Effect of inhibitors of S-Adenosylmethionine decarboxylase on polyamine content and growth of L1210 cells

Biochemistry ◽  
1988 ◽  
Vol 27 (5) ◽  
pp. 1408-1415 ◽  
Author(s):  
Anthony E. Pegg ◽  
Daniel B. Jones ◽  
John A. Secrist
Keyword(s):  
Adenosylmethionine Decarboxylase ◽  
L1210 Cells ◽  
Polyamine Content ◽  
Effect Of Inhibitors

scholarly journals Effect of inhibitors of S-adenosylmethionine decarboxylase on the contents of ornithine decarboxylase and S-adenosylmethionine decarboxylase in L1210 cells

1988 ◽  
Vol 254 (1) ◽  
pp. 45-50 ◽  
Author(s):  
R Madhubala ◽  
J A Secrist ◽  
A E Pegg
Keyword(s):  
Ornithine Decarboxylase ◽  
Protein A ◽  
Protein Translation ◽  
Adenosylmethionine Decarboxylase ◽  
Enzyme Content ◽  
L1210 Cells ◽  
Mrna Content ◽  
Important Regulator ◽  
Substantial Decline ◽  
Effect Of Inhibitors

Treatment of L1210 cells with either of two inhibitors of S-adenosylmethionine decarboxylase (AdoMetDC), namely 5′-deoxy-5′-[N-methyl-N-[2-(amino-oxy)ethyl])aminoadenosine or 5′-deoxy-5′-[N-methyl-N-(3-hydrazinopropyl)]aminoadenosine, produced a large increase in the amount of ornithine decarboxylase (ODC) protein. The increased enzyme content was due to a decreased rate of degradation of the protein and to an increased rate of synthesis, but there was no change in its mRNA content. The inhibitors led to a substantial decline in the amounts of intracellular spermidine and spermine, but to a big increase in the amount of putrescine. These results indicate that the content of ODC is negatively regulated by spermidine and spermine at the levels of protein translation and turnover, but that putrescine is much less effective in bringing about this repression. Addition of either spermidine or spermine to the cells treated with the AdoMetDC inhibitors led to a decrease in ODC activity, indicating that either polyamine can bring about this effect, but spermidine produced effects at concentrations similar to those found in the control cells and appears to be the physiologically important regulator. The content of AdoMetDC protein (measured by radioimmunoassay) was also increased by these inhibitors, and a small increase in its mRNA content was observed, but this was insufficient to account for the increase in protein. A substantial stabilization of AdoMetDC occurred in these cells, contributing to the increased enzyme content, but an increase in the rate of translation cannot be ruled out.

scholarly journals Regulation of S-adenosylmethionine decarboxylase activity by alterations in the intracellular polyamine content

1992 ◽  
Vol 288 (2) ◽  
pp. 511-518 ◽  
Author(s):  
L M Shantz ◽  
I Holm ◽  
O A Jänne ◽  
A E Pegg
Keyword(s):  
Cho Cells ◽  
Translational Regulation ◽  
Decarboxylase Activity ◽  
Origin Of Replication ◽  
Sv40 Promoter ◽  
Polyamine Biosynthesis ◽  
Adenosylmethionine Decarboxylase ◽  
Mammalian Cell Lines ◽  
Sv40 Origin ◽  
Polyamine Content

The effects of addition of exogenous spermidine and spermine and of two inhibitors of polyamine biosynthesis, alpha-difluoromethylornithine (DFMO), which decreases spermidine concentrations, and n-butyl-1,3-diaminopropane, which depletes spermine, on the expression of S-adenosylmethionine decarboxylase (AdoMetDC) activity were studied in mammalian cell lines (HT29, CHO and COS-7). AdoMetDC levels were inversely related to the polyamine content, and spermine was the more potent repressor of AdoMetDC activity, but only spermidine affected the amount of AdoMetDC mRNA. Transfection of COS-7 cells or CHO cells with plasmid constructs containing a chloramphenicol acetyltransferase (CAT) reporter gene driven by portions of the AdoMetDC promoter region indicated that CAT expression was altered by spermidine, but not by spermine, suggesting that there is a spermidine-responsive element in this promoter. Transient transfection of COS-7 cells with pSAMh1, a plasmid containing the AdoMetDC cDNA in a vector with the SV40 promoter and origin of replication, led to a large increase in AdoMetDC expression. Although treatment of COS-7 cells with n-butyl-1,3-diaminopropane greatly increased endogenous AdoMetDC activity, the spermine depletion brought about by this inhibitor did not stimulate AdoMetDC expression from pSAMh1. The pSAMh1 cDNA is missing 72 nucleotides from the 5′ end of the AdoMetDC mRNA, and it is possible that translational regulation by spermine involves this region. The expression of AdoMetDC from pSAMh1 in COS-7 cells was greatly inhibited by DFMO treatment, although endogenous AdoMetDC activity was increased. The expression of other plasmids containing the SV40 origin of replication was also inhibited by DFMO in COS-7 cells, but not in CHO cells. DFMO treatment did not interfere with the expression of plasmids driven by the RSV promoter. These results suggest that low spermidine levels interfere with the replication of plasmids containing the SV40 origin of replication.

