Probing the mechanism of transport and compartmentalisation of polyamines in mammalian cells

Background: Many mammalian cells possess an active polyamine uptake system but little is known about the molecular mechanism of this transporter. The fate of polyamines taken up from the medium and the relationship to polyami ne homeostasis remains to be fully established. The aim of this study was t o develop a range of modified polyamines, particularly ligands incorporatin g a fluorophore, to explore the structural tolerances of the polyamine tran sport system and to probe the intracellular location of polyamines acquired from the medium. Results: We synthesised a wide range of polyamine analogues incorporating c ytotoxic agents, fluorescent chromophores and bulky substituents. All of th ese analogues have been shown to be good competitive inhibitors of spermidi ne uptake in a range of mammalian cells, Direct evidence for uptake of the fluorescent polyamine analogues and their subcellular distribution was obta ined from confocal laser scanning fluorescence microscopy, which showed tha t they accumulated in granular structures within the cytoplasm and not in t he nucleus. We demonstrated that their uptake is through the polyamine tran sport system by showing that pretreatment with DFMO, a potent inhibitor of polyamine biosynthesis, led to enhanced uptake, and cells deficient in the polyamine transport system did not accumulate these polyamine analogues. Conclusions: The polyamine transport system has a surprisingly broad struct ural tolerance. Fluorophore-containing polyamine analogues derived from the extracellular pool are located in granular structures within the cytoplasm and not to any great extent in the nuclei of mammalian cells. These observ ations might be consistent with a mechanism involving receptor-mediated end ocytosis, and the granular 'structures' seen might reflect polyamine compar tmentalisation within vesicles.