Effect of forced N-myc gene expression on src gene expression in neuroblastoma cells

神经母细胞瘤细胞中强制N-myc基因表达对src基因表达的影响

• 批准号: 04670197
• 负责人: SIMIZU Bunsiti
• 金额: $ 1.34万
• 依托单位: Chiba 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: 1992
• 资助国家: 日本
• 起止时间: 1992 至 1993
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-04670197/
• 关键词: Neuroblastoma; N-myc; src

NB69N which is a clonal subline of neuroblastoma cell line NB69, has a neuroblastic characteristic and an ability to differentiate by retinoic acid. S1 nuclease protection assay showed that NB69N expressed not only nonneuronal c-src but neuronal c-srcN1 and c-srcN2 mRNA.Because NB69N cell has a single copy of N-myc gene and does not express N-myc mRNA, we introduced N-myc expression vector into the NB69N cells by an electroporation method. We cloned 7 N-myc transfectants (NB69N41 - 47) and one control transfectant NB69NR.In 7 N-myc transfectants, the exogenous N-myc DNA was integrated in genomic DNA, and N-myc mRNA and protein products from the introduced N-myc were expressed. NB69N cells had no advantage of growth activity in a normal culture medium, but acquired a resistance to serum starvation. Furthermore, NB69N47 cells were able to form 6 times colonies to compare with NB69NR in soft agar. No difference of c-src mRNA splicing and expression levels was observed between NB69N47 and NB69NR by S1 nuclease protection assays. The forced expression of N-myc gene possibly conferred a malignant potential to the neuroblastoma cells independent of the c-src mRNA expression.

NB69N是神经母细胞瘤细胞系NB69的克隆亚系，具有成神经细胞特征，并能通过维甲酸进行分化。S1核酸酶保护实验表明，NB69N不仅表达非神经元c-src，还表达神经元c-srcN1和c-srcN2 mRNA。由于NB69N细胞只有一个N-myc基因拷贝，不表达N-myc mRNA，我们采用电穿孔法将N-myc表达载体导入NB69N细胞。我们克隆了7个N-myc的转染物（NB69N41 - 47）和一个对照转染物NB69NR。在7个N-myc转染物中，外源N-myc DNA被整合到基因组DNA中，并表达了引入N-myc的N-myc mRNA和蛋白产物。NB69N细胞在正常培养基中没有生长活性的优势，但获得了对血清饥饿的抗性。NB69N47细胞在软琼脂培养基中形成的菌落是NB69NR的6倍。S1核酸酶保护实验显示，NB69N47和NB69NR之间c-src mRNA剪接和表达水平无差异。N-myc基因的强制表达可能使神经母细胞瘤细胞具有独立于c-src mRNA表达的恶性潜能。

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