Comparison of the cytotoxic effect of prednimustine in cultured cells with high or low levels of metallothionein.

The purpose of this investigation was to evaluate the ability of the cysteinyl-rich protein metallothionein (MT) to protect cells against the cytotoxic effects of prednimustine, an ester of chlorambucil and prednisolone. The cells studied were MT-rich substrains of murine fibroblasts (C1 1D100) and human epithelial cells (HE100), both demonstrated in an earlier report to exhibit an approximate 3-fold increase in resistance to chlorambucil compared to their parent lines (C1 1D and HE) (Endresen et al. 1983). Both in cloning and in growth rate studies the MT-rich strains proved to be significantly more resistant also to prednimustine. E.g. in cloning studies D0 for C1 1D cells (D0 = the dose of drug reducing survival to 1/e) was 8.7 micrograms/ml prednimustine, whereas D0 for C1 1D100 was 12.4 micrograms/ml, representing an approximate 1.5-fold increase in resistance, P less than 0.001, t-test. Other cloning studies revealed that prednimustine had a significantly higher cell killing activity in the resistant cells than equimolar concentrations of its components, single or in combination. Following treatment with 3H, 14C-prednimustine (3H in the prednisolone moiety, 14C in the chlorambucil moiety) and subsequent gel filtration, about 40% of the cytosolic chlorambucil eluted with MT. However, no intact prednimustine was recovered in the MT fractions. The data indicate that the MT-rich cells possess increased resistance to prednimustine due to a sequestration by MT of the alkylating moiety. Since the interaction probably does not take place until after hydrolysis, it is possible that the intact conjugate may bypass this cellular defence mechanism.