Cellular Polyamine Concentrations Research Articles

Impact of heavy metal lead stress on polyamine levels in Halomonas BVR 1 isolated from an industry effluent

In living systems, environmental stress due to biotic and abiotic factors triggers the production of myriad metabolites as a potential mechanism for combating stress. Among these metabolites are the small polycationic aliphatic amine molecules - polyamines, which are ubiquitous in all living organisms. In this work, we demonstrate a correlation between cellular concentration of three major polyamines (putrescine, spermidine and spermine) with lead exposure on bacteria for a period of 6–24 h. We report that indigenously isolated Halomonas sp. strain BVR 1 exhibits lead induced fluctuations in their cellular polyamine concentration. This response to lead occurs within 6 h post metal treatment. During the same time interval there was a surge in the growth of bacteria along with an enhancement in the putrescine levels. We conclude that in Halomonas sp. strain BVR 1, an early response is seen with respect to modulation of polyamines as a result of lead treatment and hypothesize that endogenous polyamines contribute towards scavenging lead in these bacteria.

A Nascent Peptide Signal Responsive to Endogenous Levels of Polyamines Acts to Stimulate Regulatory Frameshifting on Antizyme mRNA

The protein antizyme is a negative regulator of cellular polyamine concentrations from yeast to mammals. Synthesis of functional antizyme requires programmed +1 ribosomal frameshifting at the 3' end of the first of two partially overlapping ORFs. The frameshift is the sensor and effector in an autoregulatory circuit. Except for Saccharomyces cerevisiae antizyme mRNA, the frameshift site alone only supports low levels of frameshifting. The high levels usually observed depend on the presence of cis-acting stimulatory elements located 5' and 3' of the frameshift site. Antizyme genes from different evolutionary branches have evolved different stimulatory elements. Prior and new multiple alignments of fungal antizyme mRNA sequences from the Agaricomycetes class of Basidiomycota show a distinct pattern of conservation 5' of the frameshift site consistent with a function at the amino acid level. As shown here when tested in Schizosaccharomyces pombe and mammalian HEK293T cells, the 5' part of this conserved sequence acts at the nascent peptide level to stimulate the frameshifting, without involving stalling detectable by toe-printing. However, the peptide is only part of the signal. The 3' part of the stimulator functions largely independently and acts at least mostly at the nucleotide level. When polyamine levels were varied, the stimulatory effect was seen to be especially responsive in the endogenous polyamine concentration range, and this effect may be more general. A conserved RNA secondary structure 3' of the frameshift site has weaker stimulatory and polyamine sensitizing effects on frameshifting.

Crystallization and preliminary crystallographic studies of PotA, a membrane-associated ATPase of the spermidine-preferential uptake system inThermotoga maritima

A membrane-associated ATPase, PotA, is a component of the spermidine-preferential uptake system in prokaryotes that plays an important role in normal cell growth by regulating the cellular polyamine concentration. No three-dimensional structures of membrane-associated ATPases in polyamine-uptake systems have been determined to date. Here, the crystallization and preliminary X-ray diffraction analysis of PotA from Thermotoga maritima are reported. Diffraction data were collected and processed to 2.7 Å resolution from both native and selenomethionine-labelled crystals. Preliminary crystallographic analysis revealed that the crystals belonged to the hexagonal space group P3₁12 (or P3₂12), with unit-cell parameters a=b=88.9, c=221.2 Å, α=90, β=90, γ=120°, indicating that a dimer was present in the asymmetric unit.

