| Research Abstract |
Neuroblastoma (NB) is the most common pediatric solid malignant tumor derived from the sympathetic nervous system. Unlike the improvements in survival in many childhood malignancies, high-risk neumblastoma is still one of the most difficult tumors to cure, with only 30% long-term survival despite intensive multi-modal therapy. New treatments and a better understanding of drug resistance mechanisms are required for improvement of the survival rates. A noteworthy finding of neuroblastoma research is that mutations of p53 tumor suppressor have been reported less than 2% of NBs out of 340 tested (Tweddle DA et al., Cancer Res 61 : 8-13, 2001). Instead of mutation, cytoplasmic sequestration of p53 has been proposed as an alternative mechanism of inactivation in NB cells. The sequestration was first reported in frozen tumor samples using immuno-histodremical techniques (Moll UM et al., PNAS 92 : 4407-11, 1995) and later in NB oil lines by immuno-fluorescence and cell fractionation experiment
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s (Moll UM et al., MCB 16 : 1126-37, 1996). However, the p53 localization before and after DNA damage stimulation, p53 stabilization and activation by phosphorylation, binding to the p53-binding consensus sequences in vitro and in vivo, and up-regulation of p53-dowmstream molecules in NB cells have remained to be determined.
Therefore, we employed a panel of cell lines to determine whether the p53-dependent cell death in NB cells is caused by apoptotic cellular function, and we further studied the molecular mechanism of apoptosis induced via p53-dependent pathway. We provide lines of evidence that a p53-dependent stress, doxorubicin (Doxo) administration, causes accumulation of p53 in nucleus of NB cells and phosphorylation of several serine residues (serine 15, serine 20, and serine 46) in both Doxo-sensitive and -resistant NB cell lines. Up-regulation of a p53-downstream molecule, p2lCip 1/Waf1 and accumulation of Noxa, a pro-apoptotic Bcl-2 family molecule, in mitochondria are observed in only Doxo-sensitive NB cells. Mitochondrial dysfunction including cytochrome c release and membrane potential dysregulation was caused in the sensitive cells, resulting in the activation of the intrinsic caspase pathway. However, the accumulation in nucleus and phosphorylation of p53 were inert to induce the Noxa accumulation and the mitochondrial dysfunction in p53-resistant NB cells. Surprisingly, inactivation of the p53 by over-expression of MDM2 resulted in the canceling of up-regulation of Noxa in mitochondria and reduced the apoptotic cell death. Taken together, MDM2/p53 pathway seems to play an important role in NB cells by Noxa regulation in mitochondria. Less
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