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Identification and characterization of centrosome re-duplication suppressor gene(s)

中心体重复复制抑制基因的鉴定和表征

基本信息

批准号：
17590286
负责人：
IZUMI Hideki
金额：
$ 1.66万
依托单位：
Hiroshima University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2005
资助国家：
日本
起止时间：
2005 至 2006
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-17590286/
关键词：
centrosome re-duplication suppressor mouse A9 cell line chromosome がん細胞 FISH S期 M期

项目摘要

The aim of this study is to identify centrosome re-duplication suppressor gene(s) by chromosome-transfer technology. We carried out the following experiments.(1) At first, we analyzed centrosome status of mouse A9 cells. Mouse A9 cells are normal origin but makes tumors in nude mice. Thus, A9 cells are precancerous cell line. We examined centrosome staus in A9 cells by immunostaining, and found that centrosome number is normal. We next expose hydroxyurea (HU), a DNA synthesis inhibitor, to A9 cells. The exposed cells were arrested at G1-S transition in cell cycle and were inhibited DNA synthesis. However, centrosome duplication cycle was dysregulated in A9 cells; multi-round of centrosome duplication occurred under HU treatment. Thus, A9 cells have a potent centrosome re-duplication activity.(2) Next, we tried to examine centrosome re-duplication activity in A9 cells transferred with a human chromosome 1, 5, 8, 10, 11, 13, 15, 16, 17, and 18, respectively. As a result, we found that centrosome re-duplication activity was suppressed in A9-Ch. 8, A9-Ch. 15, and A9-Ch. 17 cells, respectively. These results suggest that the gene(s), which is related in suppression of centrosome re-duplication, is located on chromosome 8, chromosome 15, and chromosome 17. Especially, A9-Ch. 8 cells have high centrosome re-duplication suppressor activity. Therefore, we determined to isolate centrosome re-duplication suppressor gene(s) from chromosome 8.(3) As it is found that human osteosarsoma cell line, U2OS has centrosome re-duplication activity, we transferred chromosome 8 into U2OS cells and examined centrosome re-duplication activity. As a result, we found that chromosome 8 had a centrosome re-duplication suppressor activity. Now, we tried to isolate the candidate gene by radiation hybrid mapping.

本研究的目的是利用染色体转移技术鉴定中心体重复抑制基因。我们进行了以下实验。(1)首先，我们分析了小鼠A9细胞的中心体状态。小鼠A9细胞是正常来源，但在裸鼠中产生肿瘤。因此，A9细胞是癌前细胞系。我们用免疫组化方法检测了A9细胞的中心体状态，发现中心体数目正常。接下来，我们将DNA合成抑制剂羟基脲（HU）暴露于A9细胞。细胞周期阻滞于G1-S期，DNA合成受到抑制。然而，在A9细胞中，中心体复制周期失调; HU处理下发生多轮中心体复制。因此，A9细胞具有有效的中心体再复制活性。(2)接下来，我们试图检查分别用人染色体1、5、8、10、11、13、15、16、17和18转移的A9细胞中的中心体再复制活性。结果，我们发现，中心体再复制活动被抑制在A9-Ch.8，A9-Ch.15，和A9-Ch.17细胞，分别。这些结果表明，与抑制中心体重复有关的基因位于8号、15号和17号染色体上。特别是A9-Ch.8细胞具有高的中心体重复抑制活性。因此，我们决定从8号染色体上分离中心体再复制抑制基因。(3)由于发现人骨肉瘤细胞系U2 OS具有中心体再复制活性，我们将8号染色体转入U2 OS细胞中，并检测其中心体再复制活性。结果，我们发现8号染色体具有中心体重复抑制活性。现在，我们试图通过辐射杂交定位来分离候选基因。