p27Kip1 accumulation is associated with retinoic-induced neuroblastoma differentiation:: evidence of a decreased proteasome-dependent degradation
p27Kip1 accumulation is associated with retinoic-induced neuroblastoma differentiation:: evidence of a decreased proteasome-dependent degradation
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DOI：
10.1038/sj.onc.1203231
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发表时间：
2000-01-06
期刊：
影响因子：
8
通讯作者：
Della Ragione, F
中科院分区：
文献类型：
--
作者：
Borriello, A;Della Pietra, V;Della Ragione, F
人类神经母细胞瘤的发生是由于神经嵴交感肾上腺祖细胞分化程序的停滞。然而，神经母细胞瘤，以及它们的衍生细胞系，保持终末分化的潜力。我们研究了维甲酸（一种在临床试验中引入的化疗分子）诱导神经母细胞瘤细胞系分化的分子机制。我们的研究结果表明，视黄酸依赖的生长停滞的LAN-5神经母细胞瘤细胞系是与一个非常大的积累（> 10倍）的p27（Kip 1）蛋白，一种细胞周期蛋白依赖性激酶抑制剂;该蛋白结合并抑制细胞周期蛋白依赖性激酶2、4和6的活性，从而阻碍pRb和p107磷酸化，p27（Kip 1）的积累是视黄酸处理后的早期现象（12-24 h），并导致大量游离p27（Kip 1）形式的时间依赖性积累。此外，视黄酸处理引起细胞周期蛋白依赖性激酶5水平和活性的增加;然而，免疫沉淀研究证明不存在与p27（kip 1）的相互作用。未观察到G1期细胞周期发动机的其他组分的明显变化。脉冲追踪实验显示，维甲酸处理的LAN-5中的p27（Kip 1）半衰期显著延长，而p27（Kip 1）基因表达及其信使RNA的翻译效率没有增强。在体内降解的p27（Kip 1）是敏感的两个高度特异性的蛋白酶体抑制剂，LLnL和lactacystin，而钙蛋白酶抑制剂II ALLM和半胱氨酸蛋白酶抑制剂E64没有修改的蛋白质水平。LLnL治疗引起了一个非常快速的（2小时）建立的Cdk抑制剂的内容和积累的更高分子量的抗p27（Kip 1）免疫反应性带，这可能代表泛素化形式的蛋白质。最后，体外实验表明，从维甲酸处理的LAN-5细胞制备的提取物降解重组p27（Kip 1）的速度显着低于未处理的细胞。我们的研究结果表明，维甲酸强烈增加p27（Kip 1）le，els通过下调泛素-蛋白酶体p27（Kip 1）降解途径。
Development of human neuroblastoma is due to an arrest in the differentiation program of neural crest sympathoadrenal progenitor cells. However, neuroblastomas, as well as their derived cell lines, maintain the potentiality of terminal differentiation. We investigated the molecular mechanisms by which retinoic acid, a molecule introduced in clinical trials for chemotherapy, induces differentiation in neuroblastoma cell lines. Our findings demonstrate that the retinoic acid-dependent growth arrest of LAN-5 neuroblastoma cell line is associated to a very large accumulation (>tenfold) of p27(Kip1) protein, a cyclin-dependent kinase inhibitor; the protein binds and inhibits cyclin-dependent kinase 2, 4 and 6 activities, thus hampering pRb and p107 phosphorylation, p27(Kip1) build-up was observable as an early phenomenon (12-24 h) after retinoic exposure and resulted in a time-dependent accumulation of high quantities of a free p27(Kip1) form, Furthermore, retinoic treatment causes an increase of cyclin-dependent kinase 5 level and activity; however, immunoprecipitation studies proved the absence of interaction with p27(kip1). No noticeable variation of other components of G1 phase cell cycle engine ws observed. Pulse-chase experiments showed a remarkable elongation of p27(Kip1) half-life in retinoic-treated LAN-5, while no enhancement of p27(Kip1) gene expression and of the translational efficiency of its messenger RNA were demonstrated. In vivo degradation of p27(Kip1) was sensitive to two highly specific proteasome inhibitors, LLnL and lactacystin, while the calpain inhibitor II ALLM and the cysteine protease inhibitor E64 did not modify the level of the protein. LLnL treatment caused a very rapid (2 h) build-up of the Cdk inhibitor content and the accumulation of higher molecular weight anti-p27(Kip1) immunoreactive bands, which probably represent ubiquitinated forms of the protein. Finally, in vitro experiments demonstrated that extracts prepared from retinoic-treated LAN-5 cells degraded recombinant p27(Kip1) at a rate remarkably slower than the untreated cells. Our results indicate that retinoic acid strongly increases p27(Kip1) le, els by down-regulating the ubiquitin-proteasome p27(Kip1) degrading pathway.