scholarly journals Regulation of ornithine decarboxylase activity by spermidine and the spermidine analogue N1N8-bis(ethyl)spermidine

1987 ◽  
Vol 242 (2) ◽  
pp. 433-440 ◽  
Author(s):  
C W Porter ◽  
F G Berger ◽  
A E Pegg ◽  
B Ganis ◽  
R J Bergeron
Keyword(s):  
Ornithine Decarboxylase ◽  
Supporting Cell ◽  
Dose Dependence ◽  
Decarboxylase Activity ◽  
Response Curves ◽  
Polyamine Biosynthesis ◽  
Intact Cells ◽  
Polyamine Analogues ◽  
L1210 Cells ◽  
Minimal Treatment

Polyamine biosynthesis in intact cells can be exquisitely controlled with exogenous polyamines through the regulation of rate-limiting biosynthetic enzymes, particularly ornithine decarboxylase (ODC). In an attempt to exploit this phenomenon as an antiproliferative strategy, certain polyamine analogues have been identified [Porter, Cavanaugh, Stolowich, Ganis, Kelly & Bergeron (1985) Cancer Res. 45, 2050-2057] which lower ODC activity in intact cells, have no direct inhibitory effects on ODC, are incapable of substituting for spermidine (SPD) in supporting cell growth, and are growth-inhibitory at micromolar concentrations. In the present study, the most effective of these analogues, N1N8-bis(ethyl)SPD (BES), is compared with SPD in its ability to regulate ODC activity in intact L1210 cells and in the mechanism(s) by which this is accomplished. With respect to time and dose-dependence of ODC suppression, both polyamines closely paralleled one another in their response curves, although BES was slightly less effective than SPD. Conditions of minimal treatment leading to near-maximal ODC suppression (70-80%) were determined and found to be 3 microM for 2 h with either SPD or BES. After such treatment, ODC activity was fully recovered within 2-4 h when cells were re-seeded in drug-free media. By assessing BES or [3H]SPD concentrations in treated and recovered cells, it was possible to deduce that an intracellular accumulation of BES or SPD equivalent to less than 6.5% of the combined cellular polyamine pool was sufficient to invoke ODC regulatory mechanisms. Decreases in ODC activity after BES or SPD treatment were closely paralleled by concomitant decreases in ODC protein. Since cellular ODC mRNA was not similarly decreased by either BES or SPD, it was concluded that translational and/or post-translational mechanisms, such as increased degradation of ODC protein or decreased translation of ODC mRNA, were probably responsible for regulation of enzyme activity. Experimental evidence indicated that neither of these mechanisms seemed to be mediated by cyclic AMP or ODC-antizyme induction. On the basis of the consistent similarities between BES and SPD in all parameters studied, it is concluded that the analogue most probably acts by the same mechanisms as SPD in regulating polyamine biosynthesis.

scholarly journals Amine-specificity of the inactivating ornithine decarboxylase modification in Physarum polycephalum

1982 ◽  
Vol 205 (3) ◽  
pp. 551-557 ◽  
Author(s):  
J L A Mitchell ◽  
G K Mitchell ◽  
D D Carter
Keyword(s):  
Ornithine Decarboxylase ◽  
Translational Control ◽  
Feedback Mechanism ◽  
Decarboxylase Activity ◽  
Polyamine Biosynthesis ◽  
Polyamine Analogues ◽  
Ornithine Decarboxylase Activity ◽  
Sensitivity Studies

The enzyme catalysing the polyamine-stimulated modification of Physarum ornithine decarboxylase in vivo was partially purified and its activity on purified ornithine decarboxylase was examined with respect to its specificity for various amines. Spermidine, spermine and several polyamine analogues strongly promoted this reaction in vitro (apparent Km in the 0.1-0.5 mM range), whereas putrescine (apparent Km 5.33 mM) and several related diamines were not nearly as effective. In agreement with this, sensitivity studies performed in vivo also suggested that cellular spermidine, and not putrescine, is critical in modulating ornithine decarboxylase activity by this post-translational control. Unlike putrescine, or other diamines, 1,3-diaminopropane demonstrated a functional similarity to the polyamines in stimulating this reaction. This study has demonstrated a method whereby non-physiological amines capable of depressing ornithine decarboxylase activity by this natural feedback mechanism can be readily identified for further evaluation of their potential use in the experimental and medical control of polyamine biosynthesis.

scholarly journals Investigations of the mechanism by which mammalian cell growth is inhibited by N1N12-bis(ethyl)spermine

