Characterization ofp53 function in human neuroblastoma cell lines

Human neuroblastoma is the most common solid tumor affecting children under the age of five. The p53 tumor suppressor gene is mutated in over half of all human cancers. However, neuroblastoma cells contain wildtype p53, suggesting that it may be inactivated by other cellular mechanisms. Thus, we investigated the molecular basis for this defect. Neuroblastoma cells are placed into two molecular categories: non N-myc amplified and N-myc amplified. We chose two N-myc amplified lines, NGP and IMR5, and two non-N-myc amplified lines, SK-NSH and SY5Y. To study the function of p53 in these cells, we monitored the p53 protein levels in the nucleus and cytoplasm, p53 sequence-specific DNA binding activity, and apoptosis after exposure of the cells to gamma irradiation. Tumor suppression assays were performed with each cell line. P53 suppressed colony formation in SK-NSH and SY5Y, but did not suppress colony formation in NGP and IMR5. Western blotting indicated that p53 was present in the nucleus of all of the cell lines and was elevated upon irradiation. Depending upon the cell line, p53 was present as a singlet, doublet, or triplet. Upon irradiation, p53 from SK-NSH and SY5Y was activated for DNA binding, whereas p53 from NGP and IMR5 was not activated for DNA binding. FACS analysis demonstrated that all cell lines underwent apoptosis upon irradiation. Since p53 is a thermolabile protein, we hypothesized that lowering the ambient temperature of cellular growth would enable proper re-folding of p53 and restore function in N-myc amplified cells. Our experiments showed that p53 from NGP and IMR5 cells grown at 32°C had enhanced DNA binding ability upon irradiation. No increase in apoptosis was observed in cells grown at 32°C after irradiation. The level of downstream protein p21 was elevated in NGP cells grown at 32°C. P21 partially suppressed colony formation in NGP cells, suggesting that downstream cell cycle proteins were functional