Enzymes Of Polyamine Metabolism Research Articles

A Charge-Deficient Analogue of Spermine with Chelating Properties

1,12-Diamino-3,6,9-triazadodecane, a new isosteric and charge-deficient analogue of spermine, is synthesized. Unlike spermine, the new analogue is an excellent chelator of Cu2+ ions. Possible applications of this compound for studying enzymes of polyamine metabolism and cellular functions of spermine are discussed. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2005, vol. 31, no. 3; see also http://www.maik.ru.

Polyamine metabolism in a member of the phylum Microspora (Encephalitozoon cuniculi): effects of polyamine analogues.

The uptake, biosynthesis and catabolism of polyamines in the microsporidian parasite Encephalitozoon cuniculi are detailed with reference to the effects of oligoamine and arylamine analogues of polyamines. Enc. cuniculi, an intracellular parasite of mammalian cells, has both biosynthetic and catabolic enzymes of polyamine metabolism, as demonstrated in cell-free extracts of mature spores. The uptake of polyamines was measured in immature, pre-emergent spores isolated from host cells by Percoll gradient. Spermine was rapidly taken up and metabolized to spermidine and an unknown, possibly acetamidopropanal, by spermidine/spermine N(1)-acetyltransferase (SSAT) and polyamine oxidase (PAO). Most of the spermidine and the unknown product were found in the cell incubation medium, indicating they were released from the cell. bis(Ethyl) oligoamine analogues of polyamines, such as SL-11144 and SL-11158, as well as arylamine analogues [BW-1, a bis(phenylbenzyl) 3-7-3 analogue] blocked uptake and interconversion of spermine at micromolar levels and, in the case of BW-1, acted as substrate for PAO. The Enc. cuniculi PAO activity differed from that found in mammalian cells with respect to pH optimum, substrate specificity and sensitivity to known PAO inhibitors. SL-11158 inhibited SSAT activity with a mixed type of inhibition in which the analogue had a 70-fold higher affinity for the enzyme than the natural substrate, spermine. The interest in Enc. cuniculi polyamine metabolism and the biochemical effects of these polyamine analogues is warranted since they cure model infections of Enc. cuniculi in mice and are potential candidates for human clinical trials.

Catabolism of polyamines.

Owing to the establishment of cells and transgenic animals which either lack or over-express acetylCoA:spermidine N(1)-acetyltransferase a major progress was made in our understanding of the role of polyamine acetylation. Cloning of polyamine oxidases of mammalian cell origin revealed the existence of several enzymes with different substrate and molecular properties. One appears to be identical with the polyamine oxidase that was postulated to catalyse the conversion of spermidine to putrescine within the interconversion cycle. The other oxidases are presumably spermine oxidases, because they prefer free spermine to its acetyl derivatives as substrate. Transgenic mice and cells which lack spermine synthase revealed that spermine is not of vital importance for the mammalian organism, but its transformation into spermidine is a vitally important reaction, since in the absence of active polyamine oxidase, spermine accumulates in blood and causes lethal toxic effects. Numerous metabolites of putrescine, spermidine and spermine, which are presumably the result of diamine oxidase-catalysed oxidative deaminations, are known as normal constituents of organs of vertebrates and of urine. Reasons for the apparent contradiction that spermine is in vitro a poor substrate of diamine oxidase, but is readily transformed into N(8)-(2-carboxyethyl)spermidine in vivo, will need clarification.Several attempts were made to establish diamine oxidase as a regulatory enzyme of polyamine metabolism. However, diamine oxidase has a slow turnover. This, together with the efficacy of the homeostatic regulation of the polyamines via the interconversion reactions and by transport pathways renders a role of diamine oxidase in the regulation of polyamine concentrations unlikely. 4-Aminobutyric acid, the product of putrescine catabolism has been reported to have antiproliferative properties. Since ornithine decarboxylase and diamine oxidase activities are frequently elevated in tumours, it may be hypothesised that diamine oxidase converts excessive putrescine into 4-aminobutyric acid and thus restricts tumour growth and prevents malignant transformation. This function of diamine oxidase is to be considered as part of a general defence function, of which the prevention of histamine and cadaverine accumulation from the gastrointestinal tract is a well-known aspect.

Molecular characterization of 5'-deoxy-5'-methylthioadenosine phosphorylase-deficient mutant clones of murine lymphoma cell line R1.1.

