Inhibition of putrescine uptake by polypyridinium quaternary salts in B16 melanoma cells treated with difluoromethylornithine

Minchin, Rodney F.; Martin, Roger L.; Summers, L. A.; Ilett, Kenneth F.

doi:10.1042/bj2620391

Cited by 16 publications

(10 citation statements)

References 13 publications

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“…Structure-activity studies from our laboratory [15] and from that ofPorter et al [I91 show that ligands interacting with the polyamine-transport system(s) require specific structural features. Using a series of polypyridinium quaternary salts that competitively inhibit putrescine transport, we show here that the structural requirement for interaction with putrescine uptake differs to that for interaction with spermidine uptake.…”

Section: Discussionmentioning

confidence: 99%

“…Comparisons between means were made using the Student's t-test assuming significance at P <0.05. Inhibition constants (Ki) were estimated as described elsewhere [15].…”

Section: Methodsmentioning

confidence: 99%

“…We investigated the inhibition of putrescine and spermidine uptake by a series of polypyridinium analogs which we have previously reported to competitively inhibit putrescine accumulation in B16 cells [15]. Each of the compounds tested exhibited classical competitive inhibition kinetics for both Table 3.…”

Section: Inhibition Of Polyamine Uptake Hy Polypyridinium Saltsmentioning

confidence: 99%

“…After 24 h, the cells were washed and treated for a further 24 h with F2MeOrn in complete medium, or with fresh medium alone. The uptake of putrescine or spermidine was then measured by incubating the cells at 37°C with radiolabeled polyamine (0.05 mCi/well) and [3H]sucrose (extracellular marker; 0.2 mCi/well), as described elsewhere [15]. For the Na'-dependence studies, control or F2MeOrn-treated cells were incubated with ['4C]polyamine in 20 mM Tris/HCl buffer (pH 7.4) containing 0.5 mM CaCI,, 0.5 mM MgS04 and either 1 SO mM NaCl or 150 mM LiCI.…”

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confidence: 99%

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Evidence for the existence of distinct transporters for the polyamines putrescine and spermidine in B16 melanoma cells

Minchin

Raso

Martin

et al. 1991

European Journal of Biochemistry

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The uptake of intracellular putrescine and spermidine was examined in B16 melanoma cells. It was found that difluoromethylornithine preferentially induced putrescine transport (28-fold) compared to that for spermidine (3.5-fold). Putrescine uptake was partially Na' dependent, whereas spermidine uptake was not. Inhibition studies with the two polyamines showed that putrescine was a poor competitive inhibitor of spermidine uptake, exhibiting a Ki of 69-7.5 pM, whereas the estimated K, for putrescine uptake was only 5.36 pM. By contrast, spermidine inhibition of putrescine transport produced a non-linear Eadie-Scatchard plot suggesting that putrescine was taken up by a spermidine-sensitive and a spermidine-insensitive process. The estimated spermidine Ki for inhibition of the spermidine-sensitive process was 0.12.5 pM. Using a series of polypyridinium quaternary salts to inhibit transport, no correlation between inhibition of putrescine uptake and inhibition of spermidine uptake was seen. Finally, the photoaffinity label, 1 ,12-di(N5-azido-2-nitrobenzoy1)spermine selectively inactivated the putrescine transporter(s) without affecting spermidine uptake. From these observations, it was concluded that multiple polyamine transporters are present on B16 melanoma cells and that separate, distinct transporter(s) account for the uptake of putrescine and spermidine in this cell-line following induction with difluoromethylornithine. The present of different transporters for the two polyamines indicates that expression of uptake activity for putrescine and spermidine may be under separate cellular control.The uptake of extracellular polyamines has been shown to occur in many different cell types [I -81. The system responsible for uptake is highly inducible by a number of different stimuli [l, 2, 5, 9, 101 and may also be regulated by the extent of cell differentiation [ll]. Several recent studies have shown that the uptake of polyamines into mammalian cells can not be explained by a single transport system [8,12, 131. One of these studies has used various inhibitors of putrescine and spermidine uptake to show the existence of a non-homogeneous uptake system [12] while another has demonstrated non-Michaelis-Menten uptake behaviour that suggests the presence of multiple transporters [8]. Further support for multiple transporters has come from studies with cells cloned for the human polyamine transporter [14]. In this work, clones with different uptake characteristics were isolated and identified. However, it is still unclear whether different transporters exist for each of the polyamines, whether the activities of these transporters are under common cellular control, and to what extent polyamine uptake by the different systems contributes to overall intracellular polyamine homeostasis.We have previously shown [1.5] that the uptake of putrescine into B16 melanoma cells is highly inducible by the ornithine decarboxylase inhibitor difluoromethylornithine (F,MeOrn). This increase in uptake is a common phenomenon Correspondence to ...

