Search

The polyamines, spermidine and spermine, are abundant organic cations participating in many important cellular processes. We have previously shown that the rate-limiting enzyme of polyamine catabolism, spermidine/spermine N-acetyltransferase (SSAT), has an alternative mRNA splice variant (SSATX) which undergoes degradation via nonsense-mediated mRNA decay (NMD) pathway, and that the intracellular polyamine level regulates the ratio of the SSATX and SSAT splice variants. The aim of this study was to investigate the effect of SSATX level manipulation on SSAT activity in cell culture, and to examine the in vivo expression levels of SSATX and SSAT mRNA. Silencing SSATX expression with small interfering RNA led to increased SSAT activity. Furthermore, transfection of SSAT-deficient cells with mutated SSAT gene (which produced only trace amount of SSATX) yielded higher SSAT activity than transfection with natural SSAT gene (which produced both SSAT and SSATX). Blocking NMD in vivo by protein synthesis inhibitor cycloheximide resulted in accumulation of SSATX mRNA, and like in cell culture, the increase of SSATX mRNA was prevented by administration of polyamine analog N, N-diethylnorspermine. Although SSATX/total SSAT mRNA ratio did not correlate with polyamine levels or SSAT activity between different tissues, increasing polyamine levels in a given tissue led to decreased SSATX/total SSAT mRNA ratio and vice versa. Taken together, the regulated unproductive splicing and translation of SSAT has a physiological relevance in modulating SSAT activity. However, in addition to polyamine level there seems to be additional factors regulating tissue-specific alternative splicing of SSAT. [ABSTRACT FROM AUTHOR]

The study of polyamine flux, i.e. the circulating flow of polyamines through the interconnected biosynthetic and catabolic pathways, is of considerable interest because of the established links between the polyamine metabolism and many diseases, such as cancer and diabetes. To study polyamine flux in detail, a novel method based on following the label incorporation from the C, N-labeled polyamine precursors, arginine, methionine and ornithine, into polyamines by LC-MS/MS was implemented. This methodology was tested on three distinct cell lines with different spermidine/spermine- N-acetyltransferase (SSAT) expression levels, i.e. non-transgenic, transgenic and knockout. These trials allowed the identification of the critical conditions for the successful polyamine flux measurement, such as the functional time frame of label incorporation, until plateau phase with the selected precursor is reached. The novel LC-MS/MS-based method for polyamine flux overcame the limitations of previous existing methodologies, with baseline separation of the different polyamine species and the exact quantification of the incorporated label. Moreover, the obtained results clearly show that the increased SSAT expression is associated with accelerated polyamine flux. [ABSTRACT FROM AUTHOR]

Enhanced polyamine catabolism via polyamine acetylation-oxidation elevates the oxidative stress in an organism due to increased production of reactive oxygen species (ROS). We studied a transgenic mouse line overexpressing the rate limiting enzyme in the polyamine catabolism, spermidine/spermine N-acetyltransferase (SSAT) that is characterized by increased putrescine and decreased spermidine and spermine pools. In order to protect the mice from the chronic oxidative stress produced by the activation of polyamine catabolism, the hepatic expression of the transcription factor p53 was found threefold elevated in the transgenic mice. In addition, the prolonged activation of p53 accelerated the aging of transgenic mice and reduced their lifespan (50%). Aging was associated with decreased antioxidant enzyme activities. In the transgenic mice the activities of catalase and Cu, Zn-superoxide dismutase (SOD) were 42 and 23% reduced respectively, while the expression of CYP450 2E1 was 60% decreased and oxidative stress measured as protein carbonyl content was tenfold elevated. In the transgenic mice, the age-related repression of the different antioxidant enzymes served as a protection against the hepatotoxic effects of carbon tetrachloride and thioacetamide. [ABSTRACT FROM AUTHOR]