1993 ◽  
Vol 291 (1) ◽  
pp. 131-137 ◽  
Author(s):  
L Albanese ◽  
R J Bergeron ◽  
A E Pegg
Keyword(s):  
Mitochondrial Dna ◽  
Cell Growth ◽  
Cell Lines ◽  
Ornithine Decarboxylase ◽  
Polyamine Analogues ◽  
Proliferative Effect ◽  
L1210 Cells ◽  
Inhibition Of Growth ◽  
Variant Cell ◽  
Mammalian Cell Growth

N1N12-Bis(ethyl)spermine (BESM) and related compounds are powerful inhibitors of cell growth that may have potential as anti-neoplastic agents [Bergeron, Neims, McManis, Hawthorne, Vinson, Bortell and Ingeno (1988) J. Med. Chem. 31, 1183-1190]. The mechanism by which these compounds bring about their effects was investigated by using variant cell lines in which processes thought to be altered by these agents are perturbed. Comparisons between the response of these cells and of their parental equivalents to BESM, N1N11-bis(ethyl)norspermine, N1N14-bis(ethyl)homospermine and N1N8-bis(ethyl)spermidine were then made. It was found that D-R cells, an L1210-derived line that over-expresses ornithine decarboxylase, were not resistant to these compounds. This indicates that the decrease in ornithine decarboxylase is not critical for the action of the compounds on cell growth. Furthermore, although polyamine levels were decreased in the D-R cells, the content was not totally depleted, indicating that such depletion is also not essential for the anti-proliferative effect. Two cell lines lacking mitochondrial DNA (human 143B206 cells and chicken DU3 cells) did not differ in sensitivity to BESM from their parental 143BTK- and DU24 cells. Furthermore, the inhibition of respiration in L1210 cells in response to BESM developed more slowly than the inhibition of growth. Thus it appears that the inhibitions of mitochondrial DNA synthesis and of mitochondrial respiration are also not primary factors in the anti-proliferative effects of these polyamine analogues. The inhibition of growth did, however, correlate with the intracellular accumulation of the analogues. It appears that the bis(ethyl)polyamine derivatives act by binding to intracellular target molecules and preventing macromolecular synthesis. The decline in normal polyamines may facilitate such binding, but is not essential for growth arrest.

scholarly journals Role of pyridoxal phosphate in mammalian polyamine biosynthesis. Lack of requirement for mammalian S-adenosylmethionine decarboxylase activity

1977 ◽  
Vol 166 (1) ◽  
pp. 81-88 ◽  
Author(s):  
A E Pegg
Keyword(s):  
Ornithine Decarboxylase ◽  
Pyridoxal Phosphate ◽  
Vitamin B ◽  
Decarboxylase Activity ◽  
Deficient Diet ◽  
Polyamine Biosynthesis ◽  
Adenosylmethionine Decarboxylase ◽  
Ornithine Decarboxylase Activity

1. Polyamine concentrations were decreased in rats fed on a diet deficient in vitamin B-6. 2. Ornithine decarboxylase activity was decreased by vitamin B-6 deficiency when assayed in tissue extracts without addition of pyridoxal phosphate, but was greater than in control extracts when pyridoxal phosphate was present in saturating amounts. 3. In contrast, the activity of S-adenosylmethionine decarboxylase was not enhanced by pyridoxal phosphate addition even when dialysed extracts were prepared from tissues of young rats suckled by mothers fed on the vitamin B-6-deficient diet. 4. S-Adenosylmethionine decarboxylase activities were increased by administration of methylglyoxal bis(guanylhydrazone) (1,1′-[(methylethanediylidine)dinitrilo]diguanidine) to similar extents in both control and vitamin B-6-deficient animals. 5. The spectrum of highly purified liver S-adenosylmethionine decarboxylase did not indicate the presence of pyridoxal phosphate. After inactivation of the enzyme by reaction with NaB3H4, radioactivity was incorporated into the enzyme, but was not present as a reduced derivative of pyridoxal phosphate. 6. It is concluded that the decreased concentrations of polyamines in rats fed on a diet containing vitamin B-6 may be due to decreased activity or ornithine decarboxylase or may be caused by an unknown mechanism responding to growth retardation produced by the vitamin deficiency. In either case, measurements of S-adenosylmethionine decarboxylase and ornithine decarboxylase activity under optimum conditions in vitro do not correlate with the polyamine concentrations in vivo.

scholarly journals The role of polyamine biosynthesis in hematopoietic precursor cell proliferation in mice

