Resistance against cis-dichlorodiammineplatinum in cultured cells with a high content of metallothionein

Resistance against the antitumor drug cis-dichlorodiammineplatinum ( cis-DDP) was tested in cultured cells: one human epithelial line derived from normal skin (HE), one mouse fibroblast line derived from L cells (Cl lD), as well as Cd-resistant substrains of both cell lines (designated HE 100 and Cl lD 100, respectively). The substrains are resistant to 100 μmol Cd/liter and contain large amounts of cytoplasmic Cd-binding proteins which, in the present study, were confirmed by spectrophotometry and amino acid analysis as being metallothioneins (MT). Counting of cells at certain intervals during a growth period with cis-DDP treatment (0.5–60 μmol/liter) showed a resistance against the drug in both MT-containing strains (HE 100 and Cl lD 100), e.g., after 4 days treatment with 5 μmol/liter the number of HE cells was 8% of controls, whereas 32% of the HE 100 cells survived ( p < 0.001, t test). The resistance in the MT-containing strain was confirmed by colony formation techniques applicable to Cl lD and Cl lD 100 cells both in soft agar and on a plastic substratum. There was a significant difference in survival ( p < 0.005, F test) over a dose range of 3–60 μmol/liter. Gel filtration showed that 70% of the Pt in cytosols from cis-DDP-treated resistant cells appeared in the MT fraction. Less than 5% was recovered in the corresponding fractions from nonresistant cells. Determination of the cell cycle phase distribution did not reveal any differences between the corresponding cell strains, nor were glutathione levels increased. A three- to fourfold increase of total sulfhydryl content in the resistant strains was accounted for by MT. This investigation makes it probable that endogeneously synthesized MT contributes to the resistance against cis-DDP.