CHARACTERIZATION OF A COS CELL-LINE DEFICIENT IN POLYAMINE TRANSPORT

In the present study, we describe the isolation and characterization o f a COS cell line deficient in polyamine uptake that may provide an im portant tool for the molecular cloning of polyamine transporter(s). Th e cells were selected by isolation for resistance against the cytotoxi c agent, methylglyoxal bis(guanylhydrazone) (MGBG), which is entering the cells using the same transport system as the polyamines. The isola ted cell line was capable of growing in the presence of 100 mu M MGBG, which totally inhibited the growth of the wild-type cells. The transp ort of putrescine and spermidine was markedly decreased in the COS-MGB G(r) cells. The decrease in putrescine transport was mainly a result o f 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 parenta l and the COS-MGBG(r) cells when grown without MGBG. In the presence o f MGBG, both cell lines exhibited an increase in putrescine content. T reatment with MGBG also resulted in a decrease in spermidine and sperm ine contents in the wild-type cells. In the COS-MGBG(r) cells, on the other hand, there were no statistically significant effects on the spe rmidine and spermine contents by MGBG treatment. In the wild-type cell s, depletion of polyamines, e.g., by treatment with the ornithine deca rboxylase inhibitor 2-difluoromethylornithine (DFMO), stimulated the u ptake of polyamines (3-7-fold), whereas in the COS-MGBG(r) cells the e ffect of DFMO treatment on polyamine transport was only minor. In cont rast to the growth-medium of the wild-type cells, large amounts of pol yamines accumulated in the medium of the COS-MGBG(r) cells, presumably indicating that COS cells normally excrete polyamines and then salvag e them using the polyamine transport system.