One hallmark of Ewing's sarcoma/peripheral neuroectodermal tumors is the 
presence of the Ews/Fli-1 chimeric oncogene. Interestingly, infection of 
neuroblastoma tumor cell lines with Ews/Fli-1 switches the differentiation 
program of neuroblastomas to Ewing's sarcoma/peripheral neuroectodermal tumors. 
Here we examined the status of cytoplasmically sequestered wt-p53 in 
neuroblastomas after stable expression of Ews/Fli-1. Immunofluorescence revealed 
that in the neuroblastoma-Ews/Fli-1 infectant cell lines, p53 went from a 
punctate-pattern of cytoplasmic sequestration to increased nuclear localization. 
Western blot analysis revealed that PARC was down-regulated in one neuroblastoma 
cell line but not expressed in the second. Therefore, decreased PARC expression 
could not fully account for relieving p53 sequestration in the neuroblastoma 
tumor cells. Neuroblastoma-Ews/Fli-1 infectant cell lines showed marked 
increases in p53 protein expression without transcriptional up-regulation. 
Interestingly, p53 was primarily phosphorylated, without activation of its 
downstream target p21(WAF1). Western blot analysis revealed that whereas MDM2 
gene expression does not change, p14(ARF), a negative protein regulator of MDM2, 
increases. These observations suggest that the downstream p53 pathway may be 
inactivated as a result of abnormal p53. We also found that p53 has an extended 
half-life in the neuroblastoma-Ews/Fli-1 infectants despite the retention of a 
wild-type sequence in neuroblastoma-Ews/Fli-1 infectant cell lines. We then 
tested the p53 response pathway and observed that the neuroblastoma parent cells 
responded to genotoxic stress, whereas the neuroblastoma-Ews/Fli-1 infectants 
did not. These results suggest that Ews/Fli-1 can directly abrogate the p53 
pathway to promote tumorigenesis. These studies also provide additional insight 
into the relationship among the p53 pathway proteins.