5'-Deoxy-5'-methylthioadenosine phosphorylase (MTAP), the polyamine and purine metabolic enzyme, is ubiquitously expressed in normal human tissues and cells, but is deficient in a variety of human and murine malignant cell lines. MTAP-deficient mutants were previously selected from murine lymphoma cell line R1.1 that contains abundant MTAP activity, to analyze the metabolic consequences of MTAP deficiency. Two mutants, one with partial deficiency (F clone) and the other with complete deficiency (H5 clone), were found to have the MTAP protein by immunoblotting. However, the molecular mechanism of enzyme deficiency in these mutants has not been established. In this study, we cloned the mouse cDNA and analyzed MTAP mRNA in the two mutants by reverse transcription-polymerase chain reaction (RT-PCR) followed by direct sequencing. Both mutants have a single nucleotide substitution at the third base of codon 223, which results in a change of cysteine to tyrosine (C223Y). The MTAP mRNA level determined by RT-PCR was significantly lower in the mutant with complete deficiency than in wild-type (WT) R1.1 cells, whereas the mRNA level in the mutant with partial deficiency was comparable to that in WT R1.1 cells. C223Y mutation may cause conformational alteration of the methylthioribose-binding site and decrease the substrate binding. Thus, C223Y may account for the partial deficiency in the F clone, but the complete deficiency with a remarkably low MTAP mRNA level in the H5 clone may be a result of transcriptional abnormality in addition to C223Y mutation.

Polyamines and prostatic cancer.

The importance of polyamines in prostatic growth and differentiation has prompted studies to evaluate the clinical relevance of the ornithine decarboxylase/polyamine system in prostatic cancer. These studies show that differences in biological behaviour of prostatic (cancer) cells are associated with changes in polyamine levels and/or the activity of their metabolic enzymes. Faulty antizyme regulation of polyamine homoeostasis may play an important role in the growth and progression of prostatic carcinoma. Treatment of human prostate carcinoma cells with inhibitors of polyamine metabolic enzymes or polyamine analogues induces cell growth arrest or (apoptotic) cell death. Our recent in vitro studies using conformationally restricted polyamine analogues show that these compounds inhibit cell growth, probably by inducing antizyme-mediated degradation of ornithine decarboxylase. Sensitivity of human prostate cancer cells for these compounds was increased in the absence of androgens. These results suggest that these analogues might have chemotherapeutic potential in case prostatic cancer has become androgen-independent. Pilot data in an in vivo model show that these analogues have effects on tumour cell proliferation, vascularity, blood perfusion and tissue hypoxia. Overall, these studies show that polyamines may serve as important biomarkers of prostatic malignancy and provide a promising target for chemotherapy of prostatic cancer.

The polyamine oxidase inactivator MDL 72527.

Polyamine oxidase is a FAD-dependent amine oxidase, which is constitutively expressed in nearly all tissues of the vertebrate organism. In 1985, N1,N4-bis(2,3-butadienyl)-1,4-butanediamine (MDL 72527) was designed as a selective enzyme-activated irreversible inhibitor of polyamine oxidase (EC 1.5.3.11). It inactivates, at micromolar concentration and time-dependently, the enzyme in cells, as well as in all organs of experimental animals, without inhibiting other enzymes of polyamine metabolism. MDL 72527 served during nearly two decades as a unique tool in the elucidation of the physiological roles of polyamine oxidase. The compound has anticancer and contragestational effects, and it improves the anticancer effect of the ornithine decarboxylase inactivator (D,L)-2-(difluoromethyl)ornithine (DFMO). Profound depletion of the polyamine pools of tumour cells and effects on different components of the immune defence system are responsible for the anticancer effects of MDL 72527/DFMO combinations. Recently a direct cytotoxic effect of MDL 72527 at concentrations above those required for polyamine oxidase inactivation was observed. The induction of apoptosis by MDL 72527 was ascribed to its lysosomotropic properties. Therapeutic potentials of the apoptotic effect of MDL 72527 need to be explored. Polyamine oxidase is the last enzyme of the polyamine interconversion pathway that awaits the detailed elucidation of its structure and regulation. MDL 72527 should be useful as a lead in the development of inactivators which are selective for the isoforms of polyamine oxidase. Isozyme-selective inhibitors will give more profound insights into and reveal a diversity of specific functions of polyamine oxidase.

