Correlation of changes in transglutaminase activity and polyamine content of neoplastic tissue during the metastatic process

Transglutaminase activity and the levels of the polyamines putrescine, spermidine and spermine were measured in two transplantable rat sarcomata: P8 which metastasises consistently to the lung, and P7 which metastasises infrequently. With the P7 sarcoma no metastases were detected following implantation; similarly, no significant changes occurred in the levels of transglutaminase activity, putrescine, spermidine or spermine during tumour growth. However, with the P8 sarcoma at approx. 30 days after implantation there was a marked decrease in transglutaminase activity, mirrored exactly by a 20-fold increase in the levels of acid-soluble putrescine. Measurement of covalently-bound polyamines in the P8 sarcoma indicated a significant and corresponding decrease in the levels of bound putrescine. The timing of these changes coincided with the time at which the P8 sarcoma was shown to have metastasised, and suggests that the changes observed may be related to this phenomenon.     

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.     

Tissue-specific changes in polyamine levels of neural tissue and fat body in adult crickets

The three major polyamines—putrescine, spermidine, and spermine—were studied and changes of their levels were examined in extracts of cerebral ganglia and fat body from adult Acheta domesticus. In nervous tissue, only spermidine and spermine were present and spermine was two- to three-fold more abundant than spermidine. The polyamine levels were high up to day 3, decreased on day 4, and then remained relatively unchanged up to day 10. The spermidine/spermine ratios decreased during the imaginal life. Higher spermidine titres were observed in the neural tissue of egg-laying females compared to virgin females. In the fat body, putrescine was detected together with spermidine and spermine. Spermidine and spermine levels were two-fold higher than putrescine. Fat body of virgin females contained two times more polyamines than male fat body. Low at emergence, spermidine and spermine concentrations peaked on days 2–3 only in females, and egg-laying was characterized by an increase of putrescine and spermidine titres. Starvation did not change polyamine contents, implying homeostatic regulation of the intracellular polyamine metabolism. These data showing tissue specific changes in polyamine levels during the imaginal life of Acheta domesticus point to the physiological importance of polyamines as possible intracellular regulators during adult insect development. © 1993 Wiley-Liss, Inc.     

Specific interaction of spermine with cibacron blue F3GA

The polyamines, spermine, spermidine, and putrescine, have been shown to bind to Cibacron blue F3GA generating a difference spectrum with a maximum at 685 nm and a minimum at 585 nm, which is characteristic of ionic interactions between the dye and the polyamines. The difference spectral signal vanishes when the charges on the amino groups of the polyamines are neutralized. The magnitude of perturbation of the dye spectrum by the polyamines and, by inference, the capacity to bind to the dye, decrease in the order spermine > spermidine > putrescine. For spermine, the spectral signal of the dye-spermine complex is dependent on the charge state of an aminium group with a pK_a = 8.2.     

Correlation of spermine levels with ovary senescence and with fruit set and development inPisum sativum L.

Separation and quantitation of polyamines from unpollinated pea (Pisum sativum L.) ovaries and young fruits induced by application of gibberellic acid to unpollinated ovaries showed, in both cases, a decrease in putrescine and spermidine levels between anthesis and 4 d later. By contrast, spermine levels increased prior to the onset of senescence of the unpollinated ovaries (3 d post anthesis) and decreased during fruit development. Low levels of putrescine, spermidine and spermine were also observed in young fruits obtained by self-pollination and by treatment of unpollinated ovaries with 2,4-dichlorophenoxyacetic acid. In-vitro culture of ovary explants in a medium containing spermine showed that a reduction of the growth of gibberellic acid-treated unpollinated ovaries was associated with a rise in the level of spermine in the fruits. The results obtained indicate that changes in spermine levels are involved in the control of ovary senescence and of fruit set and development.Abbreviations BA benzyladenine - 2,4-D 2,4-dichlorophen-oxyacetic acid - GA₃ gibberellic acid - HPLC high-performance liquid chromatography     

Seasonal changes of polyamines in habitat adaptation of different ecotypes of reed plants

