INHIBITION OF N-MYC EXPRESSION IN NEUROBLASTOMA CELL LINES

N-myc是一种核癌基因，通常在肿瘤细胞中过表达。 神经母细胞瘤虽然N-myc蛋白是DNA结合蛋白， 与c-myc相似，它在正常细胞和肿瘤细胞中的功能是 未知我们正在利用“反义概念”来研究 N-myc在神经母细胞瘤和神经上皮瘤细胞系中的表达 这个致癌基因该方法是双重的：1）向细胞培养物中添加 与部分N-myc互补的小的单侧寡核苷酸 mRNA的表达载体转染细胞;和2）用含有mRNA的表达载体转染细胞。 反义方向的N-myc基因的一部分。使用前者 方法，我们已经能够表明，ohgonucleotide 15-mer 与含有N-myc mRNA起始位点的区域互补的是 能够在细胞中浓缩并抑制N-myc的产生 蛋白此外，这些寡核苷酸抑制细胞DNA合成， 一种名为Ki 67的核蛋白。这种蛋白质可能是一种辅助因子 DNA聚合酶α，是分离的细胞中DNA合成所必需的。 原子核。持续给予神经母细胞瘤反义寡聚体 导致细胞生长减少。朱鹭的生长抑制不是 伴随c-myc蛋白的减少，可能继发于 神经外胚层培养物中常见的典型混合形态。 为了证实这些发现，我们使用了第二种方法， 转染，通过构建含有反义N-myc的附加型载体 其以高拷贝数在染色体外复制。获得的结果 该载体支持用反义寡核苷酸得到的发现-即， N-myc抑制导致培养物生长速率降低; 由于对生长的直接影响，而是由于形态学的改变， 通常在体外神经外胚层培养物中观察到异质性。 这些结果具有关于生成模式的含义， 混合形态培养物的维持和N-myc的参与 在这个过程中。

N-myc is a nuclear oncogene which is generally overexpressed in neuroblastoma. Although N-myc protein is DNA binding and is structurally similar to c-myc, its function in both normal and neoplastic cells is unknown. We are making use of the "antisense concept" to study the role of N-myc in neuroblastoma and neuroepithelioma cell lines which overexpressed this oncogene. This approach is two-fold: 1) addition to cell cultures of small single-sided oligonucleotides complementary to a portion of N-myc MRNA; and 2) transfection of cells with expression vectors containing a portion of the N-myc gene in antisense orientation. Using the former approach, we have been able to show that ohgonucletoide 15-mers complementary to an area containing the initiation site of N-myc MRNA are capable of concentrating in cells and inhibiting production of N-myc protein. In addition, these oligos inhibit cellular DNA synthesis and production of a nuclear protein termed Ki67. This protein may be a cofactor for DNA polymerase alpha and is required for DNA synthesis in isolated nuclei. Continued administration of antisense oligomer to neuroblastoma cells leads to reduced cell growth. 'Ibis growth inhibition is not accompanied by a reduction in c-myc protein and may be secondary to loss of the typical mixed morphologies normally seen in neuroectodermal cultures. In order to confirm these findings, we utilized the second approach, vector transfection, by constructing an antisense N-myc containing episomal vector which replicates extra-chromosomally at high copy number. Results obtained with this vector support the findings made with antisense oligos - namely, that N-myc suppression leads to a reduced growth rate of the culture; not due to a direct affect on growth, but to an alteration in the morphologic heterogeneity normally observed in neuroectodermal cultures in vitro. These results have implications concerning the mode of generation and maintenance of the mixed morphology cultures and the involvement of N-myc in this process.