Temperature Dependence Studies of Adriamycin Uptake and Cytotoxicity1

In order to learn whether a direct relationship exists between cellular uptake and cytotoxicity of Adriamycin, we have compared the tempera ture dependencies of these two processes in LI 210 cells. We find that the equilibrium concentration of drug taken inside the cells varies smoothly with temperature between 37°Cand 0°C. Even at ()"(', however, there is still measurable uptake of the drug into cells. The cytotoxicity index (cloning in soft agar), on the other hand, does not parallel the uptake temperature dependence. Cytotoxicity rapidly diminishes as the temperature of drug exposure is lowered; at all temperatures below about 20°C, Adriamycin is not active. In contrast, other cytotoxic anticancer drugs like mitomycin C, bleomycin, and ARK 73-21 (a platinum analogue) retain cytotoxic potency at low temperatures. The inability of Adriamycin to kill cells at low temperature persists even at very high drug concentra tions where substantial quantities of drug enter the cells. The low temperature impotence is not a result of inoperative enzymes which could metabolize Adriamycin to an alkylating species or electron donor to oxygen, since NADH and NADPH dependent reducÃ-aseactivities show linear Arrhenius behavior with no indication of low temperature inactivity. Using purified L1210 plasma membranes with bound Adriamycin as a fluorescence polarization probe, we find evidence of a phase change in the cell surface occurring at the same temperature as the loss of biological activity (=20"( ). We conclude that Adriamycin induced cytotoxicity is not dictated solely by uptake, in apparent contradiction with mechanisms requiring an intracellular target. Moreover, the loss of cytotoxicity below 20°Cappears to be linked to a structural change in the cell surface membrane, supporting a role other than transport for this membrane in transducing Adriamycin action.