Department of Medical Biochemistry and Molecular Biology, University of Saarland Medical Center, D-66421 Homburg, Germany.
Neuroscience. 2012 Mar 29;206:25-38. doi: 10.1016/j.neuroscience.2012.01.001. Epub 2012 Jan 8.
Arsenite treatment of human SH-SY5Y neuroblastoma cells leads to an upregulation of caspase-3/7 activity and to the fragmentation of chromatin that is accompanied by elevated p53 and c-Jun levels. Expression of a truncated mutant of p53, p53DD, which interfered with the oligomerization of p53, suppressed the arsenite-induced upregulation of caspase-3/7 activity and the fragmentation of chromatin, indicating that p53 is required for arsenite-induced cell death. These data were corroborated by knockdown experiments of p53 following expression of a p53-specific short hairpin RNA. Likewise, expression of either p53DD or knockdown of p53 prevented caspase-3/7 activation and chromatin fragmentation induced by nutlin-3, a compound that prevents the interaction between p53 and the E3 ubiquitin ligase MDM2. Transcriptional upregulation of a chromatin-embedded p53-responsive reporter gene in either arsenite or nutlin-3 stimulated neuroblastoma cells revealed that the transcriptional activity of p53 was increased under these conditions. Expression of a c-Jun-specific short hairpin RNA failed to impair arsenite-induced caspase-3/7 activation and fragmentation of chromatin. Likewise, inhibition of c-Jun target gene expression by expression of a dominant-negative mutant of c-Jun did not interfere with arsenite-induced caspase-3/7 activation and chromatin fragmentation. However, this approach successfully reduced caspase-3/7 activity induced as a result of forced expression of a constitutively active mutant of mitogen-activated protein kinase/extracellular signal-regulated kinase kinase kinase (MEKK)-1. Together, these data show that the upregulation of p53 is causally linked with arsenite-induced cell death in neuroblastoma cells, whereas the upregulation of c-Jun is not part of this apoptotic signaling cascade.
亚砷酸盐处理人 SH-SY5Y 神经母细胞瘤细胞导致 caspase-3/7 活性上调，并伴有染色质碎片化，同时 p53 和 c-Jun 水平升高。表达一种截断的 p53 突变体 p53DD，它干扰 p53 的寡聚化，抑制亚砷酸盐诱导的 caspase-3/7 活性上调和染色质碎片化，表明 p53 是亚砷酸盐诱导细胞死亡所必需的。这些数据通过表达 p53 特异性短发夹 RNA 进行 p53 敲低实验得到了证实。同样，p53DD 的表达或 p53 的敲低均可阻止 nutlin-3 诱导的 caspase-3/7 激活和染色质碎片化，nutlin-3 是一种阻止 p53 与 E3 泛素连接酶 MDM2 相互作用的化合物。在亚砷酸盐或 nutlin-3 刺激的神经母细胞瘤细胞中，嵌入染色质的 p53 反应性报告基因的转录上调表明，在这些条件下，p53 的转录活性增加。表达 c-Jun 特异性短发夹 RNA 未能损害亚砷酸盐诱导的 caspase-3/7 激活和染色质碎片化。同样，通过表达 c-Jun 的显性负突变体抑制 c-Jun 靶基因的表达，并不干扰亚砷酸盐诱导的 caspase-3/7 激活和染色质碎片化。然而，这种方法成功地降低了由于强制表达组成性激活的丝裂原活化蛋白激酶/细胞外信号调节激酶激酶激酶（MEKK-1）突变体而诱导的 caspase-3/7 活性。总之，这些数据表明，p53 的上调与神经母细胞瘤细胞中亚砷酸盐诱导的细胞死亡有因果关系，而 c-Jun 的上调不是这个凋亡信号级联的一部分。
