Fibroblast Growth Factor 2 Reactivates G1 Checkpoint in SK-N-MC Cells via Regulation of p21, Inhibitor of Differentiation Genes (Id1-3), and Epithelium-Mesenchyme Transition-Like Events
Fibroblast Growth Factor 2 Reactivates G1 Checkpoint in SK-N-MC Cells via Regulation of p21, Inhibitor of Differentiation Genes (Id1-3), and Epithelium-Mesenchyme Transition-Like Events
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DOI：
10.1210/en.2008-1797
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发表时间：
2009-09-01
期刊：
影响因子：
4.8
通讯作者：
Russo, V. C.
中科院分区：
文献类型：
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作者：
Higgins, S.;Wong, S. H. X.;Russo, V. C.
我们最近证实成纤维细胞生长因子-2促进神经母细胞瘤细胞分化，并抑制其对IGF-I的促有丝分裂反应。然而，其中涉及的机制尚不清楚。在SK-N-MC细胞中加入50 ng/ml的FGF2和/或100 ng/mlIGF-I培养48h，荧光激活的细胞分类分析显示FGF2促进细胞周期停滞于G1/G0期。逆转录聚合酶链式反应和细胞定位显示p21基因表达上调。然后，我们研究了成纤维细胞生长因子-2诱导SK-N-MC细胞分化(通过GAP43和NeuroD-6的表达)是否涉及上皮-间充质转化。实时定量聚合酶链式反应(RT2-PCR)显示参与维持上皮细胞表型和细胞-基质相互作用的基因(E-钙粘蛋白、Snail-1、MMPs)的调控。凝胶成像证实，成纤维细胞生长因子-2上调了MMP2的表达，并诱导了MMP9，已知有助于神经元分化和轴突延伸。逆转录聚合酶链式反应检测ID1-3的表达。成纤维细胞生长因子2诱导Id2，下调Id1和Id3的表达。成纤维细胞生长因子-2诱导ID2蛋白在胞核聚集，而ID1和ID3保持胞质。RNA干扰显示，id3调节分化和细胞周期(增加Neuro-D6和p21mRNA)，而dId2调节上皮-间充质转变样事件(增加E-cadherin mRNA)。总之，我们首次证明了成纤维细胞生长因子-2通过激活复杂的基因表达程序诱导神经母细胞瘤细胞分化，从而调节细胞周期、转录因子和抑制肿瘤表型。RNA干扰的使用表明ID-3是这些事件的关键调节因子，从而为这种毁灭性的儿童癌症指明了一个新的治疗靶点。(内分泌学150：4044-4055,2009)
We have recently demonstrated that fibroblast growth factor (FGF)-2 promotes neuroblastoma cell differentiation and overrides their mitogenic response to IGF-I. However, the mechanisms involved are unknown. SK-N-MC cells were cultured with FGF-2 (50 ng/ml) and/or IGF-I (100 ng/ml) up to 48 h. Fluorescence-activated cell sorting analysis indicated that FGF-2 promotes G1/G0 cell cycle phase arrest. Gene expression by RT2-PCR and cellular localization showed up-regulation of p21. We then investigated whether FGF-2-induced differentiation of SK-N-MC cells (by GAP43 and NeuroD-6 expression) involves epithelium-mesenchyme transition interconversion. Real-time PCR (RT2-PCR) showed modulation of genes involved in maintenance of the epithelial phenotype and cell-matrix interactions (E-cadherin, Snail-1, MMPs). Zymography confirmed FGF-2 up-regulated MMP2 and induced MMP9, known to contribute to neuronal differentiation and neurite extension. Id1-3 expression was determined by RT2-PCR. FGF-2 induced Id2, while down-regulating Id1 and Id3. FGF-2 induced nuclear accumulation of ID2 protein, while ID1 and ID3 remained cytoplasmic. RNA interference demonstrated that Id3 regulates differentiation and cell cycle (increased Neuro-D6 and p21 mRNA), while d Id2 modulates epithelium-mesenchyme transition-like events ( increased E-cadherin mRNA). In conclusion, we have shown for the first time that FGF-2 induces differentiation of neuroblastoma cells via activation of a complex gene expression program enabling modulation of cell cycle, transcription factors, and suppression of the cancer phenotype. The use of RNA interference indicated that Id-3 is a key regulator of these events, thus pointing to a novel therapeutic target for this devastating childhood cancer. (Endocrinology 150: 4044-4055, 2009)