Ionic interactions are essential for the biological functions of the polyamines spermidine and spermine in mammalian physiology. Here, we describe a simple gram scale method to prepare 1,12-diamino-3,6,9-triazadodecane (SpmTrien), an isosteric charge-deficient spermine analogue. The protonation sites of SpmTrien were determined at pH range of 2.2–11.0 using two-dimensional 1H-15N NMR spectroscopy, which proved to be more feasible than conventional methods. The macroscopic p Ka values of SpmTrien (3.3, 6.3, 8.5, 9.5 and 10.3) are significantly lower than those of 1,12-diamino-4,9-diazadodecane (spermine). The effects of SpmTrien and its parent molecule, 1,8-diamino-3,6-diazaoctane (Trien), on cell growth and polyamine metabolism were investigated in DU145 prostate carcinoma cells. SpmTrien downregulated the biosynthetic enzymes ornithine decarboxylase (ODC) and S-adenosyl- l-methionine decarboxylase and decreased intracellular polyamine levels, whereas the effects of Trien alone were minor. Interestingly, both SpmTrien and Trien were able to partially overcome growth arrest induced by an ODC inhibitor, α-difluoromethylornithine, indicating that they are able to mimic some functions of the natural polyamines. Thus, SpmTrien is a novel tool to influence polyamine interaction sites at the molecular level and offers a new means to study the contribution of the protonation of spermine amino group(s) in the regulation of polyamine-dependent biological processes. [ABSTRACT FROM AUTHOR]

Activation of polyamine catabolism in transgenic mice through an overexpression of spermidine/spermine N1-acetyltransferase (SSAT) results in a massive overaccumulation of the diamine putrescine in most tissues including brain. Putrescine pool in transgenic animals was strikingly expanded in every six brain regions analyzed at present. Pons (23-fold), cerebellum (37-fold), cerebrum (34-fold), and hippocampus (16-fold) showed the greatest increases in putrescine levels. Moreover, the molar ratio of putrescine to spermidine was increased in the different brain regions of the transgenic animals on an average of nearly 40-fold. Upon an exposure of the animals to pentylenetetrazol (PTZ) infusions, a compound known to induce epilepsy-like seizure activity, the SSAT transgenic mice showed significantly elevated seizure threshold to both clonic and tonic convulsions in comparison with their syngenic littermates. This difference, however, disappeared when the animals were treated with ifenprodil prior to PTZ infusions. The latter compound acts as an antagonist of N-methyl-d-aspartate receptor by binding to the polyamine site of the receptor. Overexpression of SSAT likewise appeared to protect the transgenic animals from PTZ-induced neuron loss in the hippocampus. As putrescine is known to serve as a precursor to γ-aminobutyric acid (GABA), we carried out 1H NMR analyses the results of which revealed that the levels of the inhibitory amino acid GABA and its excitatory counterpart glutamate were indistinguishable in syngenic and transgenic animals in all brain regions analyzed. The present results suggest that the frequently observed enhanced accumulation of putrescine in response to brain insults belongs to neuroprotective measures rather than being a cause of the subsequent injury. [Copyright &y& Elsevier]

Overexpression of the rate-limiting enzyme in polyamine catabolism spermidine/spermineN1-acetyltransferase (SSAT) in transgenic (Tg) mouse leads to accumulation of putrescine in the skin and permanent hair loss at the age of 3 wk. The hair follicles of these mice are replaced by dermal cysts and epidermal utriculi. Increased putrescine production is also seen in hyperproliferative cutaneous disorders such as in psoriasis. These disorders are characterized by delayed onset of epidermal differentiation characterized as reduced expression of terminal differentiation markers such as cytokeratins 1/10, and filaggrin and persisting expression of basal cell cytokeratins 5/14 in the suprabasal layers. The use of these markers in immunohistological analysis of SSAT Tg skin clearly showed signs of disturbed differentiation. To exclude the possibility that changes in differentiation originated from underlying connective tissue, we introduced SSAT gene into an established rat epidermal cell line. Organotypic cultures derived from the transfected cells displayed similar changes in their differentiation pattern as keratinocytes in Tg skin. The role of accumulated putrescine in cutaneous changes of SSAT Tg mice was verified by an experiment in which putrescine level was reduced by systemic putrescine biosynthesis inhibition. The putrescine reduction was sufficient to alleviate the cutaneous changes to such an extent that distinct hair regrowth could be seen. These results suggest that the cutaneous changes of SSAT Tg animals are due to disorders of the keratinocyte differentiation. Moreover, they strengthen the view that the proper regulation of polyamine metabolism plays an important role in the keratinocyte maturation. [ABSTRACT FROM AUTHOR]

