Abstract
We examined the DNA binding activity of mouse and human MTF-1 in whole cell extracts from cells cultured in medium containing zinc or cadmium and from untreated cells after the in vitro addition of zinc or cadmium, as well as using recombinant MTF-1 transcribed and translated in vitro and treated with various transition metals. Incubation of human (HeLa) or mouse (Hepa) cells in medium containing cadmium (5-15 microM) did not lead to a significant increase (<2-fold) in the amount of MTF-1 DNA binding activity, whereas zinc (100 microM) led to a 6-15-fold increase within 1 h. MTF-1 binding activity was low, but detectable, in control whole cell extracts and was increased (>10-fold) after the in vitro addition of zinc (30 microM) and incubation at 37 degrees C for 15 min. In contrast, addition of cadmium (6 or 60 microM) did not activate MTF-1 binding activity. Recombinant mouse and human MTF-1 were also dependent on exogenous zinc for DNA binding activity. Cadmium did not facilitate activation of recombinant MTF-1, but instead inhibited the activation of the recombinant protein by zinc. Interestingly, glutathione (1 mM) protected recombinant MTF-1 from inactivation by cadmium, and allowed for activation by zinc. It was also noted that zinc-activated recombinant MTF-1 was protected from cadmium only when bound to DNA. These results suggest that cadmium interacts with the zinc fingers of MTF-1 and forms an inactive complex. Of the several transition metals (zinc, cadmium, nickel, silver, copper, and cobalt) examined, only zinc facilitated activation of the DNA binding activity of recombinant MTF-1. These data suggest that transition metals, other than zinc, that activate MT gene expression may do so by mechanisms independent of an increase in the DNA binding activity of MTF-1.
Highlights
§ Supported in part by a postdoctoral fellowship award from the Marion Merrell Dow Scientific Education Partnership
Whole cell extracts from mouse Hepa and human HeLa cells incubated for 1 h in medium containing zinc (60 M) or cadmium (6 M) were analyzed for MTF-1 binding activity using EMSA (Fig. 1A)
Addition of zinc (30 M) to the whole cell extracts from cadmium-treated cells resulted in a temperature dependent activation of MTF-1 binding activity, which demonstrated that MTF-1 was recovered from the cadmium-treated cells
Summary
We examined the DNA binding activity of mouse and human MTF-1 in whole cell extracts from cells cultured in medium containing zinc or cadmium and from untreated cells after the in vitro addition of zinc or cadmium, as well as using recombinant MTF-1 transcribed and translated in vitro and treated with various transition metals. These data suggest that transition metals, other than zinc, that activate MT gene expression may do so by mechanisms independent of an increase in the DNA binding activity of MTF-1.
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