Putrescine does not support the migration and growth of IEC-6 cells

The migration of IEC-6 cells is inhibited when the cells are depleted of polyamines by inhibiting ornithine decarboxylase with alpha-difluoromethylornithine (DFMO). Exogenous putrescine, spermidine, and spermine completely restore cell migration inhibited by DFMO. Because polyamines are interconverted during their synthesis and catabolism, the specific role of individual polyamines in intestinal cell migration, as well as growth, remains unclear. In this study, we used an inhibitor of S-adenosylmethionine decarboxylase, diethylglyoxal bis(guanylhydrazone)(DEGBG), to block the synthesis of spermidine and spermine from putrescine. We found that exogenous putrescine does not restore migration and growth of IEC-6 cells treated with DFMO plus DEGBG, whereas exogenous spermine does. In addition, the normal distribution of actin filaments required for migration, which is disrupted in polyamine-deficient cells, could be achieved by adding spermine but not putrescine along with DFMO and DEGBG. These results indicate that putrescine, by itself, is not essential for migration and growth, but that it is effective because it is converted into spermidine and/or spermine.     

Uptake of polyamines by the unicellular green alga Chlamydomonas reinhardtii and their effect on ornithine decarboxylase activity

Uptake of exogenous polyamines by the unicellular green alga Chlamydomonas reinhardtii and their effects on polyamine metabolism were investigated. Our data show that, in contrast to mammalian cells, Chlamydomonas reinhardtii does not contain short-living, high-affinity polyamine transporters whose cellular level is dependent on the polyamine concentration. However, exogenous polyamines affect polyamine metabolism in Chlamydomonas cells. Exogenous putrescine caused a slow increase of both putrescine and spermidine and, vice versa, exogenous spermidine also led to an increase of the intracellular levels of both spermidine and putrescine. No intracellular spermine was detected under any conditions. Exogenous spermine was taken up by the cells and caused a decrease in their putrescine and spermidine levels. As in other organisms, exogenous polyamines led to a decrease in the activity of ornithine decarboxylase, a key enzyme of polyamine synthesis. In contrast to mammalian cells, this polyamine-induced decrease in ornithine decarboxylase activity is not mediated by a polyamine-dependent degradation or inactivation, but exclusively due to a decreased synthesis of ornithine decarboxylase. Translation of ornithine decarboxylase mRNA, but not overall protein biosynthesis is slowed by increased polyamine levels.     

Antizyme regulates the degradation of ornithine decarboxylase in fission yeast Schizosaccharomyces pombe. Study in the spe2 knockout strains

The mechanism of the regulatory degradation of ornithine decarboxylase (ODC) by polyamines was studied in fission yeast, Schizosaccharomyces pombe. To regulate cellular spermidine experimentally, we cloned and disrupted S-adenosylmethionine decarboxylase gene (spe2) in S. pombe. The null mutant of spe2 was devoid of spermidine and spermine, accumulated putrescine, and contained a high level of ODC. Addition of spermidine to the culture medium resulted in rapid decrease in the ODC activity caused by the acceleration of ODC degradation, which was dependent on de novo protein synthesis. A fraction of ODC forming an inactive complex concomitantly increased. The accelerated ODC degradation was prevented either by knockout of antizyme gene or by selective inhibitors of proteasome. Thus, unlike budding yeast, mammalian type antizyme-mediated ODC degradation by proteasome is operating in S. pombe.     

