Many neuroblastoma cell lines express wild type p53 protein which nevertheless remains non-functional. Non-functionality of wild type p53 in neuroblastoma is correlated to the protein's sequestration in the cytoplasm. The purpose of this project is to study the mechanism(s) involved in the cytoplasmic sequestration of p53 in neuroblastoma in order to determine whether the mechansim(s) involved play a more general role in p53 shuttling in and out of the nucleus. To date, we have learned that, in several neuroblastoma cell lines, the cytoplasmic localization of p53 is not affected by blockade of nuclear export with leptomycin, suggesting that cytoplasmic sequestration of p53 in these cell lines is not the consequence of hyperactive nuclear export. We have also observed that p53 insensitivity to leptomycin is correlated with p53 insensitivity to proteasome inhibition. Thus, the cytoplasmically sequestered p53 in these cells is resistant to proteasome degradation. At the same time, we have observed significant association of hdm2, a p53-specific ubiquitin ligase, with the cytoplasmic p53 in these cells, together with p53 ubiquitination. Steady-state and DNA-damage-induced phosphorylation of the p53 in these cells is abnormal and this may be relevant to its cytoplasmic sequestration. We are currently examining whether the nuclear localization signal of p53 in these cells is masked by non-covalent association with another protein(s). Our new data has revealed that in those lines where p53 is resistant to proteasome inhibitor and leptomycin, the protein is point mutated (even though it was wild type in the original cell lines). Further, antibody selection determines whether wt p53 appears cytosolic or nuclear in neuroblastoma N-type cells, suggesting that epitope-masking may be occurring. Lastly, hdm2 ubiquitin ligase activity is inactivated by DNA damage in neuroblastoma lines, without concomitant dissociation of p53/hdm2 complexes, suggesting that regulation of hdm2 activity mediates DNA damage-induced stabilization of wt p53 in neuroblastoma.