Polyamines and carcinogenesis

SUMMARY The naturally occurring polyamines, spermine, spermidine and the diamine putrescine are widespread in nature. They have been implicated in growth and differentiation processes. In 1967, we reported that cancer cells are rich in polyamines. Subsequently, it has been shown that polyamines are released from cancer cells and may be detected in body fluids such as urine, blood and cerebrospinal fluids. It has also been demonstrated that the increase in cellular polyamine levels is an early and an obligatory event in the process of malignant transformation. This increase in cellular polyamine concentration is due to the activation of ornithine decarboxylase (ODC), which catalyses the rate limiting step in polyamine synthesis by converting ornithine to putrescine. Assays of urinary and blood polyamines have been used to detect cancer and to determine the success of therapy. A sensitive, rapid, chemiluminescence-based method for the determination of diamines and polyamines was developed and 2.000 urine samples were tested. An interesting "gene therapy" system for injecting amine oxidases into normal and transformed cells was developed as follows: serum amine oxidase and porcine kidney damine oxidase were trapped within reconstituted Sendai virus envelopes. Chick or rat fibroblasts, transformed by Rous sarcoma virus, were more susceptible to the injected enzymes, compared to the normal culture, when macromolecular synthesis was tested. An in vitro chemosensitivity assay for the testing of the sensitivity of cancer cells from individual patients ("tailored treatment") was also developed. All these studies stress the importance of polyamines in carcinogenesis.

Degrading Polyamines for Migration

Integrins are heterodimeric transmembrane proteins that mediate cell adhesion and migration through various mechanisms. Binding of the cytoplasmic domain of the integrin α9 subunit to the spermidine/spermine- N 1 -acetyltransferase (SSAT)--which induces the breakdown of the higher-order polyamines spermidine and spermine to the lower-order polyamine putrescine--is required for migration mediated by the α9β1 integrin. deHart et al. (see commentary by Vandenberg) report that α9 recruitment of SSAT to focal adhesions affects cell migration by modulating the activity of an inward rectifier K + (Kir) channel. Both wild-type and catalytically inactive forms of in vitro-translated SSAT bound to tagged α9 in a pull-down assay. Expression of any of the catalytically inactive forms in α9-expressing Chinese hamster ovary (CHO) cells prevented migration on an α9-specific ligand in a dominant-negative fashion. This effect was specific to α9, because migration mediated by a chimeric α9α5 protein, in which the intracellular domain of α9 was replaced with the intracellular domain of α5, was not affected by expression of the catalytically inactive SSATs. Depleting intracellular spermidine concentrations, using a combination of the ornithine decarboxylase inhibitor DFMO and putrescine, inhibited migration of CHO cells that overexpressed wild-type α9 but not those expressing the α9α5 chimera. Unexpectedly, restoring spermidine concentration by addition of putrescine in DFMO-treated cells restored migration. Furthermore, in mouse embryo fibroblasts (MEFs) overexpressing α9, but not those overexpressing the chimeric α9α5, migration was reduced when the expression of polyamine oxidase (PAO), an enzyme required for polyamine metabolism downstream of SSAT, was knocked down by small interfering RNAs (siRNAs). Adding putrescine did not restore migration to the PAO-deficient cells. Thus, the results from experiments in which global intracellular polyamine concentrations were altered with pharmacological agents were complex but may indicate that local changes in cellular polyamine concentrations at the focal adhesion regulate migration. K + efflux is implicated in cell migration, and Kir channels are inhibited by spermidine and spermine; thus, SSAT may modulate polyamine concentrations to stimulate migration. Indeed, treating α9- (but not α9α4- or α9α5-) expressing MEFs with Ba 2+ to inhibit Kir channels or with a α9β1-blocking antibody decreased migration of cells on an α9-specific ligand but had no effect on adhesion of cells to nonintegrin-specific substrates. Of the eight Kir subunits detected in MEF cells, only Kir4.2 appeared to have a role in α9-mediated migration. Knockdown of Kir4.2 by a short hairpin RNA (shRNA) or inhibition of Kir channels with Ba 2+ in either α9-expressing MEFs or human microvascular endothelial cells, which natively express Kir4.2 and α9β1, impaired migration on α9 substrates. In the MEFs expressing α9, the effects of DFMO and Kir4.2 shRNA treatment were not additive, suggesting that polyamines regulate cell migration through Kir4.2. In an in vitro wound assay, α9-expressing MEF cells treated with Ba 2+ , Kir4.2 shRNA, or both migrated shorter distances than untreated cells and extended multiple leading-edge cytoplasmic projections, whereas untreated cells usually extended only one lamellopodium. Fluorescently tagged α9 and Kir4.2 colocalized to structures that looked like focal adhesions, but only at the leading edge of MEFs. These results suggest that the α9 subunit of integrins recruits SSAT to focal adhesions, where it decreases the local concentration of higher-order polyamines; this may relieve repression of Kir channels, thus initiating an outward K + flow. The authors model that the K + efflux may cause the influx of Ca 2+ , which is known to stimulate migration in other contexts. G. W. deHart, T. Jin, D. E. McCloskey, A. E. Pegg, D. Sheppard, The α9β1 integrin enhances cell migration by polyamine-mediated modulation of an inward-rectifier potassium channel. Proc. Natl. Acad. Sci. U.S.A. 105 , 7188-7193 (2008). [Abstract] [Full Text] C. A. Vandenberg, Integrins step up the pace of cell migration through polyamines and potassium channels. Proc. Natl. Acad. Sci. U.S.A. 105 , 7109-7110 (2008). [Full Text]

