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MOLECULAR BIOLOGICAL STUDIES OF APOPTOSIS ENDONUCLEASE IN HUMAN NEUROBLASTOMAS

人神经母细胞瘤细胞凋亡核酸内切酶的分子生物学研究

基本信息

批准号：
07457419
负责人：
YOKOMORI Kinji
金额：
$ 5.06万
依托单位：
UNIVERSITY OF TOKYO
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1995
资助国家：
日本
起止时间：
1995 至 1996
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07457419/
关键词：
NEUROBLASTOMA APOPTOSIS ENDONUCLEASE DNA FRAGMENTATION

项目摘要

During the research period in 1995, apoptosis induction was examined for each of 16 human neuroblastoma cell lines, using the following three apoptosis inducers, namely, actinomycin D,adriamycin, and camptothecin, . We have already found that the cleavage of DNA in apoptotic cells occurs in at least two steps, i.e., large fragmentation of DNA of 2-50 kilobase pairs and internucleosomal fragments of 180 basepairs. Depending upon the size of DNA fragmentation oberved in each tumor cell line, we found that 16 human neuroblastoma cell lines investigated were classified into the followign three categories ; (1) cell line in which even large fragmentation was not induced by any of the aforementioned three apoptotic inducers, (2) cell line in which only large fragmentation was induced followed by no further small fragmentaton, and (3) cell line in which small fragmentation into internucleosomal fragments with typical apoptotic DNA ladder formation was induced following large fragmentation.Dur … More ing the year 1996, our study was directed mainly toward identification of DNase lambda, a newly recognized key-endonuclease responsible for large fragmentation, by comparing the enzymatic activity between a pair of neruoblastoma cell lines ; one cell line (TGW) with positive DNase lambda activity and the oher one (GOTO) lacking it. We exmined whether large DNA fragmentation can be induced in vitro by autodigested nuclei of DNase lambda positive TGW cells, and confirmed the induction of large fragmentation with about 20 kb fragments.Using this autodigestion system, fragmentation induction in vitro was further exmined in GOTO cells without DNase lambda, and in SK-N-SH cells with both DNase lambda and DNase gamma (a key-endonuclease for small, internucleosomal fragmentation). As has been expected, no induction was seen in GOTO cells, while in SK-N-SH cells small fragmentation in vitro was shown.For further genetic analysis studies on DNase gamma in small DNA fragmentation b, three neuroblastoma cells with distinct biological characteristics (GOTO,TWGW,and SK-N-SH) were propagated and kept frozen. Less

在1995年的研究期间，使用以下三种凋亡诱导剂，即放线菌素D、阿霉素和喜树碱，检查了16种人神经母细胞瘤细胞系中每一种的凋亡诱导。我们已经发现，凋亡细胞中DNA的裂解至少分两步进行，即2-50kbp的DNA大片段和180bp的核小体间片段。根据在每个肿瘤细胞系中观察到的 DNA 碎片的大小，我们发现所研究的 16 个人类神经母细胞瘤细胞系分为以下三类： (1)上述三种凋亡诱导剂中的任何一种都不会诱导大碎片的细胞系，(2)仅诱导大碎片，然后不再诱导小碎片的细胞系，以及(3)大碎片后诱导小碎片形成核小体间碎片并形成典型凋亡 DNA 梯子的细胞系。在 1996 年，我们的研究针对主要是通过比较一对神经母细胞瘤细胞系之间的酶活性来鉴定 DNase lambda，这是一种新近识别的负责大片段化的关键核酸内切酶；一种细胞系 (TGW) 具有阳性 DNase lambda 活性，而另一种细胞系 (GOTO) 则缺乏该活性。我们检查了 DNase lambda 阳性 TGW 细胞的自消化细胞核是否可以在体外诱导大 DNA 片段化，并证实了约 20 kb 片段的大片段化诱导。使用该自消化系统，在没有 DNase lambda 的 GOTO 细胞和同时具有 DNase lambda 和 DNase γ 的 SK-N-SH 细胞中进一步研究了体外片段化诱导。（一种用于小的核小体间断裂的关键核酸内切酶）。正如预期的那样，在 GOTO 细胞中没有看到诱导，而在 SK-N-SH 细胞中显示出体外小片段化。为了对小 DNA 片段 b 中的 DNase γ 进行进一步的遗传分析研究，三种具有不同生物学特征的神经母细胞瘤细胞（GOTO、TWGW 和 SK-N-SH）被增殖并冷冻。较少的