| Project/Area Number |
06557137
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| Research Category |
Grant-in-Aid for Developmental Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Research Field |
医薬分子機能学
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| Research Institution | National Institute of Health |
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Principal Investigator |
UEHARA Yoshimasa National Institute of Health, Department of Bioactive Molecules, Laboratory Chief, 生物活性物質部, 室長 (50160213)
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| Co-Investigator(Kenkyū-buntansha) |
FUKAZAWA Hidesuke National Institute of Health, Department of Bioactive Molecules, senior research, 生物活性物質部, 主任研究官 (10218878)
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| Project Period (FY) |
1994 – 1995
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| Project Status |
Completed (Fiscal Year 1995)
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| Budget Amount *help |
¥7,200,000 (Direct Cost: ¥7,200,000)
Fiscal Year 1995: ¥2,700,000 (Direct Cost: ¥2,700,000)
Fiscal Year 1994: ¥4,500,000 (Direct Cost: ¥4,500,000)
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| Keywords | RAS / ANCHORAGE INDEPENENT GROWTH / ADHESION / ONCOGENIC SIGNALS / POLY (HEMA) / HERBIMYCIN A / TRANSFORMATION / TRANSCRIPTION / トランスフォメーション / ファルネシルトランスフェラーゼ / プロテインキナーゼ / 足場依存性 / スクリーニング |
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| Research Abstract |
To evaluate ras-mediated signal transduction, we constructed a transient transfection assay system that measures chloramphenicol acetyl transferase activity expressed under the transcriptional-enhancer elements responsive to ras, protein kinase C,and protein kinase A in NIH3T3 cells.Characterization of the assay system with several known activatiors and inhibitors of signal transduction pathways proved that our system could reliably evaluate agents that affect individual pathways.
we also developed a 96-well microplate assay to quantitate anchorage-independent growth of transformed cells.Wells of tissue culture microtiter plates were coated with anti-adhesive polymer poly (2-hydroxyethyl methacrylate) (poly (HEMA) ) to prevent cell attachment, and cells suspended in liquid media were seeded into the treated plates.Cell growth was assessed by tetrazolium dye reduction or by counting [^3H] thymidine incorporation into DNA.There was a close correlation between growth in poly (HEMA) -coated plates and colony formation in soft agar., i.e., fibroblasts transformed by various oncogenes proliferated in the coated plates, whereas their normal counterparts did not.Transformed cells on the nonadhesive surface were round and formed multicellular spheroids which resembled colonies in soft agar.Cells carrying either temperature-sensitive v-src or inducible Ki-ras oncogene proliferated on poly (HEMA) -coated plates only when they displayd transformed phenotype.This method should be more practical than conventional soft agar colony formation assay for measurement of anchorage-independent growth and will be useful for screening of inhibitors of oncogenic signal transduction pathways and in many other experimental situations.
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