Abstract
Metallothionein (MT) gene expression is increased in cadmium resistant Chinese hamster ovary cells (CHO CdR) upon medium (regular or serum-free) change during culturing. Among the major components of the medium, NaHCO3 was found to be able to induce MT gene expression in a dose- and time-dependent manner. The same effect was observed with other alkaline solutions, such as HEPES and NaOH. Moreover, NaHCO3 express caused the increase of cytosolic free zinc without change of total cellular zinc content, which means that the increase of zinc is released from intracellular storage. Using MT promoter-luciferase reporter gene constructs, we found that the presence of metal response elements (MREs) in the promoter region is necessary for NaHCO3-induced MT gene transcription. This finding is further supported by the observation that the binding activity between the metal-responsive transcription factor 1 (MTF-1) and the MRE were increased after NaHCO3 treatment. When CdR cells were treated with NaHCO3 in the present of the reduced thiol reagents (glutathione, dithiothreitol, cysteine or N-acetylcysteine), MT expression was reduced. On the contrary, giving oxidized thiol reagents (GSSG or 5,5-dithiobis-2-nitrobenzoic acid) had no influence on the NaHCO3-induced MT gene expression. The reduced thiol reagents apparently inhibit the release of intracellular free zinc and MTF-1 DNA binding activity that results in a reduction of MT gene expression. NaHCO3 treatment increased the intracellular reactive oxygen species and ATP level but decreased glutathione content. These results suggest that cellular alkalinization disturbs thiol redox status and increases the cytosolic zinc, which causes the MTF-1 activation and the MT gene expression. Following NaHCO3 treatment, an increase in cell proliferation was observed in CdR cells but not in the parental CHO K1 cells that do not express MT transcripts due to MT gene methylation. Using synchronized cells, an increase in cell proliferation was observed 9 h after NaHCO3 addition. Notably, proliferation of CHO K1 cells was increased when transfected with an MT gene. The effect of MT on cell growth was affirmed by treating CHO K1 cells with 5-azacytidine (Aza) to demethylate the MT gene. Proliferation increased in Aza-treated CHO K1 cells after NaHCO3 treatment. These results demonstrate that NaHCO3-induced MT gene expression causes an enhancement of cell proliferation in CHO cells.