Metallothionein in cultured human epithelial cells and synovial rheumatoid fibroblasts after in vitro treatment with auranofin

Abstract

Radioimmunoassay (RIA) and reversed-phase high-pressure liquid chromatography (HPLC) were used to investigate gold-binding proteins of possible metallothionein (MT) nature occuring upon auranofin exposure of cultured human cells.An epithelial cell line (HE) and two sub-strains were examined.The HE AFsub-strain had been made resistant to 2 μmole auranofin/1 culture medium. The resistance was associated with the appearance of gold-binding substances with gel filtration characteristics like MT.The HE 100sub-starin had been made resistant to 100 μmole Cdcl 2/1 and contained high amounts of cytosolic cd-induced MT.In addition, cultured synovial fibroblasts, derived from normal (S N) and rheumatoid,(S RA) synovial tissues, were investigated. EVidence was obtained by RIA that the low molecular weight (mol.wt.6000–7000) gold binding proteins occuring in the HE AF cells and S RA cells following auranofin exposure, were of MT nature. The relative amounts of MT in the ephitelial cell lines were: HE:HE AF:HE 100 = 1:18:100. The relative amounts in the synovial fibroblasts were: S NS RA:S RA treated with auranofin = 1:3:10. The HPLC methods used were found suitable for isolation of CD-MT in the HE 100cells, but not fot the Au-MT in the HE AF cells. By HPLC,the Cd-MT in the HE 100 cells was resolved into 3 MT-2 iso-proteins exhibiting the amino acid composition typical of MT. Judged by HPLC, the MT in these cells constituted 0.4%of the cytosolic proteins.