| Project/Area Number |
03807146
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| Research Category |
Grant-in-Aid for General Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Research Field |
Biological pharmacy
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| Research Institution | Okayama University |
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Principal Investigator |
WATAYA Yusuke Okayama University, Faculty of Pharm.Sc. Associate Professor, 薬学部, 助教授 (90127598)
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| Co-Investigator(Kenkyū-buntansha) |
NEGISHI Kazuo Okayama University, Gene Research Ca Associate Professor, 遺伝子実験施設, 助教授 (70116490)
HAYATSU Hikoya Okayama University, Faculty of Pharm.Sc. Professor, 薬学部, 教授 (10012593)
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| Project Period (FY) |
1991 – 1992
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| Project Status |
Completed (Fiscal Year 1992)
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| Budget Amount *help |
¥1,800,000 (Direct Cost: ¥1,800,000)
Fiscal Year 1992: ¥400,000 (Direct Cost: ¥400,000)
Fiscal Year 1991: ¥1,400,000 (Direct Cost: ¥1,400,000)
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| Keywords | dNTP / Imbalance / DNA Double Strand Breaks / endonuclease / Cell death / Apoptosis / Cancer / Chemotherapy / dNTPプール / エンドヌイレアーゼ / アポトーミス / dNTPプ-ル / ヌクレア-ゼ / M13ファ-ジ / ス-パ-コイル |
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| Research Abstract |
The mechanism of intracellular deoxyribonucleoside triphosphates (dNTP) pool imbalance-induced cell death in mouse FM3A cells was studied. We previously reported that 1) 5-fluoro-deoxyuridine (FdUrd) induced intracellular dNTP pool imbalance followed by DNA double strand breaks and subsequent cell death, 2) these DNA double strand breaks and cell death were inhibited by cycloheximide, an inhibitor of protein biosynthesis, 3) an endonuclease activity toward double stranded DNA was detected in the lysate ofFdUrd-treated FM3A cells but not untreated cells. In this experiment, we have purified and characterized the double-stranded endonuclease from FdUrd-treated FM3A cells. The enzyme which was purified to near homogeneity by column chromatographys. The molecular mass of the endonuclease was approximately 45 kDa by SDS-PAGE that contained DNA. The endonuclease required no divalent metal cations and cleaved naked duplex DNA at random. In addition we have investigated the DNA fragmentation induced by FdUrd. FdUrd-induced DNA fragments were separated into two classes. One was large DNA fragment with size of 100-200 kbp, and other was ladder DNA fragment with pitch of nucleosome length, approximately 180 bp. In second case the DNA fragmentation was of the type considered characteristic of apoptosis. Furthermore, we found that mRNA levels of nuclear proto-oncogenes, c-fos, c-jun and c-myc, increased in FdUrd-treated cells. We conclude that the purified endonuclease is a novel endonuclease, distinct from previously characterized mammalian endonucleases. The enzyme, which was called endonuclease K.
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