| Project/Area Number |
10670511
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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 |
Gastroenterology
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| Research Institution | JIKEI UNIVERSITY SCHOOL OF MEDICINE |
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Principal Investigator |
INAMOTO Yukio JIKEI UNIVERCITY SCHOOL OF MEDISINE, Research Assistant, 医学部, 助手 (50281382)
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| Co-Investigator(Kenkyū-buntansha) |
OGASAWARA Hayato JIKEI UNIVERCITY SCHOOL OF MEDISINE, INTERNAL MEDICINE,Research Assistant, 医学部, 助手 (20307416)
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| Project Period (FY) |
1998 – 1999
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| Project Status |
Completed (Fiscal Year 1999)
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| Budget Amount *help |
¥1,800,000 (Direct Cost: ¥1,800,000)
Fiscal Year 1999: ¥900,000 (Direct Cost: ¥900,000)
Fiscal Year 1998: ¥900,000 (Direct Cost: ¥900,000)
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| Keywords | Human colon cancer cell / Chemotherapy / Nucleotide excision repair |
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| Research Abstract |
In order to introduce UV-photoproducts, SV40 viral DNA was irradiated with UV light (peak wavelength : 253.7 nm). DNA samples were phenol extracted and precipitated with ethanol. The precipitates were dissloved in buffer containing 10 mM Tris-HCl and 1mM EDTA. Irradiated DNA showed mobility shift on agarose gel electrophoresis in a UV-dose dependent manner.
The cell extract was prepared from 1 litter culture of exponentially growing human colon cancer cell lines as described previously by Wood(Cell, 53, p97). Protein concentration was determined by using BCA kit (PIERCE) with bovine serum albmin as standards. Typical protein concertration were approximately 15-17 mg/ml. The cell extracts were stored at -80 ℃ in small aliquots at protein concentration of l5 mg/ml.
The in vitro repair snythesis was examined as mentioned below. The standard reaction mixture contained cell extract, UV- and/or drug-treated SV40 viral DNA (Form I), undamaged pBR322 plasmid as a internal control, dNTPs, [α32-P]dCTP, ATP and its regeneration system in suitable buffer.
The repair synthesis were carried out for 3h in the dark and terminated by adding EDTA. The resulting samples were phenol-extracted and ethanol-precipitaed and were dissolved in buffer. The DNAs were linealized by EcoR I and subjected to agarose gel electrophoresis and fluorography. Then the gel was dried and autoradiographed.
Nucleotide incorporation was observed in a UV-dose dependent manner to the SV-40 DNA,indicating that human cancer cell extract supported the nucleotide excision repair.
It is possible that human colon cancer repairs drug-DNA adduct induced by DNA damaging agents. We presume that DNA repair activity of cancer cells influences their sensitivity to chemotherapy. It must be of value to evaluate the DNA repair machinery as a therapeutic target of cancer.
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