INHIBITION OF N-MYC EXPRESSION IN NEUROBLASTOMA CELL LINES

神经母细胞瘤细胞系中 N-MYC 表达的抑制

• 批准号: 3874465
• 负责人: L M NECKERS
• 金额: --
• 依托单位: DIVISION OF CANCER TREATMENT
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3874465
• 关键词: antisense nucleic acid; cell growth regulation; gene expression; growth inhibitors; human tissue; mixed tissue /cell culture; neoplastic cell; neuroblastoma; oligonucleotides; oncogenes; tissue /cell culture; transfection

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.

N-myc 是一种核癌基因，通常在 成神经细胞瘤。尽管 N-myc 蛋白是 DNA 结合蛋白，并且在结构上 与 c-myc 类似，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过度表达 这个癌基因。这种方法有两个方面：1）向细胞培养物中添加 与 N-myc 的一部分互补的小单侧寡核苷酸 信使RNA； 2)用含有a的表达载体转染细胞 N-myc 基因的反义方向部分。使用前者 方法，我们已经能够证明 ohgonucletoide 15-mers 与包含 N-myc mRNA 起始位点的区域互补的是 能够集中在细胞内并抑制 N-myc 的产生 蛋白质。此外，这些寡核苷酸还抑制细胞 DNA 合成， 称为 Ki67 的核蛋白的产生。该蛋白质可能是辅助因子 DNA聚合酶α，是分离的DNA合成所必需的 原子核。持续给予神经母细胞瘤反义寡聚物 细胞导致细胞生长减少。 '朱鹮生长抑制不是 伴随着 c-myc 蛋白的减少，并且可能继发于 通常在神经外胚层培养物中看到的典型混合形态。 为了证实这些发现，我们使用了第二种方法，向量 通过构建含有附加型反义 N-myc 载体进行转染 它以高拷贝数进行染色体外复制。获得的结果 这个载体支持反义寡核苷酸的研究结果 - 即， N-myc 抑制会导致培养物生长速度降低；不是 由于对生长的直接影响，但由于形态的改变 通常在体外神经外胚层培养物中观察到异质性。 这些结果对生成模式和 混合形态培养物的维持和 N-myc 的参与 在这个过程中。