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 polyamine homeostasis remains to be fully established. The aim of this study was to develop a range of modified polyamines, particularly ligands incorporating a fluorophore, to explore the structural tolerances of the polyamine transport system and to probe the intracellular location of polyamines acquired from the medium.


RESULTS
We synthesised a wide range of polyamine analogues incorporating cytotoxic agents, fluorescent chromophores and bulky substituents. All of these analogues have been shown to be good competitive inhibitors of spermidine uptake in a range of mammalian cells. Direct evidence for uptake of the fluorescent polyamine analogues and their subcellular distribution was obtained from confocal laser scanning fluorescence microscopy, which showed that they accumulated in granular structures within the cytoplasm and not in the nucleus. We demonstrated that their uptake is through the polyamine transport 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 structural 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 observations might be consistent with a mechanism involving receptor-mediated endocytosis, and the granular 'structures' seen might reflect polyamine compartmentalisation within vesicles.
Cullis
P M
PM
Centre for Mechanisms of Human Toxicity, Department of Chemistry, Leicester University, Leicester, LE1 7RH, UK. PMC@leicester.ac.uk
Green
R E
RE
Merson-Davies
L
L
Travis
N
N
D000818:Animals; D001693:Biological Transport, Active; D016466:CHO Cells; D002451:Cell Compartmentation; D002478:Cells, Cultured; D006224:Cricetinae; D003470:Culture Media; D000518:Eflornithine / Q000494:pharmacology; D005434:Flow Cytometry; D005456:Fluorescent Dyes; D006801:Humans; D008024:Ligands; D008856:Microscopy, Fluorescence; D008956:Models, Chemical; D008958:Models, Molecular; D011073:Polyamines / Q000378:metabolism*; D013095:Spermidine / Q000378:metabolism; D014407:Tumor Cells, Cultured
D003470:Culture Media; D005456:Fluorescent Dyes; D008024:Ligands; D011073:Polyamines; D013095:Spermidine; D000518:Eflornithine