Targeting polyamines ofAspergillusnidulansby siRNA specific to fungal ornithine decarboxylase gene

Aspergillus nidulans is a filamentous, ubiquitous and opportunistic pathogenic fungus, which causes fatal invasive aspergillosis among immunocompromised patients. As a prelude to the investigations on the possible control of human fungal diseases by selective targeting of fungal polyamines that are essential for fungal growth and development by RNAi strategy, we have examined the effect of siRNA specific to a key polyamine biosynthesis gene ornithine decarboxylase (ODC) on the in vitro germinating spores of A. nidulans to assess the ability of RNA species to induce RNA-mediated gene silencing. The present findings show that siRNA can cause specific silencing effect, with the phenotypic consequences leading to significant reduction in mycelial growth, target mRNA titers and cellular polyamine concentrations. This study suggests that fungal polyamine biosynthetic genes, particularly ODC may be targeted for the control of fungal infections in humans and crop plants. To the best of our knowledge, this is the first demonstration of up-take of siRNA by germinating fungal spores from culture medium, and attempt to target a vital pathway in fungal pathogens for their control.

Physiological responses of wild type and putrescine-overproducing transgenic cells of poplar to variations in the form and concentration of nitrogen in the medium.

We determined: (a) the physiological consequences of overproduction of putrescine in transgenic poplar (Populus nigra x maximoviczii) cells expressing an ornithine decarboxylase transgene; and (b) effects of variation in nitrogen (N) concentration of the medium on cellular polyamine concentration in transgenic and non-transgenic cells. Cells grown in the presence of supplemental (to the normal concentrations of N sources in the growth medium) and reduced amounts of NH4NO3 and KNO3 were used to study effects on membrane permeability, mitochondrial respiratory activity, protein accumulation, growth rates and changes in cellular polyamine concentration. The N concentration of the MS medium was not a limiting factor for continued overproduction of putrescine in transgenic cells. However, continued supplies of NH4+ and NO3- were required to maintain homeostatic amounts of putrescine in both cell lines. The presence of high amounts of putrescine in transgenic cells had significant effects on the physiological parameters measured. Compared with non-transgenic cells, transgenic cells had greater plasma membrane permeability, less tolerance to NH4NO3, more tolerance to KNO3, and accumulated higher amounts of soluble protein.

Involvement of polyamines in the protection of taurine against the cytotoxicity of hydrazine or carbon tetrachloride in isolated rat hepatocytes

Taurine (2-aminoethanesulfonic acid) is known to protect hepatocyte injury induced by hydrazine or carbon tetrachloride. We investigated whether cellular polyamines are involved in the protective mechanism of taurine in the hepatocyte injury caused by hydrazine or carbon tetrachloride. The agents decreased cellular polyamine concentrations, but the treatment with taurine prevented this decrease. The protection of taurine against hepatic injury was not observed in hepatocytes treated with α-difluoromethylornithine (DFMO), an irreversible inhibitor of ornithine decarboxylase which is a key enzyme in polyamine biosynthesis. The protection of taurine was recovered by the addition of polyamines to DFMO-treated hepatocytes. These results suggest that cellular polyamines play an important role in the protection of taurine in hydrazine or carbon tetrachloride-induced hepatocyte injury.