Polyamine metabolism in brain tumours: diagnostic relevance of quantitative biochemistry

OBJECTIVEActivation of polyamine metabolism is closely associated with cellular proliferation. The purpose was to investigate whether the content of the polyamines putrescine, spermidine, and spermine, and the activity of the...

Androgen responsiveness and intrarenal localization of transcripts coding for the enzymes of polyamine metabolism in the mouse

Polyamines, spermidine (SPD), and spermine (SPM) are intracellular polycations required for cell growth and differentiation. Their biosynthetic precursor, the diamine putrescine (PUT), is produced by regulatory ornithine decarboxylase (ODC). Spermidine/spermine N1-acetyltransferase (SSAT) is the ODC counterpart in the degradation pathway which retroconverts SPM and SPD into PUT. Castration of male mice for 7 days resulted in a 40% decrease of the renal levels of both SSAT and ODC transcripts. Administration of 5-α-dihydrotestosterone (DHT) to castrated mice for the last 3 days before sacrifice caused the levels of ODC and SSAT mRNAs to increase by 250% and 180%, respectively. Thus activation of the retroconversion pathway of polyamine metabolism appears to contribute towards the increase in PUT production known to be caused by androgens in the mouse kidney. In situ hybridization histochemistry experiments showed that the SSAT transcript is expressed only by the epithelial cells of the straight and convoluted distal tubules of the nephron, while the expression of the ODC transcript is confined to the epithelium of the convoluted and straight portion of the proximal tubules. The separation of the biosynthetic from the degradation pathway along the nephron suggests that PUT is mostly produced in the distal tubule, where it may play a physiological role, independent of androgen action, in protecting tubular cells from the very low osmolarity to which they are exposed in this nephron segment.Key words: Adenosylmethionine decarboxylase, spermidine/spermine N1-acetyltransferase, ornithine decarboxylase, mouse kidney, polyamines.

Androgen responsiveness and intrarenal localization of transcripts coding for the enzymes of polyamine metabolism in the mouse

Polyamines, spermidine (SPD), and spermine (SPM) are intracellular polycations required for cell growth and differentiation. Their biosynthetic precursor, the diamine putrescine (PUT), is produced by regulatory ornithine decarboxylase (ODC). Spermidine/spermine N1-acetyltransferase (SSAT) is the ODC counterpart in the degradation pathway which retroconverts SPM and SPD into PUT. Castration of male mice for 7 days resulted in a 40% decrease of the renal levels of both SSAT and ODC transcripts. Administration of 5-alpha-dihydrotestosterone (DHT) to castrated mice for the last 3 days before sacrifice caused the levels of ODC and SSAT mRNAs to increase by 250% and 180%, respectively. Thus activation of the retroconversion pathway of polyamine metabolism appears to contribute towards the increase in PUT production known to be caused by androgens in the mouse kidney. In situ hybridization histochemistry experiments showed that the SSAT transcript is expressed only by the epithelial cells of the straight and convoluted distal tubules of the nephron, while the expression of the ODC transcript is confined to the epithelium of the convoluted and straight portion of the proximal tubules. The separation of the biosynthetic from the degradation pathway along the nephron suggests that PUT is mostly produced in the distal tubule, where it may play a physiological role, independent of androgen action, in protecting tubular cells from the very low osmolarity to which they are exposed in this nephron segment.

Inhibitory effects of S-methylcysteine and cysteine on the promoting potential of sodium phenobarbital on rat liver carcinogenesis.

The effects of S‐methylcysteine (SMC) and eysteine on the promotion stages of rodent hepatocar‐cinogenesis in a medium‐term bioassay previously developed by Ito were examined. Initiation was induced by a single dose of diethylnitrosamine (DEN), followed by dietary administration of the promoter sodium phenobarbital (NaPB) 2 weeks later, for 6 weeks. Partial hepatectomy was conducted on all the animals at week 3. Inhibitory potential was evaluated by analyzing two markers of carcinogenesis, namely numbers of glutathione S‐transferase placental form (GST‐P)‐positive foci, and proliferating cell nuclear antigen (PCNA). In addition, the level of ornithine decarboxylase (ODC), one of the rate‐limiting enzymes of polyamine metabolism induced by promoters, was analyzed. SMC and cysteine induced significant reduction in the areas of GST‐P‐positive foci. A significant reduction in the PCNA index was observed in the entire liver as well as in GST‐P‐positive areas. SMC also induced down‐regulation of the ODC enzyme activity. Thus, SMC and cysteine were found to inhibit the promotion stage of DEN‐induced hepatocarcinogenesis. No cocarcinogenic effects were evident on administration of either of these chemicals with NaPB.