Polyamine depletion delays apoptosis of rat intestinal epithelial cells

The polyamines spermidine, spermine, and their precursorputrescine are essential for cell growth and the regulation of the cellcycle. Recent studies suggest that excessive accumulation of polyaminesfavors either malignant transformation or apoptosis, depending on thecell type and the stimulus. This study examines the involvement ofpolyamines in the induction of apoptosis by the DNA topoisomerase Iinhibitor, camptothecin. In IEC-6 cells, camptothecin induced apoptosiswithin 6 h, accompanied by detachment of cells. Detached cells showedDNA laddering and caspase 3 induction, characteristic features ofapoptosis. Depletion of putrescine, spermidine, and spermine byDL--difluoromethylornithine (DFMO), a specific inhibitorof ornithine decarboxylase (ODC) that is the first rate-limiting enzymefor polyamine biosynthesis, decreased the apoptotic index. Delayedapoptosis was accompanied by a decrease in caspase 3 activity inpolyamine-depleted cells. Addition of putrescine restored the inductionof apoptosis as indicated by an increase in the number of detachedcells and caspase 3 activity. Polyamine depletion did not change thelevel of caspase 3 protein. Inhibition of S-adenosylmethioninedecarboxylase by a specific inhibitor [diethylglyoxalbis-(guanylhydrazone); DEGBG] led to depletion of spermidine andspermine with a significant accumulation of putrescine and induction ofODC. The DEGBG-treated cells showed an increase in apoptosis,suggesting the importance of putrescine in the apoptotic process.Addition of putrescine to DFMO-treated cell extracts did not increasecaspase 3 activity. The above results indicate that polyamine depletiondelays the onset of apoptosis in IEC-6 cells and confers protectionagainst DNA damaging agents, suggesting that polyamines might beinvolved in the caspase activating signal cascade.

     

Regulation by polyamines of ornithine decarboxylase activity and cell division in the unicellular green alga Chlamydomonas reinhardtii

Polyamines are required for cell growth and cell division in eukaryotic and prokaryotic organisms. In the unicellular green alga Chlamydomonas reinhardtii, biosynthesis of the commonly occurring polyamines (putrescine, spermidine, and spermine) is dependent on the activity of ornithine decarboxylase (ODC, EC 4.1.1.17) catalyzing the formation of putrescine, which is the precursor of the other two polyamines. In synchronized C. reinhardtii cultures, transition to the cell division phase was preceded by a 4-fold increase in ODC activity and a 10- and a 20-fold increase, respectively, in the putrescine and spermidine levels. Spermine, however, could not be detected in C. reinhardtii cells. Exogenous polyamines caused a decrease in ODC activity. Addition of spermine, but not of spermidine or putrescine, abolished the transition to the cell division phase when applied 7 to 8 h after beginning of the light (growth) phase. Most of the cells had already doubled their cell mass after this growth period. The spermine-induced cell cycle arrest could be overcome by subsequent addition of spermidine or putrescine. The conclusion that spermine affects cell division via a decreased spermidine level was corroborated by the findings that spermine caused a decrease in the putrescine and spermidine levels and that cell divisions also could be prevented by inhibitors of S-adenosyl-methionine decarboxylase and spermidine synthase, respectively, added 8 h after beginning of the growth period. Because protein synthesis was not decreased by addition of spermine under our experimental conditions, we conclude that spermidine affects the transition to the cell division phase directly rather than via protein biosynthesis.     

Crystal structure of human ornithine decarboxylase at 2.1 A resolution: structural insights to antizyme binding

The polyamines spermidine and spermine are ubiquitous and required for cell growth and differentiation in eukaryotes. Ornithine decarboxylase (ODC, EC 4.1.1.17) performs the first step in polyamine biosynthesis, the decarboxylation of ornithine to putrescine. Elevated polyamine levels can lead to down-regulation of ODC activity by enhancing the translation of antizyme mRNA, resulting in subsequent binding of antizyme to ODC monomers which targets ODC for proteolysis by the 26S proteasome. The crystal structure of ornithine decarboxylase from human liver has been determined to 2.1 A resolution by molecular replacement using truncated mouse ODC (Delta425-461) as the search model and refined to a crystallographic R-factor of 21.2% and an R-free value of 28.8%. The human ODC model includes several regions that are disordered in the mouse ODC crystal structure, including one of two C-terminal basal degradation elements that have been demonstrated to independently collaborate with antizyme binding to target ODC for degradation by the 26S proteasome. The crystal structure of human ODC suggests that the C terminus, which contains basal degradation elements necessary for antizyme-induced proteolysis, is not buried by the structural core of homodimeric ODC as previously proposed. Analysis of the solvent-accessible surface area, surface electrostatic potential, and the conservation of primary sequence between human ODC and Trypanosoma brucei ODC provides clues to the identity of potential protein-binding-determinants in the putative antizyme binding element in human ODC.     