Involvement of polyamines in epidermal growth factor (EGF), transforming growth factor (TGF)-α and -β1 action on culture of L6 and fetal bovine myoblasts

1. 1. α-Difluoromethylornithine, an irreversible inhibitor of ornithine decarboxylase significantly abolished stimulation of protein synthesis evoked by EGF, TGF-α or -β 1 in L6 and fetal bovine myoblasts. 2. 2. The participation of polyamines in early events evoked by growth factors was shown by a significant stimulation of ornithine decarboxylase and Sdenosylmethionine decarboxylase activity as well as increased concentration of spermidine and spermine in L6 cells exposed to TGF-α and EGF. 3. 3. TGF-β 1 at a high concentration (1 ng/ml) increased protein synthesis in L6 myoblasts but inhibited it in fetal bovine myoblasts. Metabolic effects of TGF-β 1 in L6 cells was associated with an enhancement of decarboxylase activities, however there were no significant changes in cellular polyamine concentrations. Presented data suggest that polyamines are involved in the signal transduction pathway of EGF, TGF-α, and -β 1 in L6 and fetal bovine myoblasts.

Towards microfluorometric quantitation of polyamines in situ

Two different fluorescence cytochemical methods, the formaldehyde-fluorescamine (FF) method and the orthophthalaldehyde (OPT) method as well as an immunocytochemical method have been developed for the localization of spermidine and spermine. Of these three methods, the FF-method is the most easy to perform. We have studied the relationship between fluorescence intensity induced by the FF-method and cellular polyamine levels measured by HPLC in MCF-7 cells and HeLa cells. The experiments were designed to obtain different cell concentrations of polyamines. Cells grown on microscope slides in Petri-dishes were partly depleted of spermidine by two days inhibition of their ornithine decarboxylase activity using alpha-difluoromethylornithine. One hr before harvest the cells were exposed to different concentrations (0-30 microM) of spermidine. Microfluorometric results and chemical determinations of spermidine and spermine were obtained from each separate slide. The cellular total polyamine (spermidine + spermine) concentration on the slides varied between 4 and 15 nmol per mg protein (MCF-7 cells) and 5 and 26 nmol per mg protein (HeLa cells) and the corresponding microfluorometric results between 60 and 115 arbitrary units (MCF-7 cells) and 80 and 160 arbitrary units (HeLa cells). Simple regression analysis showed a good linear relationship between cellular polyamine concentration and FF-fluorescence yield. The correlation coefficient for MCF-7 cells was 0.86 and for HeLa cells 0.82, significance of the correlations was p less than or equal to 0.0001. Our results add further credence to the specificity of the FF-method and indicate that the method may be useful for microfluorometric quantitation of polyamines in situ.

Feedback control of ornithine decarboxylase expression by polyamines. Analysis of ornithine decarboxylase mRNA distribution in polysome profiles and of translation of this mRNA in vitro

Cell growth and differentiation require the presence of optimal concentrations of polyamines. Ornithine decarboxylase (ODC) catalyses the first and rate-controlling step in polyamine synthesis. In studies using cultures of Ehrlich ascites-tumour cells, we have shown that the expression of ODC is subject to feedback regulation by the polyamines. A decrease in the cellular polyamine concentration results in a compensatory increase in the synthesis of ODC, whereas an increase in polyamine concentration results in suppression of ODC synthesis. These changes in ODC synthesis were attributed to changes in the efficiency of ODC mRNA translation, because the steady-state amount of ODC mRNA remained constant. We now show that the number of ribosomes associated with ODC mRNA is low, and that the increase in ODC mRNA translation takes place without a shift in the distribution of ODC mRNA towards larger polysomes. This finding indicates that the polyamines regulate the efficiency of ODC mRNA translation by co-ordinately affecting the rates of initiation and elongation. By analysing ODC mRNA translation in vitro, using a rabbit reticulocyte lysate, polyadenylated RNA from a cell line with an amplified ODC gene, and a monospecific anti-ODC antibody, we also show that spermidine, but not putrescine, exerts a direct regulatory effect on ODC synthesis.