Sequence Analysis of 139 kb in Xp22.1 Containing Spermine Synthase and the 5′ Region of PEX

Human Xp22.1 contains genes involved in mineral balance that are implicated in X-linked hypophosphatemia (XLH) in humans, its murine homologue (Hyp), and another distinct murine hypophosphatemic disorder (Gy). In XLH, a gene, PEX, has been found to be mutated in up to 83% of patients but the sequences of the promoter and 5′ end have not been characterized. To further the understanding of this genomic region, 139,454 bp in Xp22.1 have been sequenced. Our analysis confirms the three most 5′ published exons of PEX and extends through a putative PEX promoter region. The 5′ untranslated sequence of PEX and the mouse and rat equivalents are very highly homologous, implying a conserved functional significance. In addition, we mapped and analyzed another gene 5′ of PEX, spermine synthase (SpS), which encodes a ubiquitous enzyme of polyamine metabolism that may contribute to the pathophysiology ofGy.SpS consists of 11 exons spread over 54 kb. The definition of the locations of SpS and the putative promoter region of PEX will facilitate functional analysis of these genes.

Inhibition by 1α,25-Dihydroxyvitamin D3of Activin A-Induced Differentiation of Murine Erythroleukemic F5-5 Cells

1α,25-Dihydroxyvitamin D3(1α,25-(OH)2D3) and other vitamin D3(VD3) analogs enhanced the inhibitory effect of Activin A on murine erythroleukemia (MEL) cell proliferation and differentiation in a dose-dependent manner. 1α,25-(OH)2D3inhibited differentiation more potently than proliferation by one order of magnitude. The VD3analog study demonstrated either effect of VD3on MEL cells via vitamin D receptor (VDR), as evidenced from the close relationship with the reported affinities for VDR. The effects of 1α,25-(OH)2D3were preceded by the suppression of ornithine decarboxylase (ODC) activity, a rate-limiting enzyme in polyamine metabolism. Difluoromethylornithine (DFMO), an inhibitor of ODC, inhibited MEL cell proliferation, which was reversed by the simultaneous addition of putrescine, a product of ODC, but did not affect differentiation. 1α,25-(OH)2D3inhibited cell differentiation during the phenotype-expression stage as reflected by the inhibition of β-globin gene expression, while it inhibited proliferation in the commitment stage. Furthermore, it seems unlikely that the different effects of VD3on proliferation and differentiation may be a result of up-regulation of VDR or nongenomic action. In summary, it was suggested that 1α,25-(OH)2D3inhibited Activin A-induced MEL cell proliferation and differentiation by distinct mechanisms and inhibited the proliferation by inhibiting ODC activity. We demonstrated the presence of 1α,25-(OH)2D3action on leukemic cells at physiological concentration, which was distinct from the pharmacological effect of VD3reported thus far.

Inhibition of spermidine synthase gene expression by transforming growth factor-beta 1 in hepatoma cells.

We screened genes responsive to transforming growth factor-beta (TGF-beta 1) protein in a human hepatoma cell line (Hep3B) using a PCR-mediated differential display technique, in order to investigate the mechanisms involved in TGF-beta-induced growth suppression. We found a gene that was down-regulated by TGF-beta 1 to be completely identical in an approx. 620 bp segment to the gene for the enzyme spermidine synthase, which mediates the conversion of putrescine into spermidine. Both spermidine synthase mRNA expression and its enzyme activity were decreased after TGF-beta 1 treatment of Hep3B cells. The inhibition of spermidine synthase gene expression by TGF-beta 1 protein was also observed in other hepatoma cell lines. The expression of genes for other biosynthetic enzymes in polyamine metabolism (ornithine decarboxylase and S-adenosylmethionine decarboxylase) was also inhibited to the same extent as for spermidine synthase, while the gene expression of spermidine/spermine N1-acetyltransferase, a catabolic enzyme, was relatively resistant to TGF-beta 1. Spermine levels in Hep3B cells were decreased by TGF-beta 1 treatment, although the levels of spermidine and putrescine were unchanged, probably due to compensation by remaining spermidine/spermine N1-acetyltransferase activity. Exogenously added spermidine or spermine, but not putrescine, partially antagonized the growth-inhibitor effects of TGF-beta 1 on Hep3B cells. Our data suggest that down-regulation of gene expression of the enzymes involved in polyamine metabolism, including spermidine synthase, may be associated with the mechanism of TGF-beta-induced growth suppression.

