Resistance of cultured hepatoma cells to copper toxicity. Purification and characterization of the hepatoma metallothionein.

The mechanism of resistance to the toxic effects of copper was investigated using a series of copper-resistant hepatoma cell lines maintained in copper-enriched medium. Gel electrophoresis of carboxyamidated cell extracts demonstrated the presence of a pair of low molecular mass cysteine-rich proteins in wild-type and resistant cell lines. These proteins were purified to homogeneity and contained approx. 60% of the total cellular copper. Comparisons of molecular masses, pI values and amino-acid compositions for the purified hepatoma proteins with authentic rat liver metallothionein, as well as cross-reactivity with anti-rat metallothionein antibody, confirmed that the cysteine-rich hepatoma proteins were metallothioneins. The cellular concentration of these hepatoma copper-metallothioneins was proportional to both the level of metal resistance and the amount of copper accumulated by individual cell lines. Further, resistant cells removed from copper-enriched medium for 6-12 months, yet maintaining their level of resistance, showed only a slight decrease in metallothionein concentration. Thus it is proposed that the level of resistance to metal toxicity is mediated by the concentration of copper-metallothionein. It is also suggested that the steady-state level of copper metallothionein is controlled by the degree of metal exposure.