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PubMed1987

Polyamines in normal and cancer cells.

Based on available evidence, it appears that polyamines are critical for proliferation of both normal and transformed cells. Although the requirement of polyamines for DNA replication and cell proliferation is established, the molecular events in which the polyamines are essential are yet unknown. Furthermore, transformed and cancer cells, possibly because of their higher proliferative rate, appear to be more dependent on polyamine metabolism than their normal counterparts. This has been shown by the in vivo response of tumor models and human tumor xenografts in nude mice to polyamine depletion by DFMO. Although there has been associated toxicity to the host, the inhibition of cell proliferation has been higher in the implanted tumors than in the host. DFMO, a specific irreversible inhibitor of ODC, has been used extensively in studies which have shed light on the role of polyamines in cell proliferation and differentiation. DFMO has shown interesting anti-tumor effect in a number of experimental tumor models. Currently, DFMO clinical trials are being completed, and it will be of interest to see whether this polyamine inhibitor, or other newer polyamine analogs and inhibitors, will find a place in the treatment of neoplastic disorders.

Animals↗
PubMed1988

Enzyme regulation as an approach to interference with polyamine biosynthesis--an alternative to enzyme inhibition.

The progress reviewed here would seem to validate the regulatory approach to interference with polyamine biosynthesis as an antiproliferative strategy. To our knowledge, this is the first example, among anticancer drugs, of pharmacological intervention of a biochemical pathway based strictly on regulatory control. Several features of polyamine biology naturally favor this approach and may account for its relative success. These include (a) the nature of the regulatory mechanisms themselves, (b) the exquisite sensitivity of the pathway to regulatory control, (c) the rapid turnover of ODC and AdoMetDC, (d) the different structural specificity of ODC and AdoMetDC regulation versus growth-dependent functions, and (e) the direct dependence of growth on sustained polyamine biosynthesis. As such, the regulatory approach to interference with polyamine biosynthesis offers several advantages over the use of specific enzyme inhibitors (Table 10). Of these, perhaps, the more significant are the facts that more than one enzyme can be simultaneously and specifically suppressed and that compensatory mechanisms, which otherwise counter the effects of enzyme inhibitors (11), are not invoked. We are encouraged by the concurrence of in vitro mechanistic findings with the predictions of the hypothesis for the regulatory approach and by the in vitro and in vivo growth inhibitory effects of the analogs against murine leukemia. One disadvantage of the regulatory analogs, such as BESm, has been that, as with specific polyamine inhibitors such as DFMO, analog-induced polyamine depletion results