bis(benzyl)polyamine analogues are substrates for a mammalian cell-transport system which is distinct from the polyamine-transport system

Bis(benzyl)polyamine analogues (e.g. NN′-bis(3-[(phenylmethyl)amino]propyl)-1,8-diamino-octane [C6H5CH2NH-(CH2)3NH(CH2)8NH(CH2)3NHCH2C6H5]) have previously been shown to regulate polyamine biosynthesis and growth of rat hepatoma (HTC) cells. Saturable uptake of the analogues, the ability of other bis(benzyl)polyamine analogues to compete for this uptake and the trans-acceleration of this uptake in pre-loaded cells indicate that these novel compounds are accumulated through the action of a transport system in HTC cells. A mutant Chinese-hamster-ovary (CHO) cell line, CHOMG, which lacks a functional polyamine-transport system, exhibited saturable bis(benzyl)polyamine uptake identical with that observed in the parental CHO cells, which have normal polyamine transport. The uptake of the analogue by both CHOMG and CHO cells was competitively inhibited by other bis(benzyl)polyamine analogues, but was insensitive to excess spermine. Treatment with alpha-difluoromethylornithine, an inhibitor of polyamine biosynthesis, resulted in the enhancement of spermine uptake in CHO cells but did not alter the uptake of a bis(benzyl)polyamine analogue by either CHO or CHOMG cells. Thus it appears that bis(benzyl)polyamine analogues are substrates for a mammalian-cell-transport system distinct from the polyamine-transport system.

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Properties and physiological function of the polyamine transport system

AJP Cell Physiology ◽

10.1152/ajpcell.1989.257.3.c545 ◽

1989 ◽

Vol 257 (3) ◽

pp. C545-C553 ◽

Cited By ~ 46

Author(s):

T. L. Byers ◽

A. E. Pegg

Keyword(s):

Transport System ◽

Cho Cells ◽

Chinese Hamster ◽

Selection Procedure ◽

Cell Types ◽

Decarboxylase Activity ◽

Polyamine Analogues ◽

Polyamine Transport System ◽

Polyamine Transport ◽

Genes Encoding

Polyamine transport was examined in Chinese hamster ovary (CHO) cells because of the unique potential these cells hold for utilizing genetic approaches to study the mechanisms of polyamine transport, its regulation, and its function. Parental (control) CHO cells were shown to contain a polyamine transport system with characteristics consistent with polyamine-uptake properties described in other cell types. Polyamines appear to cross the plasma membrane via an energy-requiring transport system specific for putrescine, spermidine, spermine, and their analogues. A mutant line, CHOMG, selected for resistance to the toxicity of methylglyoxal bis(guanylhydrazone), was shown to lack a functional polyamine transport system. CHOMG cells provided the negative controls necessary to examine the role of polyamine transport in maintenance of intracellular polyamine levels and in the regulation of the polyamine metabolic enzymes. It was found that the repression of ornithine decarboxylase activity by polyamines and the induction of spermidine/spermine-N1 acetyltransferase by polyamine analogues including bis(ethyl)spermine derivatives required the presence of a functional polyamine transport system. The CHO-CHOMG model was also shown to provide a means for establishing the importance of the polyamine transport system in the toxicity of polyamine analogues. The inability of alpha-difluoromethylornithine-treated CHOMG cells to utilize extracellular polyamines to replenish depleted intracellular polyamine levels suggested a means by which polyamine transport-positive cells may be identified. Such a selection procedure will permit the use of CHOMG cells in the isolation of genes encoding proteins involved in polyamine transport.

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Expression of a human gene for polyamine transport in Chinese-hamster ovary cells

Biochemical Journal ◽

10.1042/bj2630745 ◽

1989 ◽

Vol 263 (3) ◽

pp. 745-752 ◽

Cited By ~ 29

Author(s):

T L Byers ◽

R Wechter ◽

M E Nuttall ◽

A E Pegg

Keyword(s):

