Metallothionein: a protein conferring resistance in vitro to tumor necrosis factor

The role of metallothionein (MT) in the cytotoxicity of tumor necrosis factor (TNF) was investigated in vitro. A human epithelial cell line (HE100) and a mouse fibroblast line (CI 1D100) had previously been cultured to become resistant to 100 microns CdCl2 and were cultured routinely in cadmium-containing medium. The MT content of these cells and the nonresistant parent cell lines (HE and CI 1D) was determined both qualitatively and quantitatively by immunochemical techniques. Immunofluorescence microscopy revealed the cadmium-resistant cell lines to be intensely rich in MT in both the nuclear and cytoplasmic compartments. This finding was confirmed by immuno-electron microscopy which also showed the labeling to be freely distributed and not membrane-bound. In comparison, a very weak labeling of the parent cell lines was observed. MT concentration, as determined by enzyme-linked immunosorbant assay, was found to be 4.05 +/- 1.13% and 3.91 +/- 0.7% of the total protein for HE100 and CI 1D100 cells, respectively. We were unable to detect MT in the parent cell lines by this technique. Dose-survival curves obtained after 3 days treatment of the cells with TNF (0.125-500 ng/ml) revealed that the MT-rich substrains were significantly resistant compared to the parent strains (P < 0.001; t tests). In growth rate studies, where cells were exposed to TNF over a dose range of 0.25-250 ng/ml for 6 days, the resistance of the MT-rich cell lines was confirmed (P < 0.002). These data indicate that MT confers resistance to the cytotoxic effects of TNF in vitro and that sensitivity to TNF may be related to the MT content of the cell.