Cell cycle-associated accumulation of TIMP-1 in the nuclei of human gingival fibroblasts
| Project/Area Number |
08877280
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| Research Category |
Grant-in-Aid for Exploratory Research
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| Allocation Type | Single-year Grants |
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| Research Field |
Functional basic dentistry
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| Research Institution | School of Dentistry, Aichi-Gakuin University |
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Principal Investigator |
HAYAKAWA Taro Department of Biochemistry, School of Dentistry, Aichi-Gakuin University, Professor, 歯学部・生化学講座, 教授 (80064822)
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| Co-Investigator(Kenkyū-buntansha) |
YAMASHITA Kyoko Department of Biochemistry, School of Dentistry, Aici-Gakuin University, Assista, 歯学部・生化学講座, 助手 (40231659)
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| Project Period (FY) |
1996 – 1997
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| Project Status |
Completed (Fiscal Year 1997)
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| Budget Amount *help |
¥1,800,000 (Direct Cost: ¥1,800,000)
Fiscal Year 1997: ¥800,000 (Direct Cost: ¥800,000)
Fiscal Year 1996: ¥1,000,000 (Direct Cost: ¥1,000,000)
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| Keywords | TIMP-1 / nuclear accumulation / cell-cycle / Gin-1 cell / anti-TIMP-1 monoclonal antibody / ヒト歯肉線維芽(Gin-1)細胞 / 核移行 / TIMP-2 / TIMP-3 / マトリックス金属プロテアーゼ(MMP) / ヒト歯肉線維芽細胞 / 細胞核 / TIMP-1サンドイッチ酵素免疫測定法 |
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| Research Abstract |
Presently we first confirmed an earlier immunohistochemical study showing that immunoreactive TIMP-1-like protein accumulated in the nuclei of human gingival fibroblasts (Gin-1 cells), with the maximum in the S phase of the cell cycle (Li et al., Nagoya J.Med.Sci.58,133-142,1995). Then we isolated this protein from a nuclear extract of Gin-1 cells demonstrated it to be identical with human recombinant TIMP-1 by Western blotting, by a sandwich enzyme immunoassay for TIMP-1, and by an assay for matrix metalloproteinase inhibition. The amount of TIMP-1 in the cytosolic fraction after the stimulation by fetal calf serum of quiescent Gin-1 cells increased continuously for 48 hours, whereas, that in the nuclear extract showed a maximum at 24 hours (S phase) and significantly decreased after that. Gin-1 cells expressed mRNAs for both TIMP-2 and TIMP-3 together with that for TIMP-1. However, neither TIMP-2 nor TIMP-3 proteins seemed accumulate in the nuclei of Gin-1 cells. These facts strongly
… More
suggest that TIMP-1 accumulates specifically in the nuclei of Gin-1 cells in a cell cycle-dependent manner.
The question arises as to whether the trafficking of TIMP-1 to the nuclei of cells is general with respect to different cell types. To answer the question, we examined several human cells as follows : fetal lung fibroblasts HFLF,diploid fibroblast cell line WI-38 cells, osteosarcoma cell line MG-63 cells and fibrosarcoma cell line HT1080 cells as fibroblastic cells, and cervix carcinoma cell line HeLa cells and hepatoma cell line HT1080 cells as fibroblastic cells, and cervix carcinoma cell line HeLa cells and hepatoma cell line HLE cells as epithelial cells.
We observed that immunoreactive TIMP-1 protein was localized in some of the nuclei of HFLF and WI-38 cells growing nonsynchronously. Most of the nuclei of WI-38 cells showed negative staining at Go phase. After replacing the culture medium with that containing 10% FCS,however, the intensity of the nuclear staining increased time-dependently showing a maximum at around 16h (presumably corresponding to S phase). These observations are essentially the same as we already observed with Gin-1 cells. However, in the sase of tumor cells such as MG-63, HT1080, HeLa and HLE cells which generally proliferate faster than normal cells and have no clear cell cycle, the majority of their nuclei showed positive staining even after starving the cells in FCS-free culture medium for 48h. These facts are in good agreement with the accepted concept that many cancer cells have lost the control mechanism that sends nutritionally limited cells into Go phase. Less
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Report
(3 results)
Research Products
(2 results)