Blood ◽  
1983 ◽  
Vol 61 (4) ◽  
pp. 740-745 ◽  
Author(s):  
E Niskanen ◽  
A Kallio ◽  
PP McCann ◽  
DG Baker
Keyword(s):  
Ornithine Decarboxylase ◽  
Colony Formation ◽  
Calf Serum ◽  
Decarboxylase Activity ◽  
Irreversible Inhibitor ◽  
Host Animal ◽  
Polyamine Biosynthesis ◽  
Diffusion Chambers

Abstract Under the influence of a selective irreversible inhibitor of ornithine decarboxylase (ODC), DL-alpha-difluoromethylornithine (DFMO), early hematopoiesis was enhanced. In the bone marrow, the absolute number of cells that give rise to spleen colonies in lethally irradiated mice (CFU-S), granulocytic colonies in diffusion chambers in mice (CFU-DG), and granulocyte-monocyte colonies in agar in vitro (CFU-C) was increased 2–4 fold. This could be abrogated by administration of putrescine, confirming the association of the stimulatory effect with polyamine biosynthesis most likely via depression of ornithine decarboxylase activity and subsequent synthesis of putrescine. Analysis of cell cycle characteristics by 3H-TdR suicide technique demonstrated that the proportion of CFU-S, CFU-DG, and CFU-C in S-phase was significantly increased. Additionally, the stimulatory effect was reflected by enhanced colony formation in diffusion chambers implanted intraperitoneally in mice receiving DFMO. This could also be eliminated by treatment of the host animal with putrescine, again suggesting that polyamine biosynthesis plays an important role at the early stages of hematopoiesis in vivo. Effect of DFMO on colony formation in vitro (CFU- C) was inhibitory and not reversible with putrescine. It could be partially eliminated by aminoguanidine, which neutralizes diamine oxidase present in fetal calf serum used in the CFU-C assay. These data suggest that the effect of DFMO in vitro was nonspecific.

scholarly journals Inhibition of human ornithine decarboxylase activity by enantiomers of difluoromethylornithine

2003 ◽  
Vol 375 (2) ◽  
pp. 465-470 ◽  
Author(s):  
Ning QU ◽  
Natalia A. IGNATENKO ◽  
Phillip YAMAUCHI ◽  
David E. STRINGER ◽  
Corey LEVENSON ◽  
...  
Keyword(s):  
Ornithine Decarboxylase ◽  
Enzyme Inhibitor ◽  
Human Colon ◽  
Common Mechanism ◽  
Decarboxylase Activity ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Polyamine Biosynthesis ◽  
Irreversible Reaction ◽  
Kd Values

Racemic difluoromethylornithine (d/l-DFMO) is an inhibitor of ODC (ornithine decarboxylase), the first enzyme in eukaryotic polyamine biosynthesis. d/l-DFMO is an effective anti-parasitic agent and inhibitor of mammalian cell growth and development. Purified human ODC-catalysed ornithine decarboxylation is highly stereospecific. However, both DFMO enantiomers suppressed ODC activity in a time- and concentration-dependent manner. ODC activity failed to recover after treatment with either l- or d-DFMO and dialysis to remove free inhibitor. The inhibitor dissociation constant (KD) values for the formation of enzyme–inhibitor complexes were 28.3±3.4, 1.3±0.3 and 2.2±0.4 μM respectively for d-, l- and d/l-DFMO. The differences in these KD values were statistically significant (P<0.05). The inhibitor inactivation constants (Kinact) for the irreversible step were 0.25±0.03, 0.15±0.03 and 0.15±0.03 min−1 respectively for d-, l- and d/l-DFMO. These latter values were not statistically significantly different (P>0.1). d-DFMO was a more potent inhibitor (IC50~7.5 μM) when compared with d-ornithine (IC50~1.5 mM) of ODC-catalysed l-ornithine decarboxylation. Treatment of human colon tumour-derived HCT116 cells with either l- or d-DFMO decreased the cellular polyamine contents in a concentration-dependent manner. These results show that both enantiomers of DFMO irreversibly inactivate ODC and suggest that this inactivation occurs by a common mechanism. Both enantiomers form enzyme–inhibitor complexes with ODC, but the probability of formation of these complexes is 20 times greater for l-DFMO when compared with d-DFMO. The rate of the irreversible reaction in ODC inactivation is similar for the l- and d-enantiomer. This unexpected similarity between DFMO enantiomers, in contrast with the high degree of stereospecificity of the substrate ornithine, appears to be due to the α-substituent of the inhibitor. The d-enantiomer may have advantages, such as decreased normal tissue toxicity, over l- or d/l-DFMO in some clinical applications.