The leaves of four reed ecotypes (Phragmites communis Trinius) growing in the desert regions of northwest China were investigated for levels of polyamines and activity of arginine decarboxylase (ADC; EC 4.1.1.19) during the growing season of 5 months. The polyamines in the leaves of all reed ecotypes consisted of putrescine, spermidine and spermine. The polyamine levels of the leaves were lower in the swamp reed than in the terrestrial reed ecotypes. Leaf polyamine levels decreased in all ecotypes over the course of the season. Compared to the swamp reed, the terrestrial reed ecotypes maintained higher ADC activity and a predominance of spermine, resulting in a lower ratio of putrescine to spermidine and spermine. It seems that the adaptation of reed plants to drought and saline habitats may be correlated with putrescine synthesis via the ADC pathway, and with a successful conversion of putrescine to spermidine and spermine.     

Participation of polyamines in the flowering of the short-day plant Pharbitis nil

The effect of the exogenous application of polyamines on the flowering induction of the short-day plant Pharbtis nil was investigated. Putrescine, spermidine and spermine applied on the cotyledons of 4-day seedlings had no significant effect on the flowering of this plant under conditions of full induction caused by a 16-hour-long inductive night. Under the conditions of partial induction caused by a 13-hour-long subinductive night, polyamines inhibit or stimulate flowering, depending on the time of application. Also, inhibitors of the biosynthesis of polyamines influenced the flowering process. Analysis of endogenous polyamines revealed significant fluctuations in their content in cotyledons during an inductive night, as well as under continuous light conditions. Particularly large changes occurred in spermidine and spermine levels. The putrescine level in induced seedlings was lower than in non-induced ones. However, induced seedlings contained a higher level of spermine and spermidine. The highest spermidine and spermine levels were observed at the 8th h of the night, although the total concentration of spermine during photoinduction was always 2–3 times lower than that of spermidine. A break in the inductive night, leading to a complete inhibition of flowering, had caused significant changes in the polyamine level by the end of the night. The results suggest that the flowering induction of Pharbitis nil took place at a low putrescine level and increased spermidine and spermine levels.     

Polyamine regulation of ornithine decarboxylase and its antizyme in intestinal epithelial cells

Ornithine decarboxylase (ODC) is feedback regulated by polyamines. ODC antizyme mediates this process by forming a complex with ODC and enhancing its degradation. It has been reported that polyamines induce ODC antizyme and inhibit ODC activity. Since exogenous polyamines can be converted to each other after they are taken up into cells, we used an inhibitor of S-adenosylmethionine decarboxylase, diethylglyoxal bis(guanylhydrazone) (DEGBG), to block the synthesis of spermidine and spermine from putrescine and investigated the specific roles of individual polyamines in the regulation of ODC in intestinal epithelial crypt (IEC-6) cells. We found that putrescine, spermidine, and spermine inhibited ODC activity stimulated by serum to 85, 46, and 0% of control, respectively, in the presence of DEGBG. ODC activity increased in DEGBG-treated cells, despite high intracellular putrescine levels. Although exogenous spermidine and spermine reduced ODC activity of DEGBG-treated cells close to control levels, spermine was more effective than spermidine. Exogenous putrescine was much less effective in inducing antizyme than spermidine or spermine. High putrescine levels in DEGBG-treated cells did not induce ODC antizyme when intracellular spermidine and spermine levels were low. The decay of ODC activity and reduction of ODC protein levels were not accompanied by induction of antizyme in the presence of DEGBG. Our results indicate that spermine is the most, and putrescine the least, effective polyamine in regulating ODC activity, and upregulation of antizyme is not required for the degradation of ODC protein.     

Increased blood spermine levels decrease the cytotoxic activity of lymphokine-activated killer cells: a novel mechanism of cancer evasion

