Abstract

In the present study, we describe the isolation and characterization of a COS cell line deficient in polyamine uptake that may provide an important tool for the molecular cloning of polymaine transporter(s). The cells were selected by isolation for resistance against the cytotoxic agent, methylglyoxal bis(guanylhydrazone) (MGBG), which is entering the cells using the same transport system as the polyamines. The isolated cell line was capable of growing in the presence of 100 μM MGBG, which totally inhibited the growth of the wild-type cells. The transport of putrescine and spermidine was markedly decreased in the COS-MGBG r cells. The decrease in putrescine transport was mainly a result of a 14-fold decrease in V max , wheras the reduced spermidine uptake was due to a 3–4-fold decrease in V max as well a 12-fold increase in K m , indicating the existence of at least two separate transport systems. No major difference in polyamine content was seen between the parental and the COS-MGBG r cells when grown without MGBG. In the presence of MGBG, both cell lines exhibited an increase in putrescine content. Treatment with MGBG also resulted in a decrease in spermidine and spermine contents in the wild-type cells. In the COS-MGBG r cells, on the other hand, there were no statistically significant effects on the spermidine and spermine contents by MGBG treatment. In the wild-type cells, depletion of polyamines, e.g., by treatment with the ornithine decarboxylase inhibitor 2-difluoromethylornithine (DFMO), stimulated the uptake of polyamines (3–7-fold), whereas in the COS-MGBG r cells the effect of DFMO treatment on polyamine transport was only minor. In contrast to the growth-medium of the wild-type cells, large amounts of polyamines accumulated in the medium of the COS-MGBG r cells, presumably indicating that COS cells normally excrete polyamines and then salvage them using the polyamine transport system.

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