Modulation of murine erythroleukemic cell differentiation by inhibitors of polyamine biosynthesis

The differentiation of murine erythroleukemic cells induced by hexamethylene bisacetamide is shown to be differently affected by two inhibitors of polyamine biosynthesis. Methyl glyoxal bis(guanyl hydrazone) (inhibitor or S-adenosyl methionine decarboxylase) inhibited this differentiation process. By using a novel experiment protocol the inhibitory effect of this drug on the induced differentiation was dissociated from pleiotropic effects on cell growth. Methyl glyoxal bis(guanyl hydrazone) only inhibited the induced differentiation if present during the first 6 h of culture of the cells with the inducer. No effect on the induced differentiation was observed if the drug was added to the culture medium 6 h after the inducer. alpha-Difluoro methylornithine (inhibitor of ornithine decarboxylase) stimulated the differentiation of these cells. Polyamine analysis demonstrated that alpha-difluoro methylornithine increased the rapidity and the amplitude of the changes in intracellular polyamines associated with this induced differentiation. The presence of methyl glyoxal bis(guanyl hydrazone) during the first 3 h with the inducer was sufficient to produce opposing changes in the intracellular polyamines. These results suggest that changes in either intracellular polyamines or the activities of polyamine biosynthetic enzymes play a regulatory role in the differentiation process induced in murine erythroleukemic cells by hexamethylene bisacetamide.     

Control of cell proliferation and differentiation by regulating polyamine biosynthesis in cultures of Brassica and its correlation with glyoxalase-I activity

Differentiation in Brussica cultures could be induced on basal medium lacking hormones, while addition of hormones (NAA, BA) resulted in profuse callusing without any differentiation. Supplementing the hormone medium with spermidine resulted in increase in the fresh weight and glyoxalase-I activity by 330% and 8-fold, respectively. Omission of hormones caused spermidine to be less effective in inducing either cell proliferation or differentiation. Methylglyoxal-bis(guanylhydrazone) (MGBG), an inhibitor of polyamine biosynthesis, had a retarding effect on callus induction and division of cells in suspension cultures but lead to differentiation and inhibited glyoxalase-I activity. The ability of spermidine to overcome MGBG enhanced differentiation was probably through the breaking of cell cycle arrest. Addition of glutathione, a coenzyme for glyoxalase-I enzyme, promoted cell division and enzyme activity both in callus and suspension cultures. pH emerged as an important factor in controlling glyoxalase-I activity and cell division. Results indicate involvement of spermidine in cell proliferation and differentiation and its correlation with glyoxalase-I activity.     

Epidermal growth factor: modulator of murine embryonic palate mesenchymal cell proliferation, polyamine biosynthesis, and polyamine transport

Polyamines (putrescine, spermidine, and spermine) are normal cellular constituents able to modulate cellular proliferation and differentiation in a number of tissues and cell types. This investigation explores the response of murine embryonic palate mesenchymal (MEPM) cells to epidermal growth factor (EGF) in terms of biosynthesis of putrescine and its transport across the plasma membrane and tests the hypothesis that polyamine transport can serve as an alternative mechanism (other than biosynthesis) for elevating intracellular polyamines during stimulation of MEPM cellular proliferation. MEPM cells treated with EGF were stimulated to proliferate and showed a dose- and time-dependent stimulation of ornithine decarboxylase (ODC) which was maximal at 4-6 hours. EGF also stimulated the initial rate of putrescine transport in a dose- and time-dependent manner. This stimulation was found to be maximal 3 hours after treatment and specific for the putrescine transport system. The kinetic parameters of putrescine transport shifted from 2.52 microM (Km) and 23.6 nmol/mg protein/15 minutes (Vmax) in nonstimulated cells to 4.48 microM (Km) and 39.8 nmol/mg protein/15 minutes (Vmax) in EGF-treated cells. This kinetic shift did not require de novo protein or RNA synthesis, as cycloheximide (10 micrograms/ml) and actinomycin D (50 micrograms/ml) had little effect on the ability of EGF to stimulate the initial rate of putrescine uptake. The rate of transport, however, was found to be inversely related to cell density. The addition of exogenous putrescine concomitantly with EGF blocked the induction of ODC, while in the presence of difluoromethylornithine (DFMO) (irreversible inhibitor of ODC) the initial rate of putrescine transport remained elevated throughout the time course studied. This stimulation of putrescine uptake caused by polyamine deprivation was reversed by exogenous putrescine and Ca++ while alpha-aminoisobutyric acid (AIB) further stimulated the rate of uptake. EGF's ability to stimulate cellular DNA synthesis was inhibited by DFMO. If DFMO-treated cells were stimulated with EGF in the presence of exogenous putrescine, this stimulatory effect was preserved. These studies indicate that the rate of polyamine transportation is highly responsive to a signal which initiates biosynthesis of polyamines. Further, this transportation system provides a compensatory mechanism allowing the cell to increase intracellular levels of polyamines when environmental conditions inhibit biosynthesis or when polyamines are abundant.     

