Enhancement of Hyperthermia-induced Cytotoxicity by Polyamines¹

Chinese hamster ovary cells growing exponentially in medium supplemented with 20% fetal calf serum were exposed to 43° hyperthermia for varying periods of time. During these intervals, the membranes of treated cells became leaky to polyamines, with intracellular polyamine levels decreasing and extracellular levels increasing as a function of time at 43°. Addition to the culture medium during treatment intervals of exogenous polyamine hydrochlorides, at concentrations of 10⁻³ m, resulted in enhanced thermal cell killing. Titration of the treatment medium with a range of polyamine concentrations demonstrated that exogenous spermidine and spermine concentrations as low as 10⁻⁵ m resulted in a substantial (greater than 1 log) increase in 43°-induced cell killing for treatment intervals of 90 min.

Dimensional analysis of these data, based on assumptions explained in the text, demonstrated that the degree of enhancement of cell kill was a direct function of the number of carboncarbon bonds, the net charge of the polyamine added, and the concentration. Potentiation was thus ranked spermine > spermidine > putrescine. It was found that the activity of ornithine decarboxylase, the rate-limiting enzyme in polyamine biosynthesis, was decreased by exposure of whole cells to 43°. The effect of exogenous polyamines on thermal cell killing, however, does not seem to act by inhibiting this enzyme. This conclusion follows from experiments which demonstrate that ornithine decarboxylase activity is inhibited by Puromycin at either 37° or 43°; however, cell survival is unaffected by Puromycin treatments concurrent with 43° doses. When Chinese hamster cultures were maintained and then treated in medium containing only 10% fetal calf serum, cells were slightly more sensitive to 43° cytotoxicity. However, exogenous polyamine addition was now much less effective at enhancing hyperthermia-produced cell killing. At equimolar concentrations, the hyperthermia-potentiating action of the polyamines was specific to these polycations, inasmuch as additions of KCl, MgCl₂, and CaCl₂ were much less effective at enhancing hyperthermic cell killing. Along with other results, these data suggest that (a) the polyamines act specifically to sensitize cells to the cytotoxic action(s) of hyperthermia and (b) this enhancement occurs primarily through polyamine action on the cell membrane.