Functions of polyamine acetylation.

Acetylation is a means to decrease the net positive charge of the polyamines and thus liberate polyamines from anionic binding sites. The acetyl derivatives can be removed from the cells by transport and catabolism. Intracellular polyamine metabolism can be formulated as a cyclic process, which explains the transformation of one polyamine into another. As a net result, this pathway metabolizes (in an energy-requiring manner) methionine to 5'-deoxy-5'-methylthioadenosine and beta-alanine, and thus appears to be futile. It is suggested that the cyclic process is necessary for the precise control of cellular polyamine concentrations, as it allows relatively rapid spermine and spermidine concentration changes, in spite of a slow basal turnover rate. For the regulation of cellular polyamine metabolism, two decarboxylases, L-ornithine decarboxylase and S-adenosyl-L-methionine decarboxylase; the cytosolic acetyl-CoA:spermidine/spermine N1-acetyltransferase; and a polyamine transport system are required. The activity of the nuclear acetyltransferase is assumed to be the rate-limiting enzyme of nuclear polyamine turnover. The complexity and high level of sophistication of polyamine regulation is strong evidence for the important functional significance of the natural polyamines.

Developmental aspects of polyamine interconversion in rat brain

In the mammalian organism putrescine is formed by two reactions: (a) decarboxylation of ornithine and (b) degradation of spermidine via the so-called interconversion pathway. The latter comprises N1-acetylation of spermidine by a cytosolic acetyltransferase. and oxidative splitting of N1-acetylspermidine to putrescine by polyamine oxidase (PAO). It has previously been shown that specific inhibition of PAO causes a time-dependent accumulation of N1-acetylspermidine in brain, which is a measure of spermidine turnover. Another consequence of PAO inhibition is the decrease of brain putrescine concentration, proportional to its normal formation from spermidine. This observation allowed us to demonstrate the increasing significance of polyamine interconversion with brain maturation. The results support our hypothesis that the mechanisms which regulate cellular polyamine concentrations change during normal brain maturation from a system in which l-ornithine decarboxylase is dominating to a more sophisticated system in which both synthetic and catabolic processes become equally important regulatory factors.In contrast with current views, the activity of S-adenosylmethionine decarboxylase rather than that of ornithine decarboxylase limits the rate of polyamine biosynthesis during early brain development. In the mature brain the total amount of putrescine, which is formed both by decarboxylation of ornithine and by degradation of spermidine, limits the rate of spermidine formation. Changes of the regulatory system analogous to those described in this work are presumably not exclusive for brain, but rather characteristic for a variety of differentiating cells.

Developmental changes of the GABA and polyamine systems in isolated neurons in cell culture.

Dissociated cells of cerebral hemispheres from 8-day-old chick embryos were cultivated for 8 days in polylysine-coated Petri dishes. Changes of DNA, RNA, total proteins, putrescine, spermidine, spermine, free amino acids and enzymes involved in polyamine and GABA metabolism were studied throughout neuronal development in culture. The presence of GABAergic neurons in the cultured cell population was demonstrated. There were time-dependent changes in cellular polyamine concentrations and the activities of the enzymes involved in polyamine metabolism. Since no significant proliferation took place after the first day in culture, the observed changes indicate a role of polyamine metabolism during neuronal differentiation; it was not possible, however, to attribute the observed changes to specific functions. This culture system seems especially useful for the study of biochemical and of functional correlations between GABA and polyamine metabolism and morphological and functional developments of neurons.

