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

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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