Mitochondrial dysfunction, either inherited or acquired, is associated with several diseases in humans. Depending on the cell type and location, cells are prone to multiple types of insults that may compromise their proper function. Generally, these insults are overcome by defensive mechanisms but sometimes they lead to sustained damage, requiring the action of scavenging and repair machineries to retain the viability of the cells. As a final measure, severely damaged cells are targeted to a controlled cell death pathway in order to not to compromise the well-being of the whole tissue. The polyamines, spermine and spermidine are essential cellular constituents, participating in many vital functions such as proliferation and differentiation, immune response and scavenging of reactive oxygen species. Therefore, dysregulation of polyamine metabolism is often associated with different pathological states. Polyamine acetylating enzyme spermidine/spermine-N(1)-acetyltransferase is induced by inflammation, drugs and by several other environmental insults. Resulting accelerated polyamine acetylation with accompanying polyamine biosynthesis induction i.e. activation of polyamine futile cycle generates excessive amount of hydrogen peroxide, hampers cell energy metabolism and induces mitochondrial dysfunction and biogenesis. Therefore, the drugs inhibiting polyamine metabolism are valuable in protecting mitochondria and cell energy metabolism. Here we review the current literature focusing on the applicability of chargedeficient polyamine analogs as drugs to modulate polyamine metabolism. Alteration of pK(a) of amino group(s) in a respective analog is achieved by fluorine substitution of hydrogen atom, hydroxylamine substitution of methylamine or by reducing the numbers of carbon atoms between amine groups to two instead of three or four.

Earlier unknown racemic β-methylspermidine (β-MeSpd) and γ-methylspermidine (γ-MeSpd) were -synthesized starting from crotononitrile or methacrylonitrile and putrescine. Lithium aluminum hydride reduction of the intermediate di-Boc-nitriles resulted in corresponding di-Boc-amines, which after deprotection gave target β- and γ-MeSpd's. To prepare α-MeSpd, the starting compound, 3-amino-1-butanol, was converted into N-Cbz-3-amino-1-butyl methanesulfonate, which alkylated putrescine to give (after deprotection of amino group) the required α-MeSpd. Novel β- and γ-MeSpd's in combination with earlier α-MeSpd are useful tools for studying enzymology and cell biology of polyamines.

The role of polyamines in carbon tetrachloride (CCl(4))-induced organ injury was studied in syngenic rats and transgenic rats with activated polyamine catabolism. In syngenic rats, administration of CCl(4) resulted in the induction of hepatic spermidine/spermine N(1)-acetyltransferase (SSAT), accumulation of putrescine, reduction in spermine level and appearance of moderate hepatic injury within 24 h. Upon treatment with CCl(4), transgenic rats overexpressing SSAT displayed induction of both hepatic and pancreatic SSAT, with subsequent accumulation of putrescine and decrease of both spermidine and spermine pools. Administration of CCl(4) in SSAT transgenic rats induced not only massive hepatic injury, but also severe acute necrotizing pancreatitis. Pretreatment of the animals with catabolically stable functional polyamine mimetic, alpha-methylspermidine (MeSpd) prevented pancreatic and hepatic injury in SSAT rats and markedly reduced liver damage in syngenic animals. As assessed by immunostaining of proliferating cell nuclear antigen, MeSpd increased the amount of regenerating hepatocytes in both genotypes. These results show that CCl(4) induces hepatic and pancreatic polyamine catabolism, and the extent of organ damage correlates with the degree of polyamine depletion. Furthermore, MeSpd protects against CCl(4)-induced hepatic and pancreatic damage and promotes tissue regeneration.