Transport System ◽

Chinese Hamster Ovary ◽

Cho Cells ◽

Chinese Hamster Ovary Cells ◽

Mutant Cell ◽

Molecular Genetic ◽

Chinese Hamster ◽

Transport Systems ◽

Polyamine Transport System ◽

Polyamine Transport

A molecular-genetic approach towards isolating mammalian polyamine-transport genes and their encoded proteins was devised involving the production of Chinese-hamster ovary (CHO) cells expressing a human polyamine-transport protein. CHO cells and a polyamine-transport-deficient CHO mutant cell line (CHOMG) were equally sensitive to the antiproliferative effects of alpha-difluoromethylornithine (DFMO), which blocked endogenous polyamine synthesis. Exposure to exogenous polyamines increased intracellular polyamine levels and reversed this DFMO-induced cytostasis in the CHO cells, but not in the CHOMG cells. CHOMG cells were therefore transfected with human DNA (isolated from HT-29 colon carcinoma cells) and cells expressing the human polyamine-transport system were identified by the ability of these cells to grow in a medium containing DFMO and polyamines. A number of different positive clones were identified and shown to have the capacity for polyamine uptake and an increased sensitivity to the toxic effects of the polyamine analogue methylglyoxal bis(guanylhydrazone). Differences in these properties between the clones are consistent with a multiplicity of polyamine-transport systems. Some clones also showed a change in growth characteristics, which may indicate a relationship between genes involved in the polyamine-transport system and in cell proliferation.

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Effects of the S-adenosylmethionine decarboxylase inhibitor, 5′-{[(Z)-4-amino-2-butenyl]methylamino)-5′-deoxyadenosine, on cell growth and polyamine metabolism and transport in Chinese hamster ovary cell cultures

Biochemical Journal ◽

10.1042/bj3030089 ◽

1994 ◽

Vol 303 (1) ◽

pp. 89-96 ◽

Cited By ~ 15

Author(s):

T L Byers ◽

R S Wechter ◽

R H Hu ◽

A E Pegg

Keyword(s):

Cell Growth ◽

Transport System ◽

Chinese Hamster Ovary ◽

Cho Cells ◽

Chinese Hamster ◽

Ovary Cell ◽

Polyamine Metabolism ◽

Decarboxylase Inhibitor ◽

Transport Activity ◽

Polyamine Transport

The regulation of polyamine transport and the roles of polyamine transport and synthesis in cell growth were investigated using cultured Chinese hamster ovary (CHO) cells and CHOMG cells which are mutants lacking polyamine-transport activity. Metabolically stable methylated polyamine analogues were used to measure polyamine accumulation, and the irreversible S-adenosyl-L-methionine decarboxylase inhibitor, 5′-([(Z)-4-amino-2-butenyl]methylamino)-5′-deoxyadenosine (AbeAdo), was used to inhibit synthesis. Exposure to AbeAdo lead to a dose-dependent decrease in growth for both cell lines, although CHOMG cells were more sensitive. Intracellular putrescine levels were greatly increased in AbeAdo-treated CHO cells and to a lesser extent in CHOMG cells, whereas intracellular spermidine and spermine levels were substantially reduced in both. Treatment with AbeAdo increased putrescine content in the culture medium to a much greater extent in CHOMG cultures indicating that a portion of the excess putrescine synthesized in response to AbeAdo treatment is excreted, but that CHO cells salvage this putrescine whereas it is lost to CHOMG cells which cannot take up polyamines. AbeAdo treatment increased polyamine transport into CHO cells despite high intracellular putrescine, suggesting that spermidine and/or spermine, and not putrescine, are the major factors regulating transport activity. The accumulation of either 1-methylspermidine or 1,12-dimethylspermine was significantly increased by AbeAdo treatment. Accumulation was increased even further when protein synthesis was blocked by cycloheximide, indicating that a short-lived protein is involved in the regulation of polyamine uptake. In the presence of cycloheximide and AbeAdo or alpha-difluoromethylornithine, methylated polyamine derivatives accumulated to very high levels leading to cell death. These results show that the polyamine-transport system plays an important role in retaining intracellular polyamines and that down-regulation of the transport system in response to increased intracellular polyamine content is necessary to prevent accumulation of toxic levels of polyamines.

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ATP13A3 is a major component of the enigmatic mammalian polyamine transport system

Journal of Biological Chemistry ◽

10.1074/jbc.ra120.013908 ◽

2020 ◽

pp. jbc.RA120.013908

Author(s):

Norin Nabil Hamouda ◽

Chris Van den Haute ◽

Roeland Vanhoutte ◽

Ragna Sannerud ◽

Mujahid Azfar ◽

...

Keyword(s):

Transport System ◽

Mammalian Cells ◽

Polyamine Biosynthesis ◽

Polyamine Transport System ◽

Polyamine Transport ◽

Mrna And Protein Expression ◽

Transport Inhibitors ◽

Pulmonary Arterial ◽

Polyamine Uptake ◽

P Type

Polyamines, such as putrescine, spermidine and spermine, are physiologically important polycations, but the transporters responsible for their uptake in mammalian cells remain poorly characterized. Here, we reveal a new component of the mammalian polyamine transport system (PTS) using CHO-MG cells, a widely used model to study alternative polyamine uptake routes and characterize polyamine transport inhibitors for therapy. CHO-MG cells present polyamine uptake deficiency and resistance to a toxic polyamine biosynthesis inhibitor MGBG (methylglyoxal bis- (guanylhydrazone)), but the molecular defects responsible for these cellular characteristics remain unknown. By genome sequencing of CHO-MG cells, we identified mutations in an unexplored gene, ATP13A3, and found disturbed mRNA and protein expression. ATP13A3 encodes for an orphan P5B-ATPase (ATP13A3), a P-type transport ATPase that represents a candidate polyamine transporter. Interestingly, ATP13A3 complemented the putrescine transport deficiency and MGBG resistance of CHO-MG cells, whereas its knockdown in wild-type cells induced a CHO-MG phenotype, demonstrating a decrease in putrescine uptake and MGBG sensitivity. Taken together, our findings identify ATP13A3 as a major component of the mammalian PTS that confers sensitivity to MGBG and that has been previously genetically linked with pulmonary arterial hypertension.

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Polyamine transport system-targeted nanometric micelles assembled from epipodophyllotoxin-amphiphiles

Chemical Communications ◽

10.1039/c9cc07883e ◽

2019 ◽

Vol 55 (99) ◽

pp. 14968-14971 ◽

Cited By ~ 2

Author(s):

Julien Alliot ◽

Ioanna Theodorou ◽

Frédéric Ducongé ◽

Edmond Gravel ◽

Eric Doris

Keyword(s):

Transport System ◽

Biologically Active ◽

Drug Conjugates ◽

Amphiphilic Drug ◽

Polyamine Transport System ◽

Polyamine Transport

Micelle-forming amphiphilic drug conjugates were synthesized starting from a biologically active epipodophyllotoxin derivative which was covalently inserted in between a hydrophilic targeting spermine unit, and a hydrophobic stearyl chain.

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Streptomycin Uptake via an Inducible Polyamine Transport System in Escherichia coli

European Journal of Biochemistry ◽

10.1111/j.1432-1033.1978.tb12316.x ◽

1978 ◽

Vol 86 (2) ◽

pp. 345-351 ◽

Cited By ~ 31

Author(s):

Joachim-Volker HOLTJE

Keyword(s):

Escherichia Coli ◽

Transport System ◽

Polyamine Transport System ◽

Polyamine Transport

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Antizyme induction by polyamine analogues as a factor of cell growth inhibition

Biochemical Journal ◽

10.1042/bj20011612 ◽

2002 ◽

Vol 366 (2) ◽

pp. 663-671 ◽

Cited By ~ 36

Author(s):

John L.A. MITCHELL ◽

Aviva LEYSER ◽

Michelle S. HOLTORFF ◽

Jill S. BATES ◽

Benjamin FRYDMAN ◽

...

Keyword(s):

Cell Growth ◽

Growth Inhibition ◽

Feedback Inhibition ◽

Conformationally Constrained ◽

Polyamine Analogues ◽

Polyamine Transport ◽

Mammalian Cell Growth ◽

Htc Cells ◽

Cell Growth And Viability ◽

Selection Of

The polyamines spermidine and spermine and their diamine precursor putrescine are essential for mammalian cell growth and viability, and strategies are sought for reducing polyamine levels in order to inhibit cancer growth. Several structural analogues of the polyamines have been found to decrease natural polyamine levels and inhibit cell growth, probably by stimulating normal feedback mechanisms. In the present study, a large selection of spermine analogues has been tested for their effectiveness in inducing the production of antizyme, a key protein in feedback inhibition of putrescine synthesis and cellular polyamine uptake. Bisethylnorspermine, bisethylhomospermine, 1,19-bis-(ethylamino)-5,10,15-triazanonadecane, longer oligoamine constructs and many conformationally constrained analogues of these compounds were found to stimulate antizyme synthesis to different levels in rat liver HTC cells, with some producing far more antizyme than the natural polyamine spermine. Uptake of the tested compounds was found to be dependent on, and limited by, the polyamine transport system, for which all these have approximately equal affinity. These analogues differed in their ability to inhibit HTC cell growth during 3days of exposure, and this ability correlated with their antizyme-inducing potential. This is the first direct evidence that antizyme is induced by several polyamine analogues. Selection of analogues with this potential may be an effective strategy for maximizing polyamine deprivation and growth inhibition.

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