Polyamine levels and ODC activity in intestinal-type and diffuse-type gastric carcinoma.

Gastric carcinomas are divided into two types according to Lauren's classification: intestinal and diffuse types. Polyamines (putrescine, spermidine, and spermine) are polycations involved in neoplastic growth of gastrointestinal mucosa. A key role is also played by ornithine decarboxylase, the rate limiting enzyme in polyamine metabolism. Our aim was to investigate whether there were differences between the two types of tumor in polyamine metabolism. Twenty-seven patients with gastric carcinoma entered the study. Seventeen carcinomas were classified as diffuse type and 10 as intestinal type. Polyamine levels were evaluated by high-performance liquid chromatography. Ornithine decarboxylase activity was measured by a radiometric technique. Polyamine levels and ornithine decarboxylase activity were significantly higher in intestinal type samples than diffuse type samples. A similarity of polyamine levels in intestinal type samples with levels previously observed in patients with colorectal adenocarcinoma was also found. These findings show a different proliferative behavior of these two types of tumor, and therefore different therapeutic strategies can be hypothesized.

Effects of chronically elevated growth hormone levels on polyamine metabolism in elderly transgenic mice

The polyamines are ubiquitous, multifunctional aliphatic amines with roles in cell growth, proliferation, differentiation, and malignant development. After growth stimulation, rapid and transient changes occur in polyamine regulatory enzymes. In this respect, acute effects of growth hormone (GH) injection on polyamine metabolic enzymes have earlier been shown. The present investigation comprises studies of the effects on polyamine metabolism of constitutively elevated levels of circulating GH in elderly transgenic (tg +) mice, overexpressing bovine GH. Polyamine levels were found to be constitutively altered in the liver and kidney of tg + mice. Less changes were found in the spleen and none in the brain. The cellular uptake of polyamines in the liver from tg + mice showed an increase and considerable changes were observed in the activity of ornithine decarboxylase (ODC) in the liver and kidney and S-adenosylmethionine decarboxylase (AdoMetDC) in the liver. A conspicuous finding was the distribution pattern of ODC protein in the liver in both tg − and tg + animals. The results show that the effects of chronically elevated GH levels are organ-dependent and complex, and differ from acute GH effects. Despite high ODC activity and polyamine levels in liver, these mice did not display any malignant transformation even at an advanced age, indicating that high ODC activity is not sufficient to induce tumorigenesis in vivo.

Molecular cloning of spermidine/spermine N1-acetyltransferase from the periimplantation porcine uterus by messenger ribonucleic acid differential display: temporal and conceptus-modulated gene expression.

Using messenger RNA (mRNA) differential display, we isolated several putative differentially expressed complementary DNAs (cDNAs) from the periimplantation (days 11-12) endometrium of unilaterally pregnant pigs. Nucleotide sequence analysis revealed that one cDNA clone was 87% homologous to human spermidine/ spermine N1-acetyltransferase (SSAT) over a stretch of 201 bp and represents the porcine homologue of this cDNA. A second differentially expressed cDNA encoded the porcine equivalent of the human fragile X mental retardation gene (FMR1), whereas a third specified an open reading frame with significant homology to the Escherichia coli N-acetylglucosamine transfer protein. Because SSAT is the rate-limiting enzyme in polyamine metabolism and polyamines are required cytosolic components for cell growth and differentiation, we characterized the expression of the porcine SSAT gene as a potential marker for endometrial growth and/or differentiation during early pregnancy. Further, using the consensus sequence from human and mouse cDNAs, PCR primers were designed and used to generate a 568-bp cDNA fragment from gravid endometrium that encompassed the entire open reading frame for porcine SSAT and which was subsequently used for Northern hybridization analysis. Two distinct SSAT transcripts, a major species of 1.3 kilobase pairs (kb) and a minor species of 3.5 kb were detected in endometrium, each with similar temporal patterns of expression. The levels of SSAT mRNA were higher (P = 0.03) in gravid than in nongravid uterine endometrium of unilaterally pregnant pigs on days 11-12. Similarly, SSAT mRNAs were more abundant (P = 0.0004) in day 12 pregnant than in day 12 cyclic, and in days 30, 60, 90, and 105 pregnant pig endometria. Uterine endometrial luminal epithelial (LE), glandular epithelial (GE), and stromal (ST) cells expressed the SSAT gene, but mRNA abundance varied among cell types (LE > GE > ST). Expression of SSAT gene in ovariectomized gilts treated with estrogen (E2, 100 microg/day), progesterone (P4, 200 mg/day) or E2 + P4 for 11 days was highest (P = 0.03) in the endometria of the P4 group. In contrast, E2 (10 nM), P4 (10 nM) and E2 + P4 had no effect on SSAT mRNA abundance in uterine endometrial explants from day 12 pregnant pigs. However, steady-state SSAT mRNA levels were induced in day 12 pregnant uterine explants by conditioned medium from day 12 filamentous but not spherical conceptuses. These data demonstrate that the temporal induction of the endometrial SSAT gene during periimplantation is modulated by a factor(s) secreted by the periimplantation conceptus and suggest that this enzyme may have an important role in uterine endometrial growth, remodeling and/or differentiation during periimplantation.