Polyamine biosynthesis in Phytomonas: Biochemical characterisation of a very unstable ornithine decarboxylase

The metabolism of polyamines as well as their functions as growth regulators in plants have been extensively studied for many years. However, almost nothing is known about the biosynthesis and roles of these substances in Phytomonas spp., parasites of several plants. We have used HPLC and electrophoretic analyses to investigate the presence and metabolism of polyamines in Phytomonas Jma strain, detecting both putrescine and spermidine but not spermine. Experiments carried out by incubation of intact parasites with labelled ornithine or putrescine showed the formation of radioactive putrescine or spermidine, respectively. These results indicated that Phytomonas Jma can synthesise these polyamines through the action of ornithine decarboxylase (ODC) and spermidine synthase. On the other hand, we could not detect the conversion of arginine to agmatine, suggesting the absence of arginine decarboxylase (ADC) in Phytomonas. However, we cannot ensure the complete absence of this enzymatic activity in the parasite. Phytomonas ODC required pyridoxal 5′-phosphate for maximum activity and was specifically inhibited by α-difluoromethylornithine. The metabolic turnover of the enzyme was very high, with a half-life of 10-15 min, one of the shortest found among all ODC enzymes studied to date. The parasite proteasome seems to be involved in degradation of the enzyme, since Phytomonas ODC can be markedly stabilized by MG-132, a well known proteasome inhibitor. The addition of polyamines to Phytomonas cultures did not decrease ODC activity, strongly suggesting the possible absence of antizyme in this parasite.     

Inhibition of ornithine decarboxylase of HeLa cells by diamines and polyamines. Effect on cell proliferation

1. Ornithine decarboxylase activity is stimulated in high-density HeLa-cell cultures by dilution of or replacement of spent culture medium with fresh medium containing 10% (v/v) horse serum. 2. After stimulation, ornithine decarboxylase activity reaches a peak at 4–6h, then rapidly declines to the low enzyme activity characteristic of quiescent cultures, where it remains during the remainder of the cell cycle. 3. The stimulation of ornithine decarboxylase is eliminated by the addition of 0.5μm-spermine or -spermidine or 10μm-putrescine to the HeLa-cell cultures at the time of re-feeding with fresh medium. Much higher concentrations (1mm) of the non-physiological diamines, 1,3-diamino-propane or 1,3-diamino-2-hydroxypropane, are required to eliminate the stimulation of ornithine decarboxylase in re-fed HeLa-cell cultures. 4. A heat-labile, non-diffusible inhibitor, comparable with the inhibitory protein ornithine decarboxylase antizyme, is induced in HeLa cells by the addition of exogenous diamines or polyamines. 5. Intracellular putrescine is eliminated, intracellular spermidine and spermine are severely decreased and proliferation of HeLa cells is inhibited when cultures are maintained for 48h in the presence of the non-physiological inducer of ornithine decarboxylase antizyme, 1,3-diamino-2-hydroxypropane. Exogenous putrescine, a physiological inducer of the antizyme, does not decrease intracellular polyamines or interfere with proliferation of HeLa cells.     