Regulation of polyamine biosynthesis by antizyme and some recent developments relating the induction of polyamine biosynthesis to cell growth

This review considers the role of antizyme, of amino acids and of protein synthesis in the regulation of polyamine biosynthesis.The ornithine decarboxylase of eukaryotic ceils and ofEscherichia coli coli can be non-competitively inhibited by proteins, termed antizymes, which are induced by di-and poly- amines. Some antizymes have been purified to homogeneity and have been shown to be structurally unique to the cell of origin. Yet, the E. c o l i antizyme and the rat liver antizyme cross react and inhibit each other's biosynthetic decarboxylases. These results indicate that aspects of the control of polyamine biosynthesis have been highly conserved throughout evolution.Evidence for the physiological role of the antizyme in mammalian cells rests upon its identification in normal uninduced cells, upon the inverse relationship that exists between antizyme and ornithine decarboxylase as well as upon the existence of the complex of ornithine decarboxylase and antizyme in vivo. Furthermore, the antizyme has been shown to be highly specific; its Keq for ornithine decarboxylase is 1.4 x 1011 M-1. In addition, mammalian ceils contain an anti-antizyme, a protein that specifically binds to the antizyme of an ornithine decarboxylase-antizyme complex and liberates free ornithine decarboxylase from the complex. In B. coli , in which polyamine biosynthesis is mediated both by ornithine decarboxylase and by arginine decarboxylase, three proteins (one acidic and two basic) have been purified, each of which inhibits both these enzymes. They do not inhibit the biodegradative ornithine and arginine decarboxylases nor lysine decarboxylase. The two basic inhibitors have been shown to correspond to the ribosomal proteins S₂₀/L₂₆ and L₃₄, respectively. The relationship of the acidic antizyme to other known B. coli proteins remains to be determined.     

Coordinating cell proliferation and differentiation

Cell proliferation and differentiation are highly coordinated processes during development. Recent studies have revealed that this coordination may result from dual functions residing in the central regulators of proliferation, allowing them to also regulate differentiation. Studies have also shown that some terminally differentiated cells can be made to divide beyond their normal capacity.     

Biochemical plant responses to ozone : I. Differential induction of polyamine and ethylene biosynthesis in tobacco

