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Recent studies have shown that MEK/ERK-mediated signals play a major role in regulation of activity of p53 tumor suppressor protein. In this study, we investigated whether or not there is functional interaction between p53 and MEK/ERK pathways in epithelial breast cancer cells exposed to copper or zinc. We demonstrated that expression of wild-type p53 induced by copper or zinc significantly reduced phosphorylation of extracellular signal regulated kinase (ERK) in epithelial breast cancer MCF7 cells. Mutation or suppression of p53 in MDA-MB231 and MCF7-E6 cells, respectively, resulted in a strong ERK phosphorylation in the presence of metals. Weak ERK phosphorylation in MCF7 cells induced by copper or zinc was linked to mitochondrial disruption and apoptosis. Furthermore, inhibition of ERK through addition of PD98059 stimulated p53 activation in MCF7 cells and also led to upregulation of p53 downstream targets, p21 and Bax, which is a proapototic member of Bcl-2 family triggering mitochondrial pore opening. Moreover, blockage of the MEK/ERK pathway caused a breakdown of the mitochondrial membrane potential accompanied by an elevation in the ROS production. Disruption of p53 expression attenuated the depolarization of the mitochondrial membrane and ROS generation. Furthermore, PD98059 initiated apoptosis inducing factor (AIF) translocation from mitochondria to the nucleus in MCF7 cells; which are depleted in caspase 3. Interestingly, repression of MEK/ERK pathway did not intensify the cell stress caused by metal toxicity. Therefore, these findings demonstrate that MEK/ERK pathway plays an important role in downregulation of p53 and cell survival. Inhibition of ERK can lead to apoptosis via nuclear relocation of AIF. However, metal-induced activation of p53 and mitochondrial depolarization appears to be independent of ERK. Our data suggest that copper induces apoptosis through depolarization of mitochondrial membrane with release of AIF, and this process is MEK/ERK independent.

We have shown the protection of human central nervous system (CNS) cultures by zinc (Zn) or cadmium (Cd)-pre-induced metallothionein (MT) synthesis from radiation-induced cytotoxicity (lactate dehydrogenase (LDH) release and neuronal dendritic injury). The present study is to further define the types of cell death induced by different dose levels of radiation and investigate the effect of MT induction (by Zn or Cd) on radiation-induced apoptosis in primary human CNS and astrocyte cultures. Apoptosis was detected by fragmented DNA electrophoresis, TUNEL technique, and propidium iodide staining. Expression of MT protein was examined by immunofluorescent staining. Results showed that exposure of primary human CNS cultures to 15 and 30 Gy gamma-radiation predominantly induced apoptotic cell death, while exposure to 60 Gy gamma-radiation predominantly induced necrotic cell death. Normal primary human CNS cultures showed weak MT staining, while primary human CNS cultures exposed to Zn or Cd showed intense MT staining. The induced apoptotic cell death by exposure to 30 Gy gamma-radiation increased to a maximum level at 12 and 24 h, and was reduced significantly by Zn or Cd pre-induced MT. Using primary human astrocytes, the induction of MT protein by Zn or Cd was further confirmed. The enhanced MT expression also afforded a significant protection from 30 Gy gamma-ray-induced apoptosis in the primary human astrocytes. These results suggest that MT protected human CNS cells from apoptosis following ionizing radiation, probably through its antioxidant property.

Abstract: Previous reports have shown that metallothionein (MT) may modulate p53 activity through zinc exchange. However, little is known on a direct interaction between MT and p53 in cells. The results demonstrate an interaction between MT and p53 can occur in vitro. The complex between MT and p53 was observed in breast cancer epithelial cells with both wild and inactive type of p53. Furthermore, it was shown that wt-p53 was preferentially associated with Apo-MT. Our data suggest that co-expression of MT and p53 and their complex formation in tumor cells may be involved in regulation of apoptosis in these cells. [Copyright &y& Elsevier]

The objective of this study was to evaluate the potential role of TNF-α in the onset of acute hepatitis in the Long-Evans Cinnamon (LEC) rat, an animal model for inherited copper (Cu) toxicosis. In LEC rats, Cu is accumulated in the liver with age, and clinical signs of acute hepatitis were observed as, icterus, reduced body weight, nasal bleeding, dehydration, and reduced food intake at 12 weeks of age. Cellular changes such as apoptosis in the liver were evident in these rats with increasing age. Positive TNF-α and TNFR1 immunostainings were observed in hepatocytes and Kupffer cells in LEC rats. Hepatic levels of caspase-3 activity, TNF-α mRNA, and protein were also increased in LEC rats from 6 to 12 weeks of age as compared with control Long-Evans (LE) rats. The neutralization of TNF-α by passive immunization or the inhibition of caspase activity can block the apoptotic process initiated by TNF-α. In this study, we evaluated the effects of passive immunization of LEC rats with weekly administration of anti-rat TNF-α on Cu-induced acute hepatitis. This treatment resulted in a reduction of the percentage of apoptotic cells in the liver, decreased activity of caspase-3, and also in down-regulation of the TNF-α gene expression. Thus, these results suggest a major role for TNF-α on the pathogenesis of Cu-induced acute hepatitis in LEC rats. [Copyright &y& Elsevier]