Investigation of new molecular target of neuroblastoma therapy: The role of p53 pathway in neuroblastoma cell death

神经母细胞瘤（NB）是最常见的儿童来自交感神经系统的实体恶性肿瘤。我们研究了NB细胞中p53依赖性细胞死亡的抑制剂。该抑制剂在阿霉素耐药细胞中高度表达，并在细胞核中与p53结合，其候选抑制剂似乎是p53- e3泛素连接酶HDM2。值得注意的是，尽管HDM2以蛋白酶体依赖的方式加速p53降解，但p53和HDM2都在耐阿霉素NB细胞中积累。最近有报道称，自然产生的SNP309导致Mdm2过量产生，抑制染色质结合的p53激活其靶基因的转录（JBC, Arva NC et al., 2005）。这些结果促使我们研究SNP309 HDM2在NB细胞中的存在。然而，我们只能在SK-N-SH（敏感）细胞中检测到杂合SNP，而在IMR32（耐药）、NB-19（耐药）和NB-9（敏感）细胞中检测不到。接下来，我们对HDM2的p53结合域进行了测序，进一步验证了NB细胞中p53-HDM2的相互作用，并没有发现HDM2的p53结合域发生突变。我们利用一组细胞系来确定神经母细胞瘤（NB）细胞中p53依赖的细胞死亡是否由细胞凋亡功能引起，并进一步研究了p53依赖途径诱导细胞凋亡的分子机制。我们获得的证据表明，一种p53依赖的应激，多柔比星（Doxo）给药，导致NB细胞细胞核中p53的积累和多柔比星敏感和耐药细胞系中几个丝氨酸残基的磷酸化。仅在doxo敏感的NB细胞中观察到细胞中p53-下游分子的上调和Noxa在线粒体部分的积累。Noxa在doxo诱导的NB细胞死亡中的意义通过Noxa敲低实验得到证实。线粒体功能障碍，包括细胞色素c释放和膜电位失调，发生并导致内在caspase途径的激活（Kurata K等人，Oncogene，修订）。有趣的是，我们发现HDM2不仅与p53蛋白量的控制有关，还可能调节NB细胞中Noxa的动力学。在HDM-2过表达的SK-N-SH细胞中，p53下游通路失活，但p53积累。虽然刺激前Noxa在线粒体中积累，但阿霉素不能上调线粒体中的Noxa并诱导hdm2过表达的SK-N-SH细胞凋亡。这些结果表明，HDM2可能是调控NB细胞线粒体中p53稳定性和活性以及Noxa动力学的关键分子。我们希望进一步分析线粒体凋亡相关分子，以开发新的治疗不良神经母细胞瘤的方法。少

Neuroblastoma (NB) is the most common pediatric solid malignant tumor derived from the sympathetic nervous system. We investigated the inhibitor of p53-dependent cell death in NB cells. The candidate for the inhibitor, which was highly expressed in Doxorubicin-resistant cells and bound to p53 in nucleus, seemed to be p53-E3 ubiquitine ligase HDM2.Of note, both of p53 and HDM2 were accumulated in the Doxorubicin-resistant NB cells although HDM2 accelerates p53 degradation in a proteasome dependent manner. Recently, it has reported that overproduction of Mdm2, resulting from a naturally occurring SNP309, inhibits chromatin-bound p53 from activating the transcription of its target genes (JBC, Arva NC et al., 2005). These results prompted us to study the existence of SNP309 HDM2 in NB cells. However, we could detect the heterozygous SNP only in SK-N-SH (sensitive) but not in IMR32 (resistant), NB-19 (resistant) and NB-9 (sensitive) cells. Next, we sequenced the p53-binding domain of HDM2 t … More o verify the p53-HDM2 interaction in NB cells and could not found mutations in the p53 binding domain of HDM2We employed a panel of cell lines to determine whether the p53-dependent cell death in neuroblastoma (NB) cells is caused by apoptotic cellular function, and we further studied the molecular mechanism of apoptosis induced via the p53-dependent pathway. We obtained evidence that a type of p53-dependent stress, doxorubicin (Doxo) administration, causes accumulation of p53 in the nucleus of NB cells and phosphorylation of several serine residues in both Doxo-sensitive and-resistant cell lines. Up-regulation of p53-downstream molecules in cells and accumulation of Noxa in the mitochondrial fraction were observed only in Doxo-sensitive NB cells. Significance of Noxa in the Doxo-induced NB cell death was confirmed by Noxa-knockdown experiments. Mitochondrial dysfunction, including cytochrome c release and membrane potential dis-regulation, occurred and resulted in the activation of the intrinsic caspase pathway (Kurata K et al., Oncogene, revise). Intriguingly, we found that HDM2 not only relates to control of p53 protein amounts but also may regulate the kinetics of Noxa in NB cells. In HDM-2 over-expressed SK-N-SH cells, p53 downstream pathway was inactivated although p53 was accumulated. Although Noxa was accumulated in mitochondria before stimulation, Doxorubicin could not up-regulate Noxa in mitochondria and induce apoptosis in the HDM2-over-expressed SK-N-SH cells. These results indicate that HDM2 might be key molecule to control p53 stability and activities and to regulate Noxa kinetics in mitochondria in NB cells. We would like to do further analysis of the mitochondria apoptosis related molecules to develop new therapies for unfavorable neuroblastoma. Less