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Section: Discussionmentioning

confidence: 99%

“…Comparisons between means were made using the Student's t-test assuming significance at P <0.05. Inhibition constants (Ki) were estimated as described elsewhere [15].…”

Section: Methodsmentioning

confidence: 99%

Section: Inhibition Of Polyamine Uptake Hy Polypyridinium Saltsmentioning

confidence: 99%

mentioning

confidence: 99%

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Evidence for the existence of distinct transporters for the polyamines putrescine and spermidine in B16 melanoma cells

Minchin

Raso

Martin

et al. 1991

European Journal of Biochemistry

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“…Indeed, N 4 -alkylated spermidine derivatives are much better competitors of spermidine uptake than their N 4 -acyl counterparts in mouse leukemia cells, suggesting that charged secondary amino groups are important for interaction with the polyamine carrier (28). However, the latter argument cannot account for the fact that aliphatic ␣,-diamines with at least 6 or 7 methylene groups have an affinity comparable with that of spermidine (23,27,30,40,47). A more likely explanation for the poor affinity of polyamines bearing an acyl side chain might be the steric hindrance due to the amide group, which restricts freedom of rotation around the adjacent carbon and nitrogen atoms.…”

Section: Effect Of Desc On Prevention Of Dfmo-induced Growthmentioning

confidence: 92%

2,2′-Dithiobis(N-ethyl-spermine-5-carboxamide) Is a High Affinity, Membrane-impermeant Antagonist of the Mammalian Polyamine Transport System

Huber

Pelletier

Torossian

et al. 1996

Journal of Biological Chemistry

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Cell cycle–dependent uptake of putrescine and its importance in regulating cell cycle phase transition in cultured adult mouse hepatocytes

1991

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Previous studies in which investigators have induced the rate of polyamine uptake in vitro have used either inhibitors of polyamine biosynthesis or growth factors that induce cell proliferation. Recently, however, we have described the induction of putrescine uptake in cultured adult mouse hepatocytes and have shown that uptake is independent of both intracellular polyamine levels and proliferation. Although proliferation was not apparent in those studies, data suggested that, after isolation, the cells entered G1 of the cell cycle. In this study, we have examined whether the induction of putrescine uptake is a function of entry into the cell cycle and whether uptake activity is essential for optimal progression into the S phase. Using ribonuclease reductase subunit M1 as a marker of entry into the cell cycle, we have shown that hepatocytes enter G1 during the first 4 hr of culture. Both putrescine uptake and ornithine decarboxylase activity increased as the cells entered G1. Treatment of the cells with retinoic acid (10 to 33 mumol/L) prevented them from entering G1 and also inhibited the induction of the putrescine transporter by up to 90%. In contrast, initiation of G1 to S phase transition markedly down-regulated the activity of the transporter. Thus induction of the putrescine transporter in isolated hepatocytes appears to be a G1-specific event. Culturing the hepatocytes in the presence of 1,1'-bis[3-(1'-methyl-[4,4'-bipyridinium]-1-yl)-propyl]- 4,4'-bipyridinium, a potent competitive inhibitor of putrescine uptake, resulted in a 47% decrease in intracellular putrescine.(ABSTRACT TRUNCATED AT 250 WORDS)

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Inhibition of putrescine uptake by polypyridinium quaternary salts in B16 melanoma cells treated with difluoromethylornithine

Cited by 16 publications

References 13 publications

Evidence for the existence of distinct transporters for the polyamines putrescine and spermidine in B16 melanoma cells

Evidence for the existence of distinct transporters for the polyamines putrescine and spermidine in B16 melanoma cells

2,2′-Dithiobis(N-ethyl-spermine-5-carboxamide) Is a High Affinity, Membrane-impermeant Antagonist of the Mammalian Polyamine Transport System

Cell cycle–dependent uptake of putrescine and its importance in regulating cell cycle phase transition in cultured adult mouse hepatocytes

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