Polyamine homeostasis in arginase knockout mice

The role of ornithine decarboxylase (ODC) in polyamine metabolism has long been established, but the exact source of ornithine has always been unclear. The arginase enzymes are capable of producing ornithine for the production of polyamines and may hold important regulatory functions in the maintenance of this pathway. Utilizing our unique set of arginase single and double knockout mice, we analyzed polyamine levels in the livers, brains, kidneys, and small intestines of the mice at 2 wk of age, the latest timepoint at which all of them are still alive, to determine whether tissue polyamine levels were altered in response to a disruption of arginase I (AI) and II (AII) enzymatic activity. Whereas putrescine was minimally increased in the liver and kidneys from the AII knockout mice, spermidine and spermine were maintained. ODC activity was not greatly altered in the knockout animals and did not correlate with the fluctuations in putrescine. mRNA levels of ornithine aminotransferase (OAT), antizyme 1 (AZ1), and spermidine/spermine-N¹-acetyltransferase (SSAT) were also measured and only minor alterations were seen, most notably an increase in OAT expression seen in the liver of AI knockout and double knockout mice. It appears that putrescine catabolism may be affected in the liver when AI is disrupted and ornithine levels are highly reduced. These results suggest that endogenous arginase-derived ornithine may not directly contribute to polyamine homeostasis in mice. Alternate sources such as diet may provide sufficient polyamines for maintenance in mammalian tissues. ornithine; putrescine; spermidine; spermine; decarboxylase     

Agmatine modulates polyamine content in hepatocytes by inducing spermidine/spermine acetyltransferase.

Agmatine has been proposed as the physiological ligand for the imidazoline receptors. It is not known whether it is also involved in the homoeostasis of intracellular polyamine content. To show whether this is the case, we have studied the effect of agmatine on rat liver cells, under both periportal and perivenous conditions. It is shown that agmatine modulates intracellular polyamine content through its effect on the synthesis of the limiting enzyme of the interconversion pathway, spermidine/spermine acetyltransferase (SSAT). Increased SSAT activity is accompanied by depletion of spermidine and spermine, and accumulation of putrescine and N1-acetylspermidine. Immunoblotting with a specific polyclonal antiserum confirms the induction. At the same time S-adenosylmethionine decarboxylase activity is significantly increased, while ornithine decarboxylase (ODC) activity and the rate of spermidine uptake are reduced. This is not due to an effect on ODC antizyme, which is not significantly changed. All these modifications are observed in HTC cells also, where they are accompanied by a decrease in proliferation rate. SSAT is also induced by low oxygen tension which mimics perivenous conditions. The effect is synergic with that promoted by agmatine.     

Effects of S-adenosyl-1,8-diamino-3-thio-octane and S-methyl-5''-methylthioadenosine on polyamine synthesis in Ehrlich ascites-tumour cells.

The rate-limiting enzymes in polyamine biosynthesis, ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (AdoMetDC), are negatively regulated by the polyamines spermidine and spermine. In the present work the spermidine synthase inhibitor S-adenosyl-1,8-diamino-3-thio-octane (AdoDATO) and the spermine synthase inhibitor S-methyl-5'-methylthioadenosine (MMTA) were used to evaluate the regulatory role of the individual polyamines. Treatment of Ehrlich ascites-tumour cells with AdoDATO caused a marked decrease in spermidine content together with an accumulation of putrescine and spermine. Treatment with MMTA, on the other hand, gave rise to a marked decrease in spermine, with a simultaneous accumulation of spermidine. A dramatic increase in the activity of AdoMetDC, but not of ODC, was observed in MMTA-treated cells. This increase appears to be unrelated to the decrease in spermine content, because a similar rise in AdoMetDC activity was obtained when AdoDATO was given in addition to MMTA, in which case the spermine content remained largely unchanged. Instead, we show that the increase in AdoMetDC activity is mainly due to stabilization of the enzyme, probably by binding of MMTA. Treatment with AdoDATO had no effects on the activities of ODC and AdoMetDC, even though it caused a precipitous decrease in spermidine content. The expected decrease in spermidine-mediated suppression of ODC and AdoMetDC was most probably counteracted by the simultaneous increase in spermine. The combination of AdoDATO and MMTA caused a transient rise in ODC activity. Concomitant with this rise, the putrescine and spermidine contents increased, whereas that of spermine remained virtually unchanged. The increase in ODC activity was due to increased synthesis of the enzyme. There were no major effects on the amount of AdoMetDC mRNA by treatment with the inhibitors, alone or in combination. However, the synthesis of AdoMetDC was slightly stimulated in cells treated with MMTA or AdoDATO plus MMTA. The present study demonstrates that regulation of neither ODC nor AdoMetDC is a direct function of the polyamine structure. Instead, it appears that the biosynthesis of the polyamines is feedback-regulated by the various polyamines at many different levels.     