scholarly journals Selective regulation of S-adenosylmethionine decarboxylase activity by the spermine analogue 6-spermyne

1988 ◽  
Vol 254 (2) ◽  
pp. 337-342 ◽  
Author(s):  
C W Porter ◽  
J McManis ◽  
D Lee ◽  
R J Bergeron
Keyword(s):  
Binding Sites ◽  
Enzyme Activities ◽  
Decarboxylase Activity ◽  
Polyamine Biosynthesis ◽  
Adenosylmethionine Decarboxylase ◽  
L1210 Cells ◽  
Initial Decrease ◽  
Function Correlation ◽  
Apparent Ability ◽  
Interesting Structure

Polyamine-biosynthesis activity is known to be negatively regulated by intracellular polyamine pools. Accordingly, treatment of cultured L1210 cells with 10 microM-spermine rapidly and significantly lowered ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (AdoMetDC) activities in a sequential manner. By contrast, treatment for 48 h with 10 microM of the unsaturated spermine analogue 6-spermyne lowered AdoMetDC activity, but not ODC activity. An initial decrease in ODC activity at 2 h was attributed to a transient increase in free intracellular spermidine and spermine brought about through their displacement by the analogue. Thereafter, ODC activity recovered steadily to control values as 6-spermyne pools increased and spermidine and spermine pools decreased owing to analogue suppression of AdoMetDC activity. The apparent ability of 6-spermyne to regulate AdoMetDC, but not ODC, activity suggests an interesting structure-function correlation and demonstrates that the typical co-regulation of these enzyme activities can be dissociated. This, in turn, may reflect the existence of independent regulatory binding sites for the two enzymes.

scholarly journals Cellular characterization of a new irreversible inhibitor of S-adenosylmethionine decarboxylase and its use in determining the relative abilities of individual polyamines to sustain growth and viability of L1210 cells

1989 ◽  
Vol 259 (2) ◽  
pp. 325-331 ◽  
Author(s):  
D L Kramer ◽  
R M Khomutov ◽  
Y V Bukin ◽  
A R Khomutov ◽  
C W Porter
Keyword(s):  
Cell Growth ◽  
Supporting Cell ◽  
Enzyme Specificity ◽  
Irreversible Inhibitor ◽  
Enzyme Active Site ◽  
Adenosylmethionine Decarboxylase ◽  
Cellular Effects ◽  
L1210 Cells ◽  
Growth Inhibitory ◽  
Cell Growth And Viability

S-(5'-Deoxy-5'-adenosyl)methylthioethylhydroxylamine (AMA) is an irreversible inhibitor of S-adenosylmethionine (AdoMet) decarboxylase, which is designed to bind covalently the pyruvate residue at the enzyme active site. In the present study the cellular effects of AMA were characterized for the first time in cultured L1210 leukaemia cells. At the approximate IC50 (concn. giving 50% inhibition; 100 microM), AMA decreased spermidine and spermine by more than 80% at 48 h while increasing putrescine more than 10-fold. As an indication of enzyme specificity, growth inhibition was fully prevented with exogenous spermidine. When compared with the irreversible inhibitor of ornithine decarboxylase, alpha-difluoromethylornithine (DFMO), at similar growth-inhibitory concentrations, AMA was less cytotoxic, as determined by colony-formation efficiency. In combination with AMA, DFMO eliminated the rise in putrescine and decreased growth in an additive manner. The near-total depletion of intracellular polyamine pools achieved with the drug combination provided an opportunity to examine the relative abilities of individual polyamines to support growth and viability. Of the three exogenously supplied polyamines, only spermidine fully sustained cell growth and viability at control values during incubations totalling 120 h. By contrast, spermine supported growth at 23% of control and viability at 8%. Putrescine was similarly ineffective, supporting growth at 13% of control and viability at 7%. The data indicate that, in L1210 cells, spermidine is apparently the preferred polyamine in growth-related functions and is capable of fully supporting cell growth by itself. However, because spermine and putrescine can also support growth to some extent, maximum interference with growth and viability is best achieved by strategies which deplete all three polyamine pools.