Polyamine metabolism was examined in tobacco (Nicotiana tabacum L.) exposed to a single ozone treatment (5 or 7 hours) and then postcultivated in pollutant-free air. The levels of free and conjugated putrescine were rapidly increased in the ozone-tolerant cultivar Bel B and remained high for 3 days. This accumulation was preceded by a transient rise of l-arginine decar-boxylase (ADC, EC 4.1.1.19) activity. The ozone-sensitive cultivar Bel W3 showed a rapid production of ethylene and high levels of 1-aminocyclopropane-1-carboxylic acid after 1 to 2 hours of exposure. Induction of putrescine levels and ADC activity was weak in this cultivar and was observed when necrotic lesions developed. Leaf injury occurred in both lines when the molar ratio of putrescine to 1-aminocyclopropane-1-carboxylic acid or ethylene fell short of a certain threshold value. Monocaffeoyl-putrescine, an effective scavenger for oxyradicals, was detected in the apo-plastic fluid of the leaves of cv Bel B and increased upon exposure to ozone. This extracellular localization could allow scavenging of ozone-derived oxyradicals at the first site of their generation. Induction of either polyamine or ethylene pathways may represent a control mechanism for inhibition or promotion of lesion formation and thereby contribute to the disposition of plants for ozone tolerance.     

Regulation of hemopoietic cell differentiation and proliferation

Differentiation and proliferation of almost all hemopoietic cell lines can now be studied in vitro. Cloning techniques and suspension cultures allow the study of proliferation of the multipotential hemopoietic progenitor cell and the committed progenitors for granulocytes, macrophages, eosinophils, megakryocytes, and erythrocytes. The proliferation of each of the committed progenitor cells is controlled by specific glycoproteins and two of these have recently been purified: granulocyte-macrophage colony-stimulating factor (GM-CSF) and erythropoietin. The rate of proliferation of the GM-progenitor cells and their pattern of differentiation depends on the concentration of the hormone. At low concentrations of GM-CSF (10^?11 M) fewer progenitor cells are stimulated and macrophage colonies rather than granulocyte colonies develop. The change in the direction of granulocyte-macrophage differentiation appears to be related to (a) the concentration of GM- CSF and (b) the different sensitivity of a subpopulation of monocyte colony-forming cells which are responsive to GM-CSF even at low concentrations of the regulator. Analysis of the rate of RNA synthesis by bone marrow cells has shown that GM-CSF stimulates the mature nondividing end cells of differentiation (ie, polymorphs) as well as the progenitor cells. Although GM-CSF and erythropoietin have been radiolabeled, binding studies have been hampered by the loss of biologic activity during the labeling procedure and the heterogeneity of the target cells to which the regulators bind. Surface proteins and receptors for erythrocytes have been well characterized but the relationships between these proteins and the cell surface proteins of nucleated blood cells is not well understood. It appears that some proteins are lost from the cell surface during the development of granulocytes, which are retained on the surface of the B lymphocyte. Other proteins such as chemotactic receptors and complement receptors only appear on the mature cells. External radiolabeling of the granulocyte surface using iodogen yielded a simple profile of ¹²⁵I-labeled proteins when analyzed by sodium dodecyl sulphate polyacrylamide gel electrophoresis.     

Haematological and serum biochemical responses of chickens to hydric stress

Dehydration can be extremely damaging to the performance and welfare of indigenous chickens. The effect of water restriction on haematological and biochemical parameters was compared in Naked Neck (NNK) and Ovambo (OVB) chickens. A total of 54 8-week-old pullets each of NNK and OVB chickens with an initial average weight of 641 ± 10 g/bird were randomly assigned to three water intake treatments with three replications, each having six birds. The water restriction treatments were ad libitum, 70% and 40% of ad libitum intake. Nine experimental pens with a floor space of 3.3 m² per strain were used. Feed was provided ad libitum. Packed cell volume (PCV), erythrocyte count (RBC), mean corpuscular volume (MCV) and total leucocyte count (WBC), and biochemical parameters (uric acid (UA)), creatinine (CREAT), total protein (TP), albumin (ALB), globulin (GLOB), triglyceride (TGA), total cholesterol (TC), high- (HDLC) and low- (LDLC) density lipoprotein cholesterol and activity of alanine transaminase (ALT), alkaline phosphatase (ALP) and aspartate transaminase (AST) were determined from blood collected after 60 days of water restriction. PCV was higher (P < 0.05) in NNK than OVB chickens offered water ad libitum, but similar in birds offered 70% and 40% of ad libitum. There were no differences in RBC and MCV values between strains, but MCV was higher in birds on 40% than 70% of ad libitum water intake, irrespective of strain. Naked neck chickens had higher (P < 0.05) WBC values than OVB at 40% restriction level, but lower WBC than OVB at 70% water restriction level. UA, CREAT, TGA, TC, LDLC, TP and GLOB increased (P < 0.05) with each increment in water restriction, but the increase in CREAT and TC was more pronounced in OVB than NNK chickens. The opposite was observed for UA. ALT activity indicated that liver function was not affected by water restriction. It was concluded that the two strains can withstand up to 40% of ad libitum water restriction, but NNK tolerated water stress better than OVB chickens.     

