A novel approach to the study of metallothionein function in oxidative stress and DNA damage
Metallothioneins (MTs) have a major role in metal metabolism and may also protect DNA against oxidants. MT protein has been found localized in the nucleus during S-phase. The mRNA encoding for the MT-1 isoform is found localized around the nucleus and associated with the cytoskeleton; this is due to targeting signals within the 3'untranslated region (3'UTR). Using cells transfected with gene constructs differing in their 3'UTRs, the role of perinuclear mRNA localization in facilitating MT synthesis close to its site of function and subsequent import of protein into the nucleus has been investigated, as well as the role of MT protein in the nucleus. We transfected CHO cells, which have a low constitutive level of MT expression, with either the full MT-gene (MTMT) or with MR 5'UTR and coding region linked to the 3'UTR of glutathione peroxidase (MTGSH). Immunocytochemistry showed that MT protein was localized in the perinuclear cytoplasm in the MTMT cells whereas no distinct localization was found in the MTGSH cells. The cells were then synchronised in S-phase by serum depletion/repletion. After serum repletion, MT was found in the nucleus of MTMT cells but not in the MTGSH cells. This suggests that perinuclear localization of MT-1 mRNA and its association with the cytoskeleton is necessary for MT protein localization, particularly for the shuttling of MT protein into the nucleus during S-phase. Functional studies demonstrated that the extent of oxidative stress and DNA damage was lower in the MTMT than the MTGSH, showing that a loss of MT protein localization led to a reduced protection of the cell. Therefore, it seems that perinuclear localization of mRNAs coding for MT is necessary for subsequent transport and targeting of proteins into the nucleus and that the localization of the protein within the cell is important for its function.