Molecular cloning of spermidine/spermine N1-acetyltransferase from the periimplantation porcine uterus by messenger ribonucleic acid differential display: temporal and conceptus-modulated gene expression

Using messenger RNA (mRNA) differential display, we isolated several putative differentially expressed complementary DNAs (cDNAs) from the periimplantation (days 11-12) endometrium of unilaterally pregnant pigs. Nucleotide sequence analysis revealed that one cDNA clone was 87% homologous to human spermidine/ spermine N1-acetyltransferase (SSAT) over a stretch of 201 bp and represents the porcine homologue of this cDNA. A second differentially expressed cDNA encoded the porcine equivalent of the human fragile X mental retardation gene (FMR1), whereas a third specified an open reading frame with significant homology to the Escherichia coli N-acetylglucosamine transfer protein. Because SSAT is the rate-limiting enzyme in polyamine metabolism and polyamines are required cytosolic components for cell growth and differentiation, we characterized the expression of the porcine SSAT gene as a potential marker for endometrial growth and/or differentiation during early pregnancy. Further, using the consensus sequence from human and mouse cDNAs, PCR primers were designed and used to generate a 568-bp cDNA fragment from gravid endometrium that encompassed the entire open reading frame for porcine SSAT and which was subsequently used for Northern hybridization analysis. Two distinct SSAT transcripts, a major species of 1.3 kilobase pairs (kb) and a minor species of 3.5 kb were detected in endometrium, each with similar temporal patterns of expression. The levels of SSAT mRNA were higher (P = 0.03) in gravid than in nongravid uterine endometrium of unilaterally pregnant pigs on days 11-12. Similarly, SSAT mRNAs were more abundant (P = 0.0004) in day 12 pregnant than in day 12 cyclic, and in days 30, 60, 90, and 105 pregnant pig endometria. Uterine endometrial luminal epithelial (LE), glandular epithelial (GE), and stromal (ST) cells expressed the SSAT gene, but mRNA abundance varied among cell types (LE > GE > ST). Expression of SSAT gene in ovariectomized gilts treated with estrogen (E2, 100 microg/day), progesterone (P4, 200 mg/day) or E2 + P4 for 11 days was highest (P = 0.03) in the endometria of the P4 group. In contrast, E2 (10 nM), P4 (10 nM) and E2 + P4 had no effect on SSAT mRNA abundance in uterine endometrial explants from day 12 pregnant pigs. However, steady-state SSAT mRNA levels were induced in day 12 pregnant uterine explants by conditioned medium from day 12 filamentous but not spherical conceptuses. These data demonstrate that the temporal induction of the endometrial SSAT gene during periimplantation is modulated by a factor(s) secreted by the periimplantation conceptus and suggest that this enzyme may have an important role in uterine endometrial growth, remodeling and/or differentiation during periimplantation.

