Analysis of p53 regulation in neuroblastoma cell lines

许多神经母细胞瘤细胞系表达野生型p53蛋白，但仍无功能。野生型p53在神经母细胞瘤中的失功能与该蛋白在细胞质中的隔离有关。本项目旨在研究神经母细胞瘤中p53细胞质隔离的机制，以确定该机制是否在p53进出细胞核中发挥更普遍的作用。到目前为止，我们已经了解到，在一些神经母细胞瘤细胞系中，p53的细胞质定位不受leptomycin阻断核输出的影响，这表明这些细胞系中p53的细胞质隔离不是核输出过度活跃的结果。我们还观察到p53对leptomycin不敏感与p53对蛋白酶体抑制不敏感相关。因此，细胞质隔离的p53在这些细胞中抵抗蛋白酶体降解。同时，我们观察到这些细胞中p53特异性泛素连接酶hdm2与细胞质p53的显著关联，以及p53泛素化。在这些细胞中，稳态和dna损伤诱导的p53磷酸化是异常的，这可能与它的细胞质隔离有关。我们目前正在研究这些细胞中p53的核定位信号是否被与另一种蛋白的非共价结合所掩盖。

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.