scholarly journals CGP 48664, a potent and specific S-adenosylmethionine decarboxylase inhibitor: effects on regulation and stability of the enzyme

1997 ◽  
Vol 322 (1) ◽  
pp. 297-302 ◽  
Author(s):  
Fredrik SVENSSON ◽  
Helmut METT ◽  
Lo PERSSON
Keyword(s):  
Potent Inhibitor ◽  
Therapeutic Agents ◽  
Cellular Systems ◽  
Polyamine Metabolism ◽  
Mrna Levels ◽  
Leukaemia Cell Line ◽  
Adenosylmethionine Decarboxylase ◽  
L1210 Cells ◽  
Polyamine Transport ◽  
Mouse Leukaemia

Mammalian S-adenosylmethionine decarboxylase (AdoMetDC) catalyses a regulatory important step in the biosynthesis of polyamines and is a potential target for therapeutic agents against various parasitic diseases and proliferative disorders. In the present study we examined the effects of a newly synthesized AdoMetDC inhibitor, 4-amidinoindan-1-one 2ƀ-amidinohydrazone (CGP 48664), on polyamine metabolism in the mouse leukaemia cell line L1210. Treatment of the cells with 2 ƁM CGP 48664 led to a depletion of cellular spermidine and spermine. The putrescine content, in contrast, was markedly increased. Cells seeded in the presence of the inhibitor showed a significant decrease in growth rate, which was fully reversed by the addition of 2 ƁM spermidine or 1 ƁM spermine. The syntheses of ornithine decarboxylase and AdoMetDC were greatly increased in cells treated with CGP 48664. These increases were not correlated with similar changes in the mRNA levels, indicating the involvement of a translational mechanism. CGP 48664 was demonstrated to be a very poor competitor of spermidine uptake in the L1210 cells. L1210 cells deficient in polyamine transport were as sensitive to the antiproliferative effect of the inhibitor as were the parental cells, indicating that CGP 48664 did not enter the cells by the polyamine transport system. In addition to inhibiting AdoMetDC, CGP 48664 stabilized the enzyme against degradation. In the present study we also demonstrated that aminoguanidine (AMG), which is frequently used in cellular systems to inhibit any action of serum polyamine oxidase, apparently inhibits AdoMetDC by an irreversible mechanism that markedly stabilizes the enzyme against proteolytic degradation. CGP 48664 and the parental compound methylglyoxal bis(guanylhydrazone), which is also a potent inhibitor of AdoMetDC, contain one or two AMG-like moieties; the importance of these residues in the inhibition of AdoMetDC is discussed.

scholarly journals Inhibition of the synthesis of polyamines and macromolecules by 5′-methylthioadenosine and 5′-alkylthiotubercidins in BHK21 cells

1982 ◽  
Vol 204 (3) ◽  
pp. 697-703 ◽  
Author(s):  
Aarne Raina ◽  
Kyllikki Tuomi ◽  
Raija-Leena Pajula
Keyword(s):  
Growth Inhibition ◽  
Spermidine Synthase ◽  
Baby Hamster Kidney ◽  
Polyamine Synthesis ◽  
Adenosylmethionine Decarboxylase ◽  
Spermine Synthase ◽  
Target Sites ◽  
Polyamine Content ◽  
Exogenous Spermidine

5′-Methylthioadenosine and four 5′-alkylthiotubercidins were tested for their ability to inhibit polyamine synthesis in vitro and to decrease polyamine concentration and prevent growth of baby-hamster-kidney (BHK21) cells. 5′-Methylthioadenosine and 5′-methylthiotubercidin decreased the activity of spermidine synthase from brain to roughly the same extent, whereas brain spermine synthase was much more strongly inhibited by 5′-methylthioadenosine compared with 5′-methylthiotubercidin. These nucleoside derivatives also inhibited the growth of BHK21 cells and increased the concentration of putrescine. 5′-Methylthioadenosine decreased cellular spermine concentration, whereas 5′-methylthiotubercidin lowered the concentration of spermidine. The activities of ornithine decarboxylase and S-adenosylmethionine decarboxylase were enhanced in cells grown in the presence of 5′-methylthiotubercidin. The growth inhibition produced by these nucleoside derivatives was not reversed by exogenous spermidine or spermine. 5′-Ethylthiotubercidin, 5′-propylthiotubercidin and 5′-isopropylthiotubercidin did not appreciably inhibit spermidine or spermine synthase in vitro or decrease the cellular polyamine content, but effectively prevented the growth of BHK21 cells. All nucleoside derivatives at concentrations of 0.2–1 mm caused a rapid inhibition of protein synthesis. It is concluded that the growth inhibition produced by 5′-methylthioadenosine and 5′-alkylthiotubercidins was not primarily due to polyamine depletion but other target sites, for instance the cellular nucleotide pool, cell membranes etc. must be considered.

