Comparison of the Cytotoxic Effect of Prednimustine in Cultured Cells with High or Low Levels of Metallothionein

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Abstract
The ability of the cysteinyl-rich protein metallothionein (MT) to protect cells against the cytotoxic effects of prednimustine [an antitumor drug], an ester of chlorambucil and prednisolone, was evaluated. The cells studied were MT-rich substrains of murine fibroblasts (C1 1D100) and human epithelial cells (HE100); both previously exhibited an approximate 3-fold increase in resistance to chlorambucil compared to their parent lines (C1 1D and HE). Both in cloning and in growth rate studies the MT-rich strains were significantly more resistant also to prednimustine. In cloning studies, D0 for C1 1D cells (D0 = the dose of drug reducing survival to 1/l) was 8.7 .mu.g/ml prednimustine, whereas D0 for C1 1D100 was 12.4 .mu.g/ml, representing an approximate 1.5-fold increase in resistance, P < 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. No intact prednimustine was recovered in the MT fractions. The MT-rich cells evidently 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, the intact conjugate may bypass this cellular defense mechanism.