Copper Induces Apoptosis of Neuroblastoma Cells Via Post-translational Regulation of the Expression of Bcl-2-family Proteins and the tx Mouse is a Better Model of Hepatic than Brain Cu Toxicity.
Copper Induces Apoptosis of Neuroblastoma Cells Via Post-translational Regulation of the Expression of Bcl-2-family Proteins and the tx Mouse is a Better Model of Hepatic than Brain Cu Toxicity.
复制标题
DOI：
--
复制
发表时间：
2008
影响因子：
0.1
通讯作者：
Hsien W Chan;Tianbing Liu;G. Verdile;G. Bishop;Ryan J. Haasl;Mark A. Smith;George Perry;R. Martins;C. Atwood
中科院分区：
文献类型：
--
作者：
Hsien W Chan;Tianbing Liu;G. Verdile;G. Bishop;Ryan J. Haasl;Mark A. Smith;George Perry;R. Martins;C. Atwood
改变的铜稳态对肝细胞和神经元（这是在诸如威尔逊病（WD）等铜蓄积疾病中受影响的两种主要细胞类型）产生毒性的基本机制仍不清楚。我们以人M17神经母细胞瘤细胞为模型来研究铜毒性，发现神经元死亡存在时间和浓度依赖性诱导，即在24小时时，25μM的甘氨酸铜（Cu - glycine₂）会使细胞活力降低约50%。用25 - 50μM的甘氨酸铜处理24小时显著改变了296个基因的表达，包括8个与细胞凋亡相关的基因（BCL2相关的抗凋亡基因3、BCL2/腺病毒E1B 19kDa相互作用蛋白半胱天冬酶5、Fas诱导的细胞凋亡调节因子、V - jun肉瘤病毒17癌基因同源物、紧密连接蛋白5、前列腺素E受体3和非受体型蛋白酪氨酸磷酸酶6）。令人惊讶的是，更多“传统”凋亡基因（Bcl - 2、Bax、Bak和Bad）表达的变化不超过20%。为了测试神经母细胞瘤细胞中细胞凋亡的诱导是否通过翻译后机制，我们测量了用0 - 100μM甘氨酸铜处理24 - 48小时的M17神经母细胞瘤细胞中这些凋亡标记物的蛋白质表达。与甘氨酸处理的细胞相比，甘氨酸铜使Bcl - 2表达降低50%，但使Bax和Bak表达分别增加130%和400%。为了评估铜是否也在威尔逊病小鼠模型中诱导凋亡细胞死亡，我们测量了10个月大（在其生命末期，此时出现明显肝脏病理）表达ATP7b基因突变的小鼠（tx(J)小鼠）肝脏和大脑中这些凋亡标记物的表达。与背景小鼠相比，tx(J)小鼠肝脏中这些凋亡标记物表达的变化与铜处理的神经母细胞瘤细胞相似。相比之下，在tx(J)小鼠和背景小鼠的大脑之间几乎没有检测到凋亡蛋白表达的变化，这表明tx(J)小鼠是肝脏铜毒性的良好模型，但不是大脑铜毒性的良好模型。我们的结果表明，铜诱导的神经元凋亡不需要凋亡基因的从头合成或降解，并且老年tx(J)小鼠大脑中的铜蓄积不足以诱导凋亡。
The basic mechanism(s) by which altered Cu homeostasis is toxic to hepatocytes and neurons, the two major cell types affected in copper storage diseases such as Wilson's disease (WD), remain unclear. Using human M17 neuroblastoma cells as a model to examine Cu toxicity, we found that there was a time- and concentration-dependent induction of neuronal death, such that at 24 h there was a approximately 50 % reduction in viability with 25 muM Cu-glycine(2). Cu-glycine(2) (25:50 muM) treatment for 24 h significantly altered the expression of 296 genes, including 8 genes involved with apoptosis (BCL2-associated athanogene 3, BCL2/adenovirus E1B 19kDa interacting protein caspase 5, regulator of Fas-induced apoptosis, V-jun sarcoma virus 17 oncogene homolog, claudin 5, prostaglandin E receptor 3 and protein tyrosine phosphatase, non-receptor type 6). Surprisingly, changes in the expression of more 'traditional' apoptotic genes (Bcl-2, Bax, Bak and Bad) did not vary more than 20 %. To test whether the induction of apoptosis in neuroblastoma cells was via post-translational mechanisms, we measured the protein expression of these apoptotic markers in M17 neuroblastoma cells treated with Cu-glycine(2) (0-100 muM) for 24-48 h. Compared with glycine treated cells, Cu-glycine(2) reduced Bcl-2 expression by 50 %, but increased Bax and Bak expression by 130% and 400 %, respectively. To assess whether Cu also induced apoptotic cell death in a mouse model of WD, we measured the expression of these apoptotic markers in the liver and brain of mice expressing an ATP7b gene mutation (tx(J) mice) at 10 months of age (near the end of their lives when overt liver pathology is displayed). Changes in the liver expression of these apoptotic markers in tx(J) mice compared to background mice mirrored those of Cu treated neuroblastoma cells. In contrast, few changes in apoptotic protein expression were detected in the brain between tx(J) and background mice, indicating the tx(J) mouse is a good model of hepatic, but not brain, Cu toxicity. Our results indicate that Cu-induction of neuronal apoptosis does not require de novo synthesis or degradation of apoptotic genes, and that Cu accumulation in the aged tx(J) mouse brain is insufficient to induce apoptosis.