Increased blood polyamine levels, often observed in cancer patients, have negative impacts on patient prognosis and are associated with tumor progression. The purpose of our study was to examine the effects of polyamines on cellular immune function. Peripheral blood mononuclear cells (PBMCs) from healthy volunteers were cultured with the human natural polyamines spermine, spermidine, or putrescine, and the effects on immune cell function were examined. The correlation between post-operative changes in blood polyamine levels and lymphokine-activated killer (LAK) activity was also examined in cancer patients. Spermine decreased the adhesion of non-stimulated PBMCs to tissue culture plastic in a dose- and a time-dependent manner without affecting cell viability or activity. This decrease in adhesion capacity was accompanied by a decrease in the number of CD11a bright-positive and CD56 bright-positive cells. Upon stimulation with interleukin 2 to activate LAK cytotoxicity, PBMCs cultured overnight with 100 or 500 μM spermine showed decreased cytotoxic activity against Daudi cells (91.5 ± 1.7 and 84.9 ± 3.0%, respectively (n = 6) compared to PBMC cultured without polyamines). In a group of 25 cancer patients, changes in blood spermine levels after surgery were negatively correlated with changes in LAK cytotoxicity after surgery (r = −0.510, P = 0.008: n = 25). Increased blood spermine levels may be an important factor in the suppression of anti-tumor immune cell function.     

Cytoplasmic polyamines block the fast-activating vacuolar cation channel

The fast-activating vacuolar (FV) channel dominates the electrical characteristics of the tonoplast at physiological free Ca²⁺ concentrations. Since polyamines are known to increase in plant cells in response to stress, the regulation of FV channels by polyamines was investigated. Patch-clamp measurements were performed on whole barley ( Hordeum vulgare ) mesophyll vacuoles and on excised tonoplast patches. The trivalent polyamine spermidine and the tetravalent polyamine spermine blocked FV channels with K_d≈ 100 μM and K_d≈ 5 μM, respectively. Increasing cytosolic and vacuolar Ca²⁺ had no effect on putrescine and spermidine binding to FV channels but slightly decreased the affinity for spermine. The inhibition of FV channels by all three polyamines was not voltage-dependent. This points to a different mode of binding compared to inward rectifier K⁺ channels and Ca²⁺-permeable glutamate receptor channels from animal cells, which show rectification due to a voltage-dependent block by polyamines. In plant cells, the common polyamines (putrescine, spermidine and spermine) are likely to mediate a salt stress-induced decrease of ion flux across the vacuolar membrane by blocking FV channels.     

Androgenic control of polyamine concentrations in rat epididymis.

Unilateral orchidectomy resulted in a significant decrease in tissue content of putrescine and polyamines. However, no differences were detected when the results were expressed in terms of ng g-1 tissue. At 48 h after bilateral orchidectomy, a significant decrease in putrescine content was observed, but spermidine and spermine content were unaffected. The observed decrease in putrescine was prevented by treatment with testosterone propionate, but neither spermidine nor spermine were affected. Bilateral orchidectomy resulted in a significant decrease in the tissue content of putrescine, spermidine and spermine after 7 days. Treatment with testosterone propionate increased the content of putrescine, spermidine and spermine in the epididymis by about 200%, 92% and 34%, respectively. When results were expressed as nmol g-1, a significant decrease after castration in putrescine and spermidine, but not in spermine, was observed. Treatment with testosterone propionate restored putrescine concentration, but had no effect on spermidine and spermine concentrations. In castrated rats treated with testosterone propionate, the anti-androgen flutamide abolished the effect of the androgen on putrescine and spermidine content, but there was no effect on spermine. Acetylputrescine was not detected in the epididymis, while acetylpolyamines were detected at much lower concentrations than polyamines. After bilateral orchidectomy there was a decrease in the tissue content of all acetylpolyamines and an increase in their tissue concentration. The effect of castration on acetylpolyamine content was reversed by testosterone propionate treatment. We conclude that an active synthesis of polyamines occurs in the rat epididymis, and that this process depends upon the androgen environment. Regulation of ornithine decarboxylase activity appears to be the main step that is controlled by androgens.     

Polyamine auxotrophs of Saccharomyces cerevisiae.

Strains of yeast have been constructed that are unable to synthesize ornithine and are thereby deficient in polyamine biosynthesis. These strains were used to develop a protocol for isolation of mutants blocked directly in polyamine synthesis. There were seven mutants isolated that lack ornithine decarboxylase activity; these strains exhibited greatly decreased pool levels of putrescine, spermidine, and spermine when grown in the absence of polyamines. Three of the mutants lack S-adenosylmethionine decarboxylase activity; polyamine limitation of a representative mutant resulted in an accumulation of putrescine and a decrease in spermidine and spermine. When the mutants were cultured in the absence of polyamines, a continuously declining growth rate was observed.     