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.

Growth hormone inhibition of lipogenesis in sheep adipose tissue: requirement for gene transcription and polyamines

1994 ◽  
Vol 142 (2) ◽  
pp. 235-243 ◽  
Author(s):  
C A Borland ◽  
M C Barber ◽  
M T Travers ◽  
R G Vernon
Keyword(s):  
Growth Hormone ◽  
Adipose Tissue ◽  
Fatty Acid ◽  
Ornithine Decarboxylase ◽  
Gene Transcription ◽  
Actinomycin D ◽  
Total Activity ◽  
Decarboxylase Activity ◽  
Polyamine Biosynthesis

Abstract The chronic inhibitory effect of growth hormone (GH) on lipogenesis in sheep adipose tissue explants was investigated in an in vitro tissue culture system. In the absence of other hormones, GH caused a decrease in the rate of lipogenesis after 6 h of culture. In contrast, when lipogenesis was stimulated by the presence of insulin plus dexamethasone, GH again decreased lipogenesis but after a lag of at least 12 h. Actinomycin D, an inhibitor of gene transcription, prevented the effect of GH on lipogenesis in both the absence and presence of insulin plus dexamethasone. Actinomycin D added to tissue previously incubated for 6 h in the presence of GH alone prevented further decline in lipogenesis over the next 5 h, suggesting that transcription of a short-lived mediator protein is required for the GH effect to occur. An increase in ornithine decarboxylase activity was detected in explants exposed to GH, reaching a peak after 12 h incubation; this was prevented by actinomycin D. Methylglyoxal bis-(guanylhydrazone), an inhibitor of polyamine biosynthesis, partially alleviated the effect of GH on lipogenesis; this was reversed by addition of spermidine. However, spermidine did not reverse the effects of actinomycin D, implicating a short-lived protein in addition to ornithine decarboxylase in the action of GH. In the absence of other hormones GH had no effect on either the expressed (initial) or total activity of acetyl-CoA carboxylase, but GH prevented the increase in both expressed and total activities of the enzyme induced by insulin plus dexamethasone. Varying lipolysis and fatty acid accumulation in adipose tissue by addition of adenosine deaminase plus indomethacin or bovine serum albumin to the culture medium had no effect on lipogenesis and these agents partly alleviated GH inhibition of lipogenesis. No effect of GH was found on the amount of glycerol released by cultured tissue. GH also had no effect on fatty acid esterification. Thus the chronic inhibitory effects of GH on lipogenesis involve a protein with a very short half-life. The effect also requires polyamines but does not appear to involve changes in fatty acid concentrations in the cell. In addition GH appears to inhibit lipogenesis and to antagonise insulin-stimulation of lipogenesis by different mechanisms. Journal of Endocrinology (1994) 142, 235–243

scholarly journals Coupling of muscarinic receptors to the mobilization of intracellular Ca2+ stores in permeabilized SH-SY5Y human neuroblastoma cells

1990 ◽  
Vol 272 (1) ◽  
pp. 269-272 ◽  
Author(s):  
R J H Wojcikiewicz ◽  
S T Safrany ◽  
R A J Challiss ◽  
J Strupish ◽  
S R Nahorski
Keyword(s):  
Muscarinic Receptors ◽  
Neuroblastoma Cells ◽  
Dose Dependence ◽  
Maximal Response ◽  
Functional Coupling ◽  
Maximal Effect ◽  
Human Neuroblastoma Cells ◽  
Response Curves ◽  
Human Neuroblastoma ◽  
Intact Cells

Intracellular stores of Ca2+ were mobilized transiently by carbachol in suspensions of electrically permeabilized SH-SY5Y cells. The kinetics and the dose-dependence of this mobilization paralleled carbachol-induced increases in inositol 1,4,5-trisphosphate (InsP3) mass [for both parameters EC50 (concn. giving half-maximal response) = 60-70 microM]. Guanosine 5′-[gamma-thio]triphosphate enhanced the maximal effect and the potency of carbachol on Ca2+ mobilization and InsP3 mass, but caused separation of the dose-response curves (EC50 = 0.6 microM and 5.6 microM respectively). These data show that functional coupling of muscarinic receptors to Ca2+ mobilization can be maintained after permeabilization, reveal major effects of guanine nucleotides on agonist-induced Ca2+ mobilization and provide a basis for explanation of discrepancies between agonist potency on InsP3 concentration and Ca2+ mobilization in intact cells.

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