dNTP Pool Imbalance Induced Endonuclease : Mechanism of All Death.

dNTP 池失衡诱导的核酸内切酶：所有死亡的机制。

• 批准号: 03807146
• 负责人: WATAYA Yusuke
• 金额: $ 1.15万
• 依托单位: Okayama University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 财政年份: 1991
• 资助国家: 日本
• 起止时间: 1991 至 1992
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-03807146/
• 关键词: 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

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.

研究了细胞内脱氧核糖核苷三磷酸(DNTP)池失衡诱导小鼠FM3A细胞死亡的机制。我们曾报道：1)5-氟脱氧尿苷(FdUrd)导致细胞内dNTP池失衡，导致DNA双链断裂和随后的细胞死亡，2)这些DNA双链断裂和细胞死亡被蛋白质生物合成抑制剂放线菌酮抑制，3)在FdUrd处理的FM3A细胞的裂解物中检测到双链DNA内切酶活性，但未处理的细胞没有检测到。在本实验中，我们从FdUrd处理的FM3A细胞中纯化并鉴定了双链内切酶。该酶经柱层析纯化至接近均一的水平。经含有DNA的SDS-PAGE分析，该酶的相对分子质量约为45 kDa。该酶不需要二价金属阳离子，可随机切割裸露的双链DNA。此外，我们还研究了FdUrd诱导的DNA片段化。FdUrd诱导的DNA片段分为两类。一种为大片段DNA，大小为100-200kbp，另一种为梯状DNA片段，其节距为核小体长度约180bp。在第二例中，DNA片段化被认为是以细胞凋亡为特征的类型。此外，我们还发现，在FdUrd处理的细胞中，核原癌基因c-fos、c-jun和c-myc的mRNA水平增加。我们的结论是，纯化的内切酶是一种新的内切酶，不同于以前鉴定的哺乳动物内切酶。这种酶被称为内切酶K。

项目成果

M.Otani, S.Yoshida, A.Yoshioka-Hiramoto, T.Nakazawa, and Y.Wataya: "The dNTP Imbalance Death" Nucleic Acids Symposium Series. No.25. 111-112 (1991)

M.Otani、S.Yoshida、A.Yoshioka-Hiramoto、T.Nakazawa 和 Y.Wataya：“dNTP 不平衡死亡”核酸研讨会系列。

M.Yamato.J.Ando,K.Sakata,K.Haohigaki,Y.Wataya,A.Tsukagoshi,T.Taslizo&T.Tsuruta: "Synthesis and Anyitumor Activity of Tro polone Derivations 7.Bistropolone Containing Connecting Methylene Chain" J.Med.Chem. 35. 267-273 (1992)

M.Yamato.J.Ando、K.Sakata、K.Haohigaki、Y.Wataya、A.Tsukagoshi、T.Taslizo

H.Satake: "Action of 5-Trifluoromethyl-2^2-deoxyuridine on DNA 5ynthesis." Nucleic Acids Symposium Series. 26. 193-194 (1992)

H.Satake：“5-三氟甲基-2^2-脱氧尿苷对 DNA 5 合成的作用。”

S.Yoshida, K.Watanabe, A.Yoshioka-Hiramoto, and Y.Wataya: "The dNTP Imbalance Death" Nucleisides and Nucleotides.

S.Yoshida、K.Watanabe、A.Yoshioka-Hiramoto 和 Y.Wataya：“dNTP 不平衡死亡”核苷和核苷酸。

M.Yamato,Y.Hirota,S.Yoshida,S.Tanaka,T.Morita,J.Sakai K.Hadigaki,H.Hagatsu and Y.Wataya: "Imbalomce of Desxpribomucleoside Triphotes and DNA Double StaanlーBraks in Mouse Mammary Tumor FM3Aalls Treatel in vitro with an Antineoplastic fropolone Derirative"

M. Yamato、Y. Hirota、S. Yoshida、S. Tanaka、T. Morita、J. Sakai K. Hadigaki、H. Hagatsu 和 Y. Wataya：“小鼠乳腺肿瘤 FM3Aalls 中 Desxpribomucleoside Triphotes 和 DNA Double Staanl Braks 的不平衡抗肿瘤福罗泊酮衍生物的体外治疗"