Oxidative stress promotes ocular neovascularization

The polyamines spermidine, spermine, and putrescine are intimately involved in and required for cell growth and proliferation. There are also multiple effects of polyamines on other cellular processes that seem not to be a result of changes in protein expression. It is a daunting task to classify and understand cellular effects of endogenous polyamines. There has been no central hypothesis how these effects can occur or how spermine and spermidine could be targeted to various signal transduction cascades. However, now there is evidence that multiple effects of endogenous polyamines on different cellular processes may involve plasma membrane PI(4,5)P₂ and recent evidence of how polyamines could be targeted to specific cellular functions. J. Cell. Physiol. 221: 544–551, 2009. © 2009 Wiley‐Liss, Inc.     

Acrolein produced from polyamines as one of the uraemic toxins

It is well known that the addition of spermine or spermidine to culture medium containing ruminant serum inhibits cellular proliferation. This effect is caused by the products of oxidation of polyamines that are generated by serum amine oxidase. Among the products, we found that acrolein is a major toxic compound produced from spermine and spermidine by amine oxidase. We then analysed the level of polyamines (putrescine, spermidine and spermine) and amine oxidase activity in plasma of patients with chronic renal failure. It was found that the levels of putrescine and the amine oxidase activity were increased, whereas spermidine and spermine were decreased in plasma of patients with chronic renal failure. The levels of free and protein-conjugated acrolein were also increased in plasma of patients with chronic renal failure. An increase in putrescine, amine oxidase and acrolein in plasma was observed in all cases such as diabetic nephropathy, chronic glomerulonephritis and nephrosclerosis. These results suggest that acrolein is produced during the early stage of nephritis through kidney damage and also during uraemia through accumulation of polyamines in blood due to the decrease in their excretion into urine.     

Polyamine oxidase and tissue transglutaminase activation in rat small intestine by polyamines.

Polyamine degradation was studied in the small intestine from rats fed on a polyamine-supplemented diet. Lactalbumin diet was given to Hooded-Lister rats, with or without 5 mg rat(-1) day(-1) of putrescine or spermidine for 5 days. Polyamine oxidase activity increased with putrescine and spermidine in the diet, whereas spermidine/spermine N(1)-acetyltransferase and diamine oxidase activities were unchanged. We also studied the calcium-dependent and -independent tissue transglutaminase activities, since they can modulate intestinal polyamine levels. Both types of enzymes increased in the cytosolic fraction after putrescine (about 65%) or spermidine (80-100%). Our results indicate that exogenous polyamines stimulate intestinal polyamine oxidase and tissue transglutaminase activities, probably to prevent polyamine accumulation, when other pathways of polyamine catabolism (acetylation and terminal catabolism) are not activated.     

Transglutaminase activity is involved in polyamine-induced programmed cell death.

Natural polyamines, i.e., putrescine, spermidine, and spermine, are ubiquitous molecules essential for cell proliferation and differentiation. In the present study, the effect of polyamines on primary cultures of bovine aortic endothelial cells (BAECs), rat aortic smooth muscle cells (RASMCs), and a human melanoma cell line was examined. While in the absence of fetal calf serum (FCS) polyamines had no effect on viability, in the presence of FCS spermidine and spermine, at concentrations close to physiologic levels, induced a dose-dependent cell death, whereas putrescine was ineffective. RASMCs were significantly more sensitive than other cells. FACS analysis, oligo-nucleosome ELISA, Hoechst nuclear staining, and Annexin V-FITC quantification showed that cell death was likely due to apoptosis. Cells exposed to spermidine showed a marked increase of intracellular transglutaminase (TGase) activity ( approximately 30-fold over control). Inhibitors of polyamine oxidation or inhibitors of TGase activity prevented polyamine-induced apoptosis. Moreover, tissue TGase overexpression significantly increased cell sensitivity to polyamine, suggesting that this effect is likely related to enhanced intracellular TGase activity. These data indicate that polyamines may modulate cell viability through a novel TGase-dependent process.     

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.     

