Metallothionein Gene Expression in the Cisplatin Resistant Cells and the Effect of Cellular Endogeneous Metal

Abstract

It has been reported that the cellular Cu²⁺ ion and metallothionein (MT) expression contribute to the acquisition of cisplatin (CDDP)-resistance phenotype in the tumor cells. However, the mechanism of cellular MT induction by CDDP has not been clear. Previously, we reported that the nuclear proteins prepared from HeLa cells with the elevated MT level after treatment of 12-O-tetradecanoyl phorbol-13-acetate (TPA) or the tumor necrosis factor α (TNFα) bind to the metal responsive elements (MREs) as the regulatory DNA sequences in human MT-IIA gene promoter, in the presence of Cu²⁺ ion. In this study, the copper effect on binding of MREs to the nuclear proteins extracted from CDDP-resistant HeLa cells which were maintained for 3 months in Dulbecco’s modified Eagle (DMEM) medium supplemented with 10 % FBS and 1 μM or 5 μM CDDP. The expression level of MT-IIA mRNA in CDDP-resistant cells was compared with that of the normal HeLa cells, the resistant cells expressed more MT-IIA mRNA than the HeLa cells. The plasmid constructed the human MT-IIA promoter gene in puC19 were digested with the restriction enzymes. The bindings of linear DNA fragments produced by the enzyme digestion to the nuclear extracts prepared from the CDDP-resistant cells or the cells treated with 1 μM CDDP for 1 h in the presence of Cu²⁺ ion, was examined by the gel-mobility shift assay. It was suggested that Cu²⁺ ion may play an important role in the binding of the proteins in nuclear extracts prepared these cells to 3’-end DNA fragments of MRE_d.