Involvement of antizyme in stabilization of ornithine decarboxylase caused by inhibitors of polyamine synthesis

Contrary to previous findings, ornithine decarboxylase (ODC) was stabilized by treatment of cells with DL-alpha-difluoromethylornithine, an enzyme-activated irreversible inhibitor of ODC. Both this inhibitor and cyclohexylamine, a spermidine synthase inhibitor known to stabilize ODC, caused decreases in the antizyme/ODC ratio by increasing ODC content and conversely decreasing antizyme content. The relationship between cellular polyamine levels and antizyme content indicated that spermidine is the most important polyamine for antizyme induction. These results suggest that antizyme is involved in the mechanism underlying the stabilization of ODC by inhibitors of polyamine synthesis and support the hypothesis that cellular polyamines regulate ODC degradation via antizyme.     

Effect of ATP depletion and phenanthroline on the spermidine-mediated decay of ornithine decarboxylase in erythroleukemia cells

Addition of spermidine to Friend erythroleukemia cells caused a rapid decay of ornithine decarboxylase (ODC) activity and the accumulation of a ODC-antizyme complex. The induction of antizyme only partially accounted for the decrease of ODC activity by a direct inhibition of the enzyme. However, the antizyme induction was accompanied by a marked reduction of the half-life of ODC. Shift of the cells to an ATP-depleting medium prevented the spermidine-elicited decay of ODC activity as well as the accumulation of ODC-antizyme complex. However, ODC appeared to be stabilized even when ATP depletion was performed 40 min after spermidine addition, in the presence of high levels of antizyme. Similar results were obtained by treating the cells with phenanthroline, a heavy metal chelator and protease inhibitor. These findings indicate that ATP and some metalloprotease(s) may be involved in the degradation pathway of ODC, even in the presence of high levels of polyamines.     

Feedback regulation of polyamine synthesis in Ehrlich ascites tumor cells. Analysis using nonmetabolizable derivatives of putrescine and spormine

Ornithine decarboxylase (ODC) is subject to feedback regulation by the polyamines. Thus, addition of putrescine, spermidine or spermine to cells causes inhibition of ODC mRNA translation. Putrescine and spermine are readily converted into spermidine. Therefore, it is conceivable that the inhibition of ODC synthesis observed in putrescine- and spermine-supplemented cells is instead an effect of spermidine. To examine this possibility we have used two analogs of putrescine and spermine, namely 1,4-dimethylputrescine and 5,8-dimethylspermine, which cannot be converted into spermidine. Both analogs were found to inhibit the incorporation of [³⁵S]methionine into ODC protein to approximately the same extent, suggesting that putrescine as well as spermine exert a negative feedback control of ODC mRNA translation in the cell. In addition to suppressing ODC synthesis, both analogs were found to increase the turnover rate of the enzyme. 5,8-Dimethylspermine caused a marked decrease in the activity of S-adenosylmethionine decarboxylase (AdoMetDC). This effect was not obtained with 1,4-dimethylputrescine, indicating that spermine, but not putrescien, exerts a negative control of AdoMetDC. Treatment with 1,4-dimethylputrescine caused extensive depletion of the cellular putrescine and spermidine content, but accumulation of spermine. 5,8-Dimethylspermine treatment, on the other hand, effectively depleted the spermine content and had less effect on the putrescine and spermidine content, at least initially. Nevertheless, the total polyamine content was more extensively reduced by treatment with 5,8-dimethylspermine than with 1,4-dimethylputrescine. Accordingly, only 5,8-dimethylspermine treatment exerted a significant inhibitory effect on Ehrlich ascites tumor cell growth.     