scholarly journals Effect of 1-amino-oxy-3-aminopropane on polyamine metabolism and growth of L1210 cells

1989 ◽  
Vol 263 (1) ◽  
pp. 215-221 ◽  
Author(s):  
R Poulin ◽  
J A Secrist ◽  
A E Pegg
Keyword(s):  
Polyamine Metabolism ◽  
Reversible Inhibitor ◽  
Dependent Manner ◽  
Intact Cells ◽  
Adenosylmethionine Decarboxylase ◽  
L1210 Cells ◽  
Cell Extracts ◽  
Drug Concentrations ◽  
Dose Dependent

1-Amino-oxy-3-aminopropane (AOAP) was reported to inhibit several mammalian polyamine-biosynthetic enzymes in vitro, including ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (AdoMetDC) [Khomutov, Hyvönen, Karvonen, Kauppinen, Paalanen, Paulin, Eloranta, Pajula, Andersson & Pösö (1985) Biochem. Biophys. Res. Commun. 130, 596-602]. In order to clarify its mechanism of action in intact cells, the inhibitory properties of AOAP on the growth and polyamine metabolism of L1210 cells were compared with those seen in a variant subline (D-R cells) which overproduces ODC. As little as 20 microM-AOAP completely blocked proliferation of L1210 cells, and this effect was reversed by the concomitant addition of exogenous putrescine or spermidine. Growth of D-R cells was not affected by AOAP at concentrations up to 0.5 mM. There was no difference in the uptake of AOAP between the L1210 and the D-R cells. Exposure of L1210 or D-R cells to AOAP greatly decreased ODC activity in undialysed cell extracts, but did not decrease AdoMetDC. Activities of both enzymes were increased severalfold by AOAP treatment when activity was measured in dialysed extracts. Treatment with AOAP depleted intracellular putrescine and spermidine contents of L1210 cells, while inducing a massive accumulation of decarboxylated AdoMet. The 8-fold higher putrescine pool present in untreated D-R cells was depleted in a dose-dependent manner by AOAP, but a significant decrease in spermidine and accumulation of decarboxylated AdoMet required 10 times higher drug concentrations, and the changes were much less dramatic than in L1210 cells. These results indicate that in L1210 cells AOAP behaves primarily as a reversible inhibitor of ODC.

scholarly journals The role of polyamine depletion and accumulation of decarboxylated S-adenosylmethionine in the inhibition of growth of SV-3T3 cells treated with α-difluoromethylornithine

1984 ◽  
Vol 224 (1) ◽  
pp. 29-38 ◽  
Author(s):  
A E Pegg
Keyword(s):  
Growth Rate ◽  
Mammalian Cells ◽  
Specific Inhibitor ◽  
Normal Growth ◽  
Inhibitory Effect ◽  
Adenosylmethionine Decarboxylase ◽  
Inhibition Of Growth ◽  
Control Growth ◽  
Polyamine Content ◽  
In Cells