Spermine facilitates recovery from drought but does not confer drought tolerance in transgenic rice plants expressing <Emphasis Type="Italic">Datura stramonium</Emphasis><Emphasis Type="Italic">S</Emphasis>-adenosylmethionine decarboxylase

Polyamines are known to play important roles in plant stress tolerance but it has been difficult to determine precise functions for each type of polyamine and their interrelationships. To dissect the roles of putrescine from the higher polyamines spermidine and spermine, we generated transgenic rice plants constitutively expressing a heterologous S-adenosylmethionine decarboxylase (SAMDC) gene from Datura stramonium so that spermidine and spermine levels could be investigated while maintaining a constant putrescine pool. Whereas transgenic plants expressing arginine decarboxylase (ADC) produced higher levels of putrescine, spermidine and spermine, and were protected from drought stress, transgenic plants expressing SAMDC produced normal levels of putrescine and showed drought symptoms typical of wild type plants under stress, but the transgenic plants showed a much more robust recovery on return to normal conditions (90% full recovery compared to 25% partial recovery for wild type plants). At the molecular level, both wild type and transgenic plants showed transient reductions in the levels of endogenous ADC1 and SAMDC mRNA, but only wild type plants showed a spike in putrescine levels under stress. In transgenic plants, there was no spike in putrescine but a smooth increase in spermine levels at the expense of spermidine. These results confirm and extend the threshold model for polyamine activity in drought stress, and attribute individual roles to putrescine, spermidine and spermine.     

Induction of spermidine/spermine N1-acetyltransferase in rat tissues by polyamines.

Treatment of rats with spermidine, spermine or sym-norspermidine led to a substantial induction of spermidine/spermine N1-acetyltransferase activity in liver, kidney and lung. The increase in this enzyme, which was determined independently of other acetylases by using a specific antiserum, accounted for all of the increased acetylase activity in extracts from rats treated with these polyamines. Spermine was the most active inducer, and the greatest effect was seen in liver. Liver spermidine/spermine N1-acetyltransferase activity was increased about 300-fold within 6 h of treatment with 0.3 mmol/kg doses of spermine; activity in kidney increased 30-fold and activity in the lung 15-fold under these conditions. The increased spermidine/spermine N1-acetyltransferase activity led to a large increase in the liver putrescine content and a decline in spermidine. These changes are due to the oxidation by polyamine oxidase of the N1-acetylspermidine formed by the acetyltransferase. Our results indicated that spermidine was the preferred substrate in vivo of the acetylase/oxidase pathway for the conversion of the higher polyamines into putrescine. The induction of the spermidine/spermine N1-acetyltransferase by polyamines may provide a mechanism by which excess polyamines can be removed.     

Inhibition of spermidine and spermine synthesis leads to growth arrest of rat embryo fibroblasts in G1

Following growth stimulation of rat embryo fibroblast (REF) cells previously arrested in G₁ by serum deprivation, there occurs a large increase in the synthesis of the polyamines putrescine, spermidine and spermine. Methylglyoxal bis(guanylhydrazone) (MGBG), a potent inhibitor of S-adenosylmethionine decarboxylase can block the accumulation of both spermidine and spermine over a period of several days. Under such conditions REF cells treated with MGBG will approximately double in number and then become growth-arrested again predominantly in the G₁ phase of the cell cycle. REF cells therefore appear to contain sufficient spermidine and spermine to progress through one cell cycle before the intracellular levels of these polyamines is reduced sufficiently to arrest growth in the absence of continued polyamine synthesis. Limitation of intracellular polyamine levels is therefore not the mechanism by which deprivation of serum growth factors arrests cell growth. While continued growth is nevertheless dependent on polyamine synthesis, this cell type is capable of limited proliferation in its absence. Addition of spermidine or spermine to MGBG-arrested REF cells results in a rapid resumption of proliferation demonstrating that either polyamine can fulfill the role played by these polyamines in the growth process. Low levels of spermidine and spermine therefore arrest this cell type at a resriction point in G₁ at which it is decided whether the intracellular level of these polyamines is sufficiently high to enable a cell to enter into and complete a new cell cycle. This polyamine-sensitive restriction point is considered to be analogous to the restriction point(s) in G₁ at which serum and nutrient limitation act.