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，但仍然没有功能。神经母细胞瘤中野生型P53的非功能性与细胞质中的蛋白质隔离有关。本课题的目的是研究神经母细胞瘤中P53胞浆固存的机制(S)，以确定所涉及的机制(S)是否在P53穿梭进出细胞核中起更普遍的作用。到目前为止，我们已经了解到，在几个神经母细胞瘤细胞系中，P53的胞浆定位不受软霉素阻断核输出的影响，这表明这些细胞系中P53的胞质隔离不是核输出过度活跃的结果。我们还观察到P53对软霉素不敏感与P53对蛋白酶体抑制不敏感有关。因此，这些细胞中胞质隔离的P53对蛋白酶体的降解具有抵抗力。同时，我们观察到Hdm2，一种P53特异性泛素连接酶，与这些细胞中细胞质的P53，以及P53的泛素化显著相关。在这些细胞中，稳态和DNA损伤诱导的P53的磷酸化是异常的，这可能与其胞质隔离有关。我们目前正在研究这些细胞中P53的核定位信号是否被另一种蛋白质(S)的非共价结合所掩盖。-p，神经母细胞瘤

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 proteins 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). - p, , neuroblastoma,