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Analysis of N-myc Gene Amplification and Rearrangement in Neuroblastoma Cells and Tumors

神经母细胞瘤细胞和肿瘤中 N-myc 基因扩增和重排分析

基本信息

批准号：
02807116
负责人：
KISHIKAWA Teruaki
金额：
$ 1.02万
依托单位：
Fujita Health 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：
1990
资助国家：
日本
起止时间：
1990 至 1992
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-02807116/
关键词：
Alu Sequence Gene Amplification Neuroblastoma N-myc gene PFG 神経芽腫 DNA rearrangement Nーmyc 遺伝子増幅単位パルスフィ-ルドゲル電気泳動法 pYAC パルスフィ-ルドゲル電気〓動法 PYAC

项目摘要

N-myc gene is frequently amplified in human Neuroblastoma(Nb) cells and correlation between N-myc gene amplification and advanced Nb tumor with poor prognosis is well documented. We have been working on the mechanism of N-myc gene amplification in Nb tumors and cell lines. We analyzed 7 cell lines (IMR-32, GOTO, TGW, Ngai, NB-1, YT-nu, SKNSH) and 86 tumor samples. Amplification of N-myc gene was observed in 5 cell line (IMR-32, GOTO, TGW, Nagai, YT-nu) and 9 tumor samples (StageIII2, StageIV_A 7) and DNA rearrangement in the vicinity of N-myc gene was also observed in 5 cell lines (GOTO, TGW, Ngai, YT-nu, NB-1). We isolated the rearranged DNA from TGW cells and determined the nucleotide sequences of the rearranged region. The rearrangement occurred between the end of exon 3 of N-myc gene and Alu sequence. The rearranged point in the Alu sequence corresponds to a hot point for DNA rearrangements including Alu-Alu homologous recombination. We also characterized N-myc gene amplification in three cell lines(IMR-32, TGW, GOTO). Rearrangements in long-range regions surrounding amplified N-myc genes were examined by pulsed-field gel electrophoresis. Since rarecutting enzymes complete-ly digested DNA at the middle of the N-myc gene, we were able to construct a physical map upstream and downstream of the germline N-myc gene, and to obtain information on restriction sites surrounding amplified N-myc genes. This method enables us to envisage the organization of amplified units over a long range. Digestion patterns differed considerably among the germline and the three cell lines, but were simple in each case.We estimate that the minimal distance between neighboring N-myc genes is at least several hundred kilobases.

N-myc 基因在人神经母细胞瘤 (Nb) 细胞中频繁扩增，并且 N-myc 基因扩增与预后不良的晚期 Nb 肿瘤之间的相关性已得到充分证明。我们一直致力于Nb肿瘤和细胞系中N-myc基因扩增的机制研究。我们分析了 7 种细胞系（IMR-32、GOTO、TGW、Ngai、NB-1、YT-nu、SKNSH）和 86 个肿瘤样本。在5个细胞系（IMR-32、GOTO、TGW、Nagai、YT-nu）和9个肿瘤样本（StageIII2、StageIV_A 7）中观察到N-myc基因扩增，并且在5个细胞系（GOTO、TGW、Ngai、YT-nu、NB-1）中也观察到N-myc基因附近的DNA重排。我们从 TGW 细胞中分离出重排的 DNA，并确定了重排区域的核苷酸序列。重排发生在N-myc基因的外显子3末端和Alu序列之间。 Alu 序列中的重排点对应于 DNA 重排（包括 Alu-Alu 同源重组）的热点。我们还在三种细胞系（IMR-32、TGW、GOTO）中表征了 N-myc 基因扩增。通过脉冲场凝胶电泳检查扩增的 N-myc 基因周围长范围区域的重排。由于稀切酶完全消化 N-myc 基因中部的 DNA，因此我们能够构建种系 N-myc 基因上游和下游的物理图谱，并获得有关扩增的 N-myc 基因周围限制性位点的信息。这种方法使我们能够设想在长范围内组织扩增单元。种系和三种细胞系之间的消化模式差异很大，但每种情况都很简单。我们估计相邻 N-myc 基因之间的最小距离至少为数百千碱基。