The effect of polyamine biosynthesis inhibition on growth and differentiation of the phytopathogenic fungus Sclerotinia sclerotiorum

We studied the effects of several polyamine biosynthesis inhibitors on growth, differentiation, free polyamine levels and in vivo and in vitro activity of polyamine biosynthesis enzymes in Sclerotinia sclerotiorum. -Difluoromethylornithine (DFMO) and -difluoromethylarginine (DFMA) were potent inhibitors of mycelial growth. The effect of DFMO was due to inhibition of ornithine decarboxylase (ODC). No evidence for the existence of an arginine decarboxylase (ADC) pathway was found. The effect of DFMA was partly due to inhibition of ODC, presumably after its conversion into DFMO by mycelial arginase, as suggested by the high activity of this enzyme detected both in intact mycelium and mycelial extracts. In addition, toxic effects of DFMA on cellular processes other than polyamine metabolism might have occurred. Cyclohexylamine (CHA) slightly inhibited mycelial growth and caused an important decrease of free spermidine associated with a drastic increase of free putrescine concentration. Methylglyoxal bis-[guanyl hydrazone] (MGBG) had no effect on mycelial growth. Excepting MGBG, all the inhibitors strongly decreased sclerotial formation. Results demonstrate that sclerotial development is much more sensitive to polyamine biosynthesis inhibition than mycelial growth. Our results suggest that mycelial growth can be supported either by spermidine or putrescine, while spermidine (or the putrescine/spermidine ratio) is important for sclerotial formation to occur. Ascospore germination was completely insensitive to the inhibitors.     

Alterations in polyamine metabolism during embryonal carcinoma cell differentiation in vitro

Murine embryonal carcinoma (EC) cells can be stimulated to differentiate by several chemical inducers. Since the response of EC cells to induction is likely to occur shortly after exposure to the inducer, we report here the changes that occur in polyamine levels in a number of EC cell lines shortly after exposure to two chemical stimuli, alpha-difluoromethylornithine (DFMO) and retinoic acid (RA). Our results suggest that polyamine levels are important in determining the state of EC cell differentiation, but that reduction in these levels alone is not sufficient to induce differentiation in all the EC cell lines tested. Also, it is apparent that RA does influence levels of polyamines. However, this influence does not seem to be mediated through direct interaction with ODCase.     

Natural antisense mRNAs to hyaluronan synthase 2 inhibit hyaluronan biosynthesis and cell proliferation

