring on the vimentin cytoskeleton. We also examined GFP-chimera of class I (functional in both G1-arrest and apoptosis), class II (G1-arrest but not in apoptosis), and other class III mutants of p53 (defective in G1-arrest and apoptosis). Class I and class II mutants localized in the nuclei, in contrast to class III mutants, which significantly localized in the cytoplasm of Cos-7 cells. These results suggest that the conformation of p53 or interaction with other cellular component (s) is important in its cytoplasmic anchoring, which may have an inhibitory effect against p53-dependent apoptosis. P53 with mutant type conformation involves with cytoplasmic anchoring and may have an inhibitory effect against p53-dependent apoptosis. Biochemical investigation of apoptosis revealed that cleavage of nuclear lamin B2 was necessary for the DNA fragmentation.
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