Early developmental profile of ornithine decarboxylase in the frog, Microhyla ornata and its regulation by polyamines

Ornithine decarboxylase (ODC) activity and polyamine levels were measured during early development of the frog, Microhyla ornata. ODC activity was found to be high and it showed three major peaks during the first 60 hr of development. Putrescine and spermidine levels increased gradually during the above period with little change in spermine. Treatment of developing embryos with exogenous putrescine and spermidine prevented the normal increase in ODC activity. Spermine did not have any significant effect. Addition of ornithine also prevented the increase in ODC activity. Experiment using exogenous ornithine and alpha-methylornithine revealed that formation of putrescine and/or spermidine from ornithine is necessary for the suppression of ODC to occur. Suppression of ODC takes place even if conversion of putrescine to spermidine is blocked, indicating that putrescine, independent of its conversion to spermidine, also plays a role in ODC regulation.     

Feedback regulation of polyamine synthesis in Ehrlich ascites tumor cells. Analysis using nonmetabolizable derivatives of putrescine and spermine

Ornithine decarboxylase (ODC) is subject to feedback regulation by the polyamines. Thus, addition of putrescine, spermidine or spermine to cells causes inhibition of ODC mRNA translation. Putrescine and spermine are readily converted into spermidine. Therefore, it is conceivable that the inhibition of ODC synthesis observed in putrescine- and spermine-supplemented cells is instead an effect of spermidine. To examine this possibility we have used two analogs of putrescine and spermine, namely 1,4-dimethylputrescine and 5,8-dimethylspermine, which cannot be converted into spermidine. Both analogs were found to inhibit the incorporation of [35S]methionine into ODC protein to approximately the same extent, suggesting that putrescine as well as spermine exert a negative feedback control of ODC mRNA translation in the cell. In addition to suppressing ODC synthesis, both analogs were found to increase the turnover rate of the enzyme. 5,8-Dimethylspermine caused a marked decrease in the activity of S-adenosylmethionine decarboxylase (AdoMetDC). This effect was not obtained with 1,4-dimethylputrescine, indicating that spermine, but not putrescine, exerts a negative control of AdoMetDC. Treatment with 1,4-dimethylputrescine caused extensive depletion of the cellular putrescine and spermidine content, but accumulation of spermine. 5,8-Dimethylspermine treatment, on the other hand, effectively depleted the spermine content and had less effect on the putrescine and spermidine content, at least initially. Nevertheless, the total polyamine content was more extensively reduced by treatment with 5,8-dimethylspermine than with 1,4-dimethylputrescine. Accordingly, only 5,8-dimethylspermine treatment exerted a significant inhibitory effect on Ehrlich ascites tumor cell growth.     

Characterization of ornithine decarboxylase and regulation by its antizyme in Thermus thermophilus

Ornithine decarboxylase (ODC), the key enzyme of polyamine biosynthesis was highly purified from the thermophilic bacterium Thermus thermophilus. The enzyme preparation showed a single band on SDS-polyacrylamide gel electrophoresis, a pH optimum of 7.5 and a temperature optimum at 60°C. The native enzyme which is phosphorylated could, upon treatment with alkaline phosphatase, lose all activity. The inactive form could be reversibly activated by nucleotides in the order of NTP>NDP>NMP. When physiological polyamines were added to the purified enzyme in vitro, spermine or spermidine activated ODC by 140 or 40%, respectively, while putrescine caused a small inhibition. The basic amino acids lysine and arginine were competitive inhibitors of ODC, while histidine did not affect the enzyme activity. Among the phosphoamino acids tested, phosphoserine was the most effective activator of purified ODC. Polyamines added at high concentration to the medium resulted in a delay or in a complete inhibition of the growth of T. thermophilus, and in a decrease of the specific activity of ornithine decarboxylase. The decrease of ODC activity resulted from the appearance of a non-competitive inhibitor of ODC, the antizyme (Az). The T. thermophilus antizyme was purified by an ODC-Sepharose affinity column chromatography, as well as by immunoprecipitation using antibodies raised against the E. coli antizyme. The antizyme of E. coli inhibited the ODC of T. thermophilus, and vice versa. The fragment of amino acids 56-292 of the E. coli antizyme, produced as a fusion protein of glutathione S-transferase, did not inhibit the ODC of E. coli or T. thermophilus.