We report the identification of a natural antisense mRNA of hyaluronan synthase 2 that we have chosen to designate as HASNT (for HA synthase 2 antisense) in human and mouse. HASNT is transcribed from the opposite strand of the HAS2 gene locus and is represented by several independent expressed sequence tags in human. Portions of the mouse Hasnt gene were identified through an exon-trapping approach. Sequence conservation is extremely low between human and mouse HASNT, and it is not clear whether these mRNAs contain functional open reading frames. HASNT has an alternate splice site in both human and mouse. This splice site is located at an identical position within the gene in both species and results in mRNAs of two different lengths. In each species, the antisense portion of the HASNT gene is complementary to the first exon of HAS2, which represents the 5'-untranslated region. To study the biological activity of HASNT, two human expressed sequence tag clones, representing long and short HASNT splice variants, were cloned into a tetracycline-inducible vector and were stably transfected into human osteosarcoma U2-OS Tet-on cells. The long and short HASNT-expressing cells had a reduction in HAS2 mRNA levels up to 94 and 86%, respectively, whereas hyaluronan biosynthesis was inhibited by 40 and 37%, respectively. Cell proliferation was reduced throughout the time frame of the experiment. Exogenous high molecular mass hyaluronan failed to rescue the suppressed cell proliferation, whereas adenoviral-mediated overexpression of hyaluronan synthase 3, which stimulated endogenous hyaluronan biosynthesis, was able to rescue. Collectively, our data suggest that natural antisense mRNAs of HAS2 are able to regulate HAS2 mRNA levels and hyaluronan biosynthesis in a cell culture model system and may have an important and novel regulatory role in the control of HAS2, HA biosynthesis, and HA-dependent cell functions in vivo.     

Calcium and cell cycle progression: possible effects of external perturbations on cell proliferation.

Exit from the phase of cellular division appears to be driven by a calcium signal that triggers a cascade of events leading to the completion of mitosis. Here we propose a model that relates the dynamics of cytosolic calcium to progression through mitosis, G1 and G2 phases of the cell cycle. To this end, the assumption has been made that the transient rise ir cytosolic calcium concentration during mitosis is induced by inositol(1,4,5)triphosphate (IP3), which in turn is released at high levels of mitosis-promoting factor (MPF). On this basis, a system of ordinary differential equations is proposed to simulate the evolution of ten cell-cycle-specific molecular species, including cyclins A and B, MPF, IP3, Ca2+, the CaMKII holoenzyme, and the ubiquitination complex. The influence on the cell proliferation capacity exerted by external perturbations, like calcium microinjections, depletion of intracellular calcium stores, electromagnetic fields, or stimulation/inhibition of different calcium currents through the plasma membrane, can be studied by appropriate modulation of the parameters involved in the signal transduction pathway.     

CFTR modulates lung secretory cell proliferation and differentiation

We have permanently reversed the lethal phenotype in the cystic fibrosis (CF) transmembrane conductance regulator (CFTR)-deficient (knockout) mouse after in utero gene therapy with an adenovirus containing the cftr gene. The gene transfer targeted somatic stem cells in the developing lung and intestine, and these epithelial surfaces demonstrated permanent developmental changes after treatment. The survival statistics from the progeny of heterozygote-heterozygote matings after in utero cftr gene treatment demonstrated an increased mortality in the homozygous normal pups, indicating that overexpression during development was detrimental. The lungs of these pups revealed accelerated secretory cell proliferation and differentiation. The extent of proliferation and differentiation in the secretory cells of the lung parenchyma after in utero transfer of the cftr gene was evaluated with morphometric and biochemical analyses. These studies provide further support of the regulatory role of the cftr gene in the development of the secretory epithelium.     

Physical forces modulate cell differentiation and proliferation processes

Currently, the predominant hypothesis explains cellular differentiation and behaviour as an essentially genetically driven intracellular process, suggesting a gene‐centrism paradigm. However, although many living species genetic has now been described, there is still a large gap between the genetic information interpretation and cell behaviour prediction. Indeed, the physical mechanisms underlying the cell differentiation and proliferation, which are now known or suspected to guide such as the flow of energy through cells and tissues, have been often overlooked. We thus here propose a complementary conceptual framework towards the development of an energy‐oriented classification of cell properties, that is, a mitochondria‐centrism hypothesis based on physical forces‐driven principles. A literature review on the physical–biological interactions in a number of various biological processes is analysed from the point of view of the fluid and solid mechanics, electricity and thermodynamics. There is consistent evidence that physical forces control cell proliferation and differentiation. We propose that physical forces interfere with the cell metabolism mostly at the level of the mitochondria, which in turn control gene expression. The present perspective points towards a paradigm shift complement in biology.