Polyamines in Mammalian Biology and Medicine

POLYAMINES IN MAMMALIAN BIOLOGY AND MEDICINE* H. GUY WILUAMS-ASHMAm AND ZOE N. CANEUAKISt Spermine, spermidine, and putrescine are three aliphatic amines of widespread biological occurrence whose molecular structures (fig. 1) were established over 50 years ago. Yet until past the middle of this century these polyamines were seldom allotted more than a line or two in the textbooks and were generally considered only as curios of physiological chemistry. In the interim, research on polyamines has blossomed into an astonishingly vigorous enterprise. H3N(CHj)4NH3 Putrescine H3N(CH2)4NH2(CH2)3NH3 Spermidine H3N(CH2)3NH2(CH2)4NH2(CH2)3NH3 Spermine Fig. 1.—The molecular structure ofthe three major polyamines in mammalian cells. All of the primary and secondary amine groups are shown in the protonated form, as occurs under physiological conditions. Some major results of recent investigations may be highlighted (Note 1). AU prokaryotes and eukaryotes manufacture putrescine and spermidine , whereas spermine production is largely confined to nucleated eukaryotic cells. A number of pathways for the biosynthesis and ?Studies from the authors' laboratories were supported by NIH research grants HD04592 and CA-16359. We are grateful to Ms. D. WaIz for excellent help in the preparation of the manuscript. This essay is dedicated to the memory of Chandrakant V. Dave. tBen May Laboratory for Cancer Research, Departments of Biochemistry and of Pharmacological and Physiological Sciences, University of Chicago, Chicago, Illinois 60637. fVeterans Administration Hospital, West Haven, Connecticut 06516, and Departments of Pharmacology and Medicine, Yale University School of Medicine, New Haven, Connecticut 06510.© 1979 by The University of Chicago. 0031-5982/79/2203-0072$01.00 Perspectives in Biology and Medicine · Spring 1979 \ 421 metabolism of polyamines have been uncovered in animals, plants, and microorganisms. Various polyamine biosynthetic enzymes are exquisitely regulated by innumerable agents that stimulate cell growth and differentiation, with resulting fluctuations in cellular polyamine concentrations . In cell-free systems, polyamines form tight, noncovalent complexes with many biomolecules, and especially with various nucleic acids, thereby influencing many reactions involved in the formation, turnover, and postsynthetic modification of DNA, RNA, and protein molecules. Spermine, spermidine, and putrescine also directly modulate, or participate in, other diverse metabolic processes and exert stabilizing effects on cell and organelle membranes. And clinical studies suggest that alterations in polyamines in some human body fluids may aid in the diagnosis or prognostic evaluation of certain diseases. Despite all this, many challenging questions in polyamine physiology remain unanswered. Here we shall dwell on several of these problems in the context of mammalian biology and medicine, with emphasis on neglected lines of research. I. Conformationally Flexible Polycations As its epithet implies, spermine was originally discovered in semen. Notwithstanding the large amounts of polyamines in the seminal plasma ofcertain species, these substances are preeminently ofintracellular significance in mammals. Polyamine levels in blood plasma and many other body fluids except semen are extremely low. Spermidine and spermine concentrations in most tissues lie within the range of 0.1-2 /¿mol per gram fresh weight; notable exceptions are the pancreas and the prostate gland in which higher values are encountered in some creatures. The spermidine/spermine ratio is frequently, but not invariably, highest in cells undergoing rapid growth or differentiation, and tends to drop in many tissues with advancing age or as a result of involution of many hormone-dependent organs precipitated by hormonal deprivation. Intracellular putrescine concentrations are strikingly lower than those of spermidine and spermine: values ofless than 0. 1 /nmol of putrescine per gram are usually observed in resting mammalian cells, and hardly ever exceed thrice this value in stimulated or malignant tissues. The levels and turnover rates of the three amines in higher animal cells can fluctuate enormously—often independently ofeach other and sometimes in a polyphasic fashion—in response to hormones, drugs, viral infections, and other factors. Assessment of polyamine functions in mammalian cells would obviously be facilitated by knowledge of their intracellular distributions. Unfortunately , technical difficulties have stood in the way of obtaining trustworthy data. Many attempts to study the associations of polyamines 422 I H. Guy Williams-Ashman andZoe N. Canellakis ¦ Polyamines in Biology andMedicine with subcellular fractions of homogenized tissues have been vitiated by uncontrollable redistribution artifacts. The only chemically reactive groups...