Polyamines are essential molecules supporting the structure, conformation, and function of nucleic acids and proteins. We studied stereoisomers of alpha,alpha'-dimethylated spermine [(R,R)-Me(2)Spm, (S,S)-Me(2)Spm, (R,S)-Me(2)Spm] for their ability to provoke DNA condensation and protect DNA from damage. (R,R)- and (R,S)-Me(2)Spm displayed more efficient condensing ability than spermine, with significantly lower EC(50) (concentration for 50% compaction) values (p < or = 0.01). However, spermine exerted slightly more duplex stabilization than Me(2)Spm. Condensation resulted in nanoparticles with hydrodynamic radii between 39.6 and 48.4 nm, and electron microscopy showed the presence of toroids and spheroids. Natural polyamines and stereoisomers of Me(2)Spm protected DNA against DNase digestion and oxidative stress in vitro and against etoposide and oxidative stress in DU145 cells but afforded little protection against UV-C irradiation. Our findings indicate that Me(2)Spm stereoisomers are efficient DNA packaging agents with potential applications in gene delivery. Our study also reveals stereospecificity in DNA interaction and protection against cellular stress.

The natural polyamines are ubiquitous multifunctional organic cations which play important roles in regulating cellular proliferation and survival. Here we present a novel approach to investigating polyamine functions by using optical isomers of MeSpd (alpha-methylspermidine) and Me2Spm (alpha,omega-bismethylspermine), metabolically stable functional mimetics of natural polyamines. We studied the ability of MeSpd and Me2Spm to alter the normal polyamine regulation pathways at the level of polyamine uptake and the major control mechanisms known to affect the key polyamine metabolic enzymes. These include: (i) ODC (ornithine decarboxylase), which catalyses the rate-limiting step of polyamine synthesis; (ii) ODC antizyme, an inhibitor of ODC and polyamine uptake; (iii) SSAT (spermidine/spermine N1-acetyltransferase), the major polyamine catabolic enzyme; and (iv) AdoMetDC (S-adenosyl-L-methionine decarboxylase), which is required for the conversion of putrescine into spermidine, and spermidine into spermine. We show that the stereoisomers differ in their cellular uptake and ability to downregulate ODC and AdoMetDC, and to induce SSAT. These effects are mediated by the ability of the enantiomers to induce +1 ribosomal frameshifting on ODC antizyme mRNA, to suppress the translation of AdoMetDC uORF (upstream open reading frame) and to regulate the alternative splicing of SSAT pre-mRNA. The unique effects of chiral polyamine analogues on polyamine metabolism may offer novel possibilities for studying the physiological functions, control mechanisms, and targets of the natural polyamines, as well as advance therapeutic drug development in cancer and other human health-related issues.

We describe synthesis of alpha-methylated analogues of the natural polyamines and their use as tools in unraveling polyamine functions. Experiments with alpha-methylated spermidine and spermine revealed that the polyamines are exchangeable in supporting cellular growth. Degradation of the analogues by polyamine oxidase disclosed hidden, aldehyde-guided stereospecificity of the enzyme.

Efficient syntheses of metabolically stable alpha-methylspermidine 1, alpha-methylspermine 2, and bis-alpha,alpha'-methylated spermine 3 starting from ethyl 3-aminobutyrate are described. The biological tolerance for these compounds was tested in wild-type mice and transgenic mice carrying the metallothionein promoter-driven spermidine/spermine N(1)-acetyltransferase gene (MT-SSAT). The efficient substitution of natural polyamines by their derivatives was confirmed in vivo with the rats harboring the same MT-SSAT transgene and in vitro with the immortalized fibroblasts derived from these animals. Enantiomers of previously unknown 1-amino-8-acetamido-5-azanonane dihydrochloride 4 were synthesized starting from enantiomerically pure (R)- and (S)-alaninols. The studies with recombinant human polyamine oxidase (PAO) showed that PAO (usually splits achiral substrates) strongly favors the (R)-isomer of 4 that demonstrates for the first time that the enzyme has hidden potency for stereospecificity.