Hypoosmotic stimulation of ornithine decarboxylase activity in the brine shrimpArtemia franciscana

In the brine shrimp, long-term (several days) osmotic and ionic regulation is controlled principally by the activity of Na+K+ATPase. However, short-term cell volume regulation in Artemia and most euryhaline crustaceans is not understood. In this study, Artemia stage 1 nauplii reared at 32 ppt salinity and exposed to hypoosmotic conditions (12 or 4 ppt) exhibited an immediate increase in the activity of ornithine decarboxylase, the rate-limiting enzyme in polyamine synthesis. ODC activity increased within 15 min relative to control and reached a peak within 2 hr of hypoosmotic exposure. The level of increased enzyme activity was directly related to the degree of hypoosmotic exposure (a three- and sevenfold increase in activity at 12 and 4 ppt salinity, respectively). This increase in activity was apparently due to the synthesis of a new enzyme, based on cycloheximide administration. Activity decreased significantly by 4 hr but remained above that of the 32 ppt control throughout the 24-hr exposure period. Levels of putrescine increased significantly within 2 hr in organisms exposed to 4 ppt; spermidine and spermine levels were not affected. When nauplii were exposed to altered ion and/or osmotic concentrations it was determined that decreased total osmotic concentration was the proximate factor responsible for ODC induction. However, reduced ion concentration resulted in the induction/activation of DFMO-insensitive ODC activity, suggesting the production of a novel enzyme in polyamine metabolism during hypotonic exposure. Changes in ODC activity and polyamines preceded acclimatory changes normally associated with salinity acclimation, further suggesting that modulation of ODC activity and subsequent polyamines synthesis, degradation, and/or transport are mechanistically related to short-term regulation of osmotic and ionic balance. © 1996 Wiley-Liss, Inc.

Polyamines in brain tumor therapy

In the search for ways to augment current brain tumor therapies many have sought to exploit the fact that adult brain tissue is virtually lacking in cell division. This endorses a special appeal to therapeutic approaches which target the dependence on cell division for brain tumor growth. Polyamines play an essential role in the proliferation of mammalian cells and depletion results in inhibition of growth. As a result, there are investigations into the feasibility of controlling tumor growth by targeting the enzymes in polyamine metabolism with specific enzyme inhibitors. DFMO, an inhibitor of putrescine synthesis, is a cytostatic agent which in combination with tritiated radioemitters or cytotoxic agents such as, MGBG or BCNU is an effective antitumor agent, but the effectiveness of DFMO in vivo is reduced by tumor cell uptake of polyamines released into the circulation by normal cells and from gut flora or dietary sources. However, DFMO therapy combined with elimination of exogenous polyamines inhibits tumor growth but also results in body weight loss, reduced protein synthesis and evidence of toxicity. Furthermore, tumor growth recurs upon termination of treatment. In contrast, competitive polyamine analogs function in the homeostatic regulation of polyamine synthesis but fail to fulfill the requirements for growth and they continue to inhibit tumor growth for several weeks after cessation of treatment. Analogs are now in clinical trials. However, their action may be highly specific and differ from one cell type to another. We suggest that the effectiveness of polyamine based therapy would be enhanced by two approaches: local delivery by intracerebral microdialysis and tumor cell killing by internal radioemitters such as tritiated putrescine or tritiated thymidine which are taken up in increased amounts by polyamine depleted tumor cells. The growth inhibition by polyamine depletion prevents the dilution of the radioactive putrescine and thymidine. The overload of radioactivity kills the growth inhibited cells so that growth cannot recur when treatment terminates.

Lithium exerts a time-dependent and tissue-selective attenuation of the dexamethasone-induced polyamine response in rat brain and liver

It has previously been shown that chronic, but not acute, lithium treatment indirectly prevents the dexamethasone-induced increase in brain polyamine-metabolizing enzymes. In the present study we determined the effects of lithium treatment on changes in cellular polyamines, 6 h after dexamethasone challenge (3 mg/kg intraperitoneally). The findings demonstrate that chronic lithium (daily intraperitoneal 2.5 mmol/kg injections for 2 weeks) treatment completely prevents the accumulation of putrescine, in parallel to its prevention of the dexamethasone-induced increase in ornithine decar☐ylase activity. A partial attenuation of this polyamine response was also observed in the liver. Only minor and inconsistent changes were observed in the concentrations of the polyamines, spermidine and spermine. Acute lithium treatment (a single injection at times ranging from 1 to 24 h prior to dexamethasone challenge) did not attenuate the dexamethasone-induced increases in brain putrescine concentration nor in ornithine decar☐ylase activity. It is suggested that prevention of the stress-induced polyamine response in the brain may be an important mechanism through which prophylactic lithium may exert its beneficial effect in manic-depressive illness.