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

Abstract

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.

Keywords: Bad; Bak; Bax; Bcl-2; Copper; Wilson's disease; apoptosis; gene expression; glycine; metal ion; neuron; protein expression; toxic milk mice.

Figure 1

Cu is toxic to human M17 neuroblastoma cells in a concentration- and time-dependent manner. The graph represents the effect of varying concentrations of Cu (5 μM, 10 μM, 20 μM, 40 μM, 50 μM and 100 μM) on the viability of M17 neuroblastoma cells at 6, 12, 24, 36 and 48 h. Viability of treated cells is expressed as a percentage of the viability of untreated cells at each time point. Data is presented as mean ± SEM (n = 6 replicates; * P < 0.05; boxes indicate significance changes of multiple points from time 0).

Figure 2

Cu induction of apoptotic protein expression in human M17 neuroblastoma cells. (A) M17 neuroblastoma cells were incubated with 0, 25, 50 and 100 μM Cu-glycine² for various times (0, 6, 12, 24 and 48 h) and the expression of Bcl-2 (A), Bax (B) and Bak (C) determined by immunoblot analysis. The expression of β-actin was measured as a loading control. Molecular weight markers are shown on the left. Experiments are representative of 3 independent experiments.

Figure 3

Apoptotic protein expression in the liver and brain of toxic milk mice. The expression of Bcl-2, Bax, Bak and Bad in the liver (A) and brain (B) of 2 txJ and 2 control mice was determined by immunoblot analysis. Relative band densities (normalized to a non-specific loading control (liver) or β-actin (brain)) are shown below each band. Apoptotic protein expression was observed in the liver but not the brain.