Characterization of a COS cell line deficient in polyamine transport.
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 polyamine 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 microM MGBG, which totally inhibited the growth of the wild-type cells. The transport of putrescine and spermidine was markedly decreased in the COS-MGBGr cells. The decrease in putrescine transport was mainly a result of a 14-fold decrease in Vmax, whereas the reduced spermidine uptake was due to a 3-4-fold decrease in Vmax as well as 12-fold increase in Km, indicating the existence of at least two separate transport systems. No major difference in polyamine content was seen between the parental and the COS-MGBGr 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-MGBGr 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-MGBGr 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-MGBGr cells, presumably indicating that COS cells normally excrete polyamines and then salvage them using the polyamine transport system.
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ABSTRACT: To determine whether intracellular uptake of spermine is necessary to induce haemoglobin synthesis in murine erythroleukaemia (MEL) DS 19 cells, we used single-step selection for resistance to N1,N12-bis(ethyl)spermine (BESM), a cytotoxic spermine analogue, to isolate clones deficient in polyamine transport. The cells were approximately 500-fold more resistant to BESM than parental cells and were unable to accumulate BESM, putrescine, spermidine or spermine. Addition of spermine to the polyamine-transport-deficient cells failed to induce haemoglobin synthesis. Hexamethylene-1,6-bisacetamide, a well-known differentiating agent, induced haemoglobin synthesis in both parental and resistant cells. Polyamine-transport-deficient cells transfected with DNA purified from the parental cell line were further selected for their ability to grow in the presence of alpha-difluoromethylornithine and putrescine. The transfectants had an active transport system for polyamines, and spermine added to their culture medium accumulated inside the cells and induced haemoglobin production. These findings indicate that intracellular spermine uptake is required to induce haemoglobin production in MEL cells.Biochemical Journal 01/1996; 312 ( Pt 3)(3):933-8. DOI:10.1042/bj3120933 · 4.78 Impact Factor
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ABSTRACT: The uptake and release of the natural polyamines putrescine, spermidine and spermine by mammalian cells are integral parts of the systems that regulate the intracellular concentrations of these biogenic amines according to needs. Although a general feature of all tissues, polyamine uptake into intestinal mucosa cells is perhaps the most obvious polyamine transport pathway of physiological and pathophysiological importance. Mutant cell lines lacking the ability to take up polyamines from the environment are capable of releasing polyamines. This indicates that uptake and release are functions of two different transport systems. The isolation of a transporter gene from a mammalian cell line is still lacking. Overaccumulation of polyamines is controlled by release and by a feedback regulation system that involves de novo synthesis of antizyme, a well known protein that also regulates the activity of ornithine decarboxylase. Recent work has demonstrated that Ca(2+)-signalling pathways are also involved. Although there is consensus about the importance of polyamine uptake inhibitors in the treatment of neoplastic disorders, a practically useful uptake inhibitor is still missing. However, the attempts to target tumours, and to increase the selectivity of cytotoxic agents by combining them with the polyamine structure, are promising. New, less toxic and more selective anticancer drugs can be expected from this approach.The International Journal of Biochemistry & Cell Biology 09/1996; 28(8):843-61. DOI:10.1016/1357-2725(96)00021-0 · 4.24 Impact Factor