Over-expressed human divalent metal transporter 1 is involved in iron accumulation in MES23.5 cells.

Elevated iron accumulation has been reported in brain regions in some neurodegenerative disorders. However, the mechanism for this is largely unknown. Divalent metal transporter 1 (DMT1) is an important divalent cation transporter. The aim of the present study is to construct recombinant adenovirus encoding human DMT1 with iron responsive element (DMT1+IRE) and infect MES23.5 dopaminergic cells in order to investigate the relationship between increased DMT1+IRE expression and iron accumulation. The human DMT1 gene was obtained by RT-PCR from tissues of human duodenum. AdDMT1+IRE was successfully constructed and identified by PCR, restriction endonuclease analyses and DNA sequencing, respectively. It was able to efficiently infect MES23.5 cells, which was confirmed by RT-PCR and Western blots. When incubated with 100 microM ferrous iron for 6h, the intracellular iron levels dramatically increased in AdDMT1+IRE infected MES23.5 cells compared to the solely adenovirus infected cells. Meanwhile, the levels of hydroxyl free radicals and malondialdehyde (MDA) in these cells increased. This led to the activation of caspase-3. The apoptosis in AdDMT1+IRE infected cells was shown with hypercondensed nuclei using Hoechst staining. Analysis of DNA extracted from these cells showed the typical "ladder pattern", indicating the formation of mono- and oligonucleosomes. These results suggested that increased DMT1+IRE expression in MES23.5 cells caused the increased intracellular iron accumulation. This resulted in the increased oxidative stress leading to ultimate cell apoptosis.