The effects of alpha-difluoromethylornithine, a specific inhibitor of ornithine decarboxylase, on cell growth rate, polyamine content and the content of decarboxylated S-adenosylmethionine in SV-3T3 transformed mouse fibroblasts were studied. DL-alpha-Difluoromethylornithine at 1 mM or higher concentrations decreased the growth rate by over 90% after 2 or more days of exposure, but the cells remained viable, although quiescent for at least 9 days. Addition of 10 microM-spermidine or -spermine or 50 microM-putrescine at any time throughout this period completely reversed the inhibition of growth. Treatment with alpha-difluoromethylornithine decreased putrescine and spermidine contents by more than 98% and that of spermine by 60%, but cells exposed to exogenous polyamines did not require complete replenishment of the polyamine pools to resume growth. In fact, a virtually normal growth rate was obtained in cells lacking putrescine, having 2% of normal spermidine content and 156% of normal spermine. These results suggest that the well-known increase in putrescine and spermidine in cells stimulated for growth is not essential for this to occur and that mammalian cells can utilize spermine as their only polyamine. A substantial reversal of the growth-inhibitory effect of alpha-difluoromethylornithine was produced by a number of polyamines not normally found in mammalian cells, including the spermidine analogues aminopropylcadaverine and sym-homospermidine, which were partially converted into their respective spermine analogues by addition of an aminopropyl group within the cell. The spermine analogue sym-norspermine was also effective, but the maximal growth rate produced by these unphysiological polyamines was only 60-70% of that produced by the normal polyamines. These results indicate that spermidine and spermine have the optimal length for activation of the cellular processes critically dependent on polyamines and should help in identifying these processes. Exposure to alpha-difluoromethylornithine leads to an enormous rise in the concentration of decarboxylated S-adenosylmethionine, which reached a peak at 530-fold after 3 days of exposure and steadily declined to 140-fold after 11 days. This increase was abolished by addition of exogenous polyamines, which rapidly decreased the activity of S-adenosylmethionine decarboxylase. The increase in decarboxylated S-adenosylmethionine is unlikely to be solely responsible for the decrease to the same extent by spermine, sym-norspermidine and sym-homospermidine, which produce 97%, 16% and 60% of the control growth rate, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)

Regulation of polyamine content in cultured fibroblasts

1982 ◽  
Vol 243 (5) ◽  
pp. C262-C269 ◽  
Author(s):  
D. R. Bethell ◽  
H. Hibasami ◽  
A. E. Pegg
Keyword(s):  
Growth Period ◽  
Transformed Cells ◽  
Biosynthetic Enzymes ◽  
Spermidine Synthase ◽  
3T3 Cells ◽  
Cultured Fibroblasts ◽  
Mouse Fibroblasts ◽  
Adenosylmethionine Decarboxylase ◽  
Spermine Synthase ◽  
Polyamine Content

The content of putrescine and of the polyamines (spermidine and spermine) and the activities of their biosynthetic enzymes were measured in 3T3 mouse fibroblasts and SV40-transformed mouse fibroblasts over the entire period from subculturing in fresh medium until confluence. The transformed cells had a substantially higher content of putrescine and spermidine than the 3T3 cells and higher activities of all of the biosynthetic enzymes. However, the ratio of spermine synthase to spermidine synthase was higher in the 3T3 cells, which correlated with their higher spermine-to-spermidine ratio. All of the biosynthetic enzymes increased in activity during cell growth. Ornithine decarboxylase increased 20-fold with a maximum at 24-36 h after culturing whereas S-adenosylmethionine decarboxylase increased 3-fold at the same time. Spermidine synthase increased 10- to 16-fold during the growth period whereas spermine synthase increased 2- to 3-fold. The relative enzyme activities and the changes in total polyamine content suggested that 1) the activity of S-adenosylmethionine decarboxylase limited the production of the polyamines and 2) the relative amounts of spermidine and spermine synthase determined the predominant polyamine that the available decarboxylated S-adenosylmethionine is used to synthesize. When 3T3 cells become quiescent at confluence, there was a substantial fall in the intracellular spermidine level because of a greatly increased excretion of spermidine into the medium. Spermine content also fell because there was an increased conversion of spermine into spermidine, which was then excreted. The specific excretion of spermidine did not occur with the transformed SV-3T3 cells.

scholarly journals Feedback regulation of S-adenosylmethionine decarboxylase synthesis

1989 ◽  
Vol 257 (3) ◽  
pp. 929-931 ◽  
Author(s):  
L Persson ◽  
A R Khomutov ◽  
R M Khomutov
Keyword(s):  
Cell Growth ◽  
Marked Decrease ◽  
Potent Inhibitor ◽  
Feedback Regulation ◽  
Adenosylmethionine Decarboxylase ◽  
Ehrlich Ascites ◽  
Ehrlich Ascites Tumour ◽  
Polyamine Content ◽  
Ascites Tumour Cells ◽  
Ehrlich Ascites Tumour Cells

Treatment of Ehrlich ascites-tumour cells with 1-amino-oxy-3-aminopropane (AOAP), a potent inhibitor of ornithine decarboxylase, resulted in a marked decrease in cellular contents of putrescine and spermidine, concomitant with an arrest of cell growth. The activity of S-adenosylmethionine decarboxylase (AdoMetDC) was greatly increased in cells treated with AOAP. This increase in AdoMetDC activity was shown to be, at least partly, caused by enhanced synthesis of the enzyme, which most likely was induced by the change in cellular polyamine content.

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