MECHANISM OF RESISTANCE TO COPPER TOXICITY IN CULTURED HEPATOMA CELLS (METALLOTHIONEIN, X-RAY SPECTROSCOPY)

Abstract

A series of four cell lines resistant to the toxic effect of copper were developed from Morris rat hepatoma cells by gradually increasing the concentration of copper in the growth medium. The EC(,50), that concentration of copper that kills and/or inhibits the growth of 50% of the cells after 72 hrs, increased four-fold over that for wild type cells in the most resistant cell line. The amount of copper accumulated by resistant cells increased proportionally with the concentration of copper in the medium in which they were maintained. Gel electrophoresis and HPLC of cell extracts demonstrated the presence of a cytoplasmic 18-kilodalton, cysteine-rich protein in both the resistant and in the wild type parent cell lines. The concentration of the 18-kDa protein was directly proportional to the level of resistance and the amount of copper accumulated by each cell line. It is proposed that this 18-kDa protein is the hepatoma metallothionein, distinct from authentic rat liver metallothionein and that resistance to copper toxicity is due to separation of the metal by this protein.;The structure of the binding site for the copper accumulated by resistant cells, which is believed to be complexed by the 18-kDa Cys-rich protein, was compared to the copper binding sites in metallothionein from canine liver (LyCuLP) with X-ray spectroscopy. The k-absorption edge spectra of the resistance cells and LyCuLP were consistent with univalent copper coordination in both samples. The Fourier transform of the EXAFS data for LyCuLP showed four resolved shells of backscattering atoms. Analysis of the data showed that each copper was coordinated by four sulfur atoms at a distance of 2.27 (+OR-) 0.02 (ANGSTROM). In addition, backscattering copper atoms at 2.74 (+OR-) 0.05, 3.32 (+OR-) 0.05, and 3.88 (+OR-) 0.05 (ANGSTROM) were indicated. Analysis of EXAFS data for the copper accumulated by resistant cells indicates that the copper is coordinated by four sulfur atoms at a distance 2.28 (+OR-) 0.02 (ANGSTROM). In addition, a copper atom is located in the outer coordination shell at 3.69 (+OR-) 0.05 (ANGSTROM). The structural arrangement of the complexed copper in these cells is very similar to that observed in purified Cu-metallothionein.