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

N-myc基因在人神经母细胞瘤（Neuroblastoma，Nb）细胞中频繁扩增，N-myc基因扩增与晚期Nb肿瘤预后不良的相关性已被充分证实。我们一直致力于N-myc基因在Nb肿瘤和细胞系中扩增的机制。我们分析了7个细胞系（IMR-32、后藤、TGW、Ngai、NB-1、YT-nu、SKNSH）和86个肿瘤样品。在5个细胞系（IMR-32、后藤、TGW、永井、YT-nu）和9个肿瘤标本（StageIII 2、StageIV_A 7）中观察到N-myc基因扩增，在5个细胞系（后藤、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，并测定了重排区的核苷酸序列。重排发生在N-myc基因外显子3末端与Alu序列之间。Alu序列中的重排点对应于DNA重排的热点，包括Alu-Alu同源重组。我们还在三个细胞系（IMR-32，TGW，后藤）中表征了N-myc基因扩增。通过脉冲场凝胶电泳检测扩增的N-myc基因周围长程区域的重排。由于N-myc基因中间的DNA被罕见的切割酶完全消化，我们能够构建生殖系N-myc基因上游和下游的物理图谱，并获得扩增的N-myc基因周围的限制性酶切位点的信息。这种方法使我们能够设想在一个长的范围内放大单位的组织。种系和三种细胞系之间的消化模式差异很大，但每种情况下都很简单。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。

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.