Analysis of dynamics of DNA damage repair protein DDB1 in oxidative stresses in N2a cells
N2a细胞氧化应激中DNA损伤修复蛋白DDB1的动态分析
基本信息
- 批准号:15591241
- 负责人:
- 金额:$ 2.11万
- 依托单位:
- 依托单位国家:日本
- 项目类别:Grant-in-Aid for Scientific Research (C)
- 财政年份:2003
- 资助国家:日本
- 起止时间:2003 至 2004
- 项目状态:已结题
- 来源:
- 关键词:
项目摘要
We established a cell line derived from HEK293 (EGFP-DDB1-HEK293-1) stably expressing EGFP-fused DDB1. Both in EGFP-DDB1-HEK293-1 cells and in HEK293 cells, repair of UV-induced DNA damages (CPDs and 6-4PPs) was compared. As a result, there was no difference in the velocity of repair of them, indicating that massive expression of DDB1 did not affect the repair of them. On the other hand, there was no difference in the velocity of cell division in both cells. Translocation of fluorescently labeled DDB1 from cytoplasm to nucleus was on real time observed in the cells of EGFP-DDB1-HEK293-1 after oxidative stresses on the stage kept at 37℃ by means of fluorescence microscopy. As the oxidative stresses, we employed addition of H_2O_2. In living cells, intensity of local fluorescence was measured on real time, and dynamics of DDB1 was analyzed. Otherwise, 20 J/m^2 UV radiation and 1〜20gy ionizing X ray radiation were also compared in dynamics of DDB1. The mount of translocated fluorescent DDB1 was largest in case of ionizing X ray radiation, and the amount of translocated fluorescence was observed in maximum level 24 hours after damages. And fluorescent DDB1 was relocated to cytoplasm 48 hours after damages. These findings indicated that DDB1 reacted with a variety of DNA damages, and that DDB1 functioned as a chaperon-like protein that transported certain signals from cytoplasm to nucleus.On the other hand, we failed to establish a cell line derived from SKN-MC neuroblastoma cells stably expressing EGFP-fused DDB1. It indicated that massive expression of full-length DDB1 might be harmful to SKN-MC neuroblastoma cells. Because DDB1 lack of its C-terminus region has no ability to bind to APP fragments, translocation of APP fragments to nucleus was expected to harm neuronal cells.
建立了稳定表达EGFP融合DDB1的HEK293(EGFP-DDB1-HEK293-1)细胞系。比较了EGFP-DDB1-HEK293-1细胞和HEK293细胞对紫外线诱导的DNA损伤修复(CPDS和6-4PPS)的影响。因此,它们的修复速度没有差异,表明大量表达DDB1并不影响它们的修复。另一方面,两种细胞的细胞分裂速度没有差异。在荧光显微镜下实时观察氧化应激37℃后,EGFP-DDB1-HEK293-1细胞内荧光标记的DDB1从胞浆向胞核的移位。对于氧化应激,我们采用了添加H_2O_2的方法。在活细胞中,实时测量了局部荧光强度,并分析了DDB1的动力学。此外,还比较了20J/m2UV辐射和1~20gy电离X射线辐射对DDB1的动力学影响。在电离X射线照射下,DDB1的移位荧光量最大,损伤后24小时的移位荧光量最大。损伤后48h,荧光DDB1重新定位到胞浆内。这些结果表明,DDB1与多种DNA损伤发生反应,并且DDB1作为伴侣蛋白将某些信号从细胞质传递到细胞核。另一方面,我们未能建立稳定表达EGFP融合DDB1的SKN-MC神经母细胞瘤细胞系。提示全长DDB1的大量表达对SKN-MC神经母细胞瘤细胞可能是有害的。由于DDB1缺乏其C-末端区域,无法与APP片段结合,APP片段移位到细胞核可能会损害神经细胞。
项目成果
期刊论文数量(8)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
Imoto K: "The total amount of DNA damage determines ultraviolet-radiation-induced cytotoxicity after uniform or localized irradiation of human cells"J Invest Dermatol. 119. 1177-1182 (2002)
Imoto K:“DNA 损伤的总量决定了人体细胞均匀或局部照射后紫外线辐射诱导的细胞毒性”J Invest Dermatol。
- DOI:
- 发表时间:
- 期刊:
- 影响因子:0
- 作者:
- 通讯作者:
Nishiwaki Y: "Trichothiodystrophy fibroblasts are deficient in the repair of UV-induced cyclobutane pyrimidine dimers and (6-4) photoproducts"J Invest Dermatol. (in press). (2004)
Nishiwaki Y:“毛发硫营养不良成纤维细胞在修复紫外线诱导的环丁烷嘧啶二聚体和 (6-4) 光产物方面存在缺陷”J Invest Dermatol。
- DOI:
- 发表时间:
- 期刊:
- 影响因子:0
- 作者:
- 通讯作者:
In situ detection of acetylaminofluorene-DNA adducts in human cells using monoclonal antibodies
- DOI:10.1016/j.dnarep.2004.05.016
- 发表时间:2004-11-02
- 期刊:
- 影响因子:3.8
- 作者:Iwamoto, TA;Kobayashi, N;Mori, T
- 通讯作者:Mori, T
Trichothiodystrophy fibroblasts are deficient in the repair of UV-induced cyclobutane pyrimidine dieters and (6-4) photoproducts
毛发硫营养不良成纤维细胞缺乏紫外线诱导的环丁烷嘧啶节食剂和 (6-4) 光产物的修复
- DOI:
- 发表时间:2004
- 期刊:
- 影响因子:0
- 作者:Nishiwaki;Y.;Kobayashi;N.;Imoto;K.;Iwamoto;T.;Yamamoto;A.;Nakamura;Y.;Sarasin;A.;Miyagawa;S.;Mori;T.
- 通讯作者:T.
Trichothiodystrophy fibroblasts are deficient in the repair of UV-induced cyclobutane pyrimidine dimers and (6-4) photoproducts.
毛发硫营养不良成纤维细胞缺乏紫外线诱导的环丁烷嘧啶二聚体和 (6-4) 光产物的修复。
- DOI:
- 发表时间:2004
- 期刊:
- 影响因子:0
- 作者:Fujita H;Takahashi H;Aihara M;Hirasawa T;Ikezawa Z;Y.Nishiwaki
- 通讯作者:Y.Nishiwaki
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NAKAMURA Yu其他文献
NAKAMURA Yu的其他文献
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