High Concentration of Cytoplasmic Metallothionein Increased Resistance to Chlorambucil in Cultured Cells with a Updated Version

Cultured cells with elevated levels of cytoplasmic metalloidionein (MT), a small-molecular-weight protein extremely rich in cysteine. showed resistance against the alkylating drug chlorambucil. One human epithelial line (HE) and one mouse fibroblast line (CI 1D) were used, as well as substrains of both cell lines (HE10oand CI 1D10o).The substrains contain large amounts of MT. Dose-survival curves obtained from cloning studies with the murine cells after exposure to Chlorambucil for 1 hr revealed that CI 1Dioo was approximately 3-fold more resistant than were CI 1D cells (Do = 32.9 and 11.6 /tg/ml, respectively); this difference was highly significant (p < 0.001 ; f test). In growth rate studies, both MT-containing strains showed resistance against Chloram bucil over the total dose range of 0.5 to 100 u.g/ml compared to the parent strains. After 4 days treatment with 25 ^g/ml, the growth inhibition of CI 1D was 67%, but only 13% in CI 1D100For HE and HEioo, 50 u.g/m\ gave growth inhibitions of 83 and 65%, respectively. Following 1and 24-hr incubations of cells with [14C]chlorambucil and subsequent ultracentrifugation and gel filtration of the cytosols, about 20 to 40% of the 14C-derived radioactivity coeluted with the MT from the resistant strains. The results indicate that intracellular MT sequesters Chlorambucil or its toxic metabolites and that the protein thus contributes to the resistance against this drug.