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，但其仍然保持非功能性。神经母细胞瘤中野生型p53的非功能性与蛋白质在细胞质中的隔离相关。本研究的目的是研究神经母细胞瘤中p53的胞质隔离机制，以确定所涉及的机制是否在p53穿梭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，在几个神经母细胞瘤细胞系，细胞质定位的p53不受封锁的核出口与链霉素，这表明在这些细胞系的细胞质隔离的p53是不是过度活跃的核出口的后果。我们还观察到p53对来普霉素的不敏感性与p53对蛋白酶体抑制的不敏感性相关。因此，这些细胞中的胞质隔离的p53对蛋白酶体降解具有抗性。与此同时，我们观察到hdm 2，一种p53特异性的泛素连接酶，与这些细胞中的胞质p53，以及p53泛素化的显著关联。稳态和DNA损伤诱导的磷酸化的p53在这些细胞是异常的，这可能是有关其胞质隔离。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质的非共价结合所掩盖。 我们的新数据显示，在p53对蛋白酶体抑制剂和来普霉素具有抗性的细胞系中，该蛋白质是点突变的（即使它在原始细胞系中是野生型）。此外，抗体选择决定了野生型p53是否出现在神经母细胞瘤N型细胞的胞质或核，这表明表位掩蔽可能发生。最后，hdm 2泛素连接酶活性失活的DNA损伤在神经母细胞瘤株，没有伴随的p53/hdm 2复合物的解离，这表明调节hdm 2活性介导的DNA损伤诱导的稳定野生型p53在神经母细胞瘤。

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 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.