Borriello A, Pietra V D, Criscuolo M, Oliva A, Tonini G P, Iolascon A, Zappia V, Ragione F D
Institute of Biochemistry of Macromolecules, Second University of Naples, via Constantinopoli 16, 80138 Naples, Italy.
Oncogene. 2000 Jan 6;19(1):51-60. doi: 10.1038/sj.onc.1203231.
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 p27Kip1 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. p27Kip1 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 p27Kip1 form. Furthermore, retinoic treatment causes an increase of cyclin-dependent kinase 5 level and activity; however, immunoprecipitation studies proved the absence of interaction with p27kip1. No noticeable variation of other components of G1 phase cell cycle engine was observed. Pulse-chase experiments showed a remarkable elongation of p27Kip1 half-life in retinoic-treated LAN-5, while no enhancement of p27Kip1 gene expression and of the translational efficiency of its messenger RNA were demonstrated. In vivo degradation of p27Kip1 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-p27Kip1 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 p27Kip1 at a rate remarkably slower than the untreated cells. Our results indicate that retinoic acid strongly increases p27Kip1 levels by down-regulating the ubiquitin-proteasome p27Kip1 degrading pathway.
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，神经母细胞瘤及其衍生的细胞系保持着终末分化的潜能。我们研究了视黄酸（一种用于化疗临床试验的分子）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，LAN-5神经母细胞瘤细胞系视黄酸依赖性生长停滞与细胞周期蛋白依赖性激酶抑制剂p27Kip1蛋白大量积累（超过十倍）有关；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的磷酸化。视黄酸处理后早期现象（12 - 24小时）即可观察到p27Kip1的积累，并导致大量游离p27Kip1形式的时间依赖性积累。此外，视黄酸处理导致细胞周期蛋白依赖性激酶5水平和活性增加；然而，免疫沉淀研究证明其与p27kip1不存在相互作用。未观察到G1期细胞周期引擎其他成分的明显变化。脉冲追踪实验表明，视黄酸处理的LAN-5中p27Kip1半衰期显著延长，而未证明p27Kip1基因表达及其信使核糖核酸翻译效率增强。p27Kip1的体内降解对两种高度特异性的蛋白酶体抑制剂LLnL和乳胞素敏感，而钙蛋白酶抑制剂II ALLM和半胱氨酸蛋白酶抑制剂E64未改变该蛋白水平。LLnL处理导致细胞周期蛋白依赖性激酶抑制剂含量迅速（2小时）积累以及高分子量抗p27Kip1免疫反应条带的积累，这可能代表该蛋白的泛素化形式。最后，体外实验表明，视黄酸处理的LAN-5细胞制备的提取物降解重组p27Kip1的速率明显慢于未处理细胞。我们的结果表明，视黄酸通过下调泛素-蛋白酶体p27Kip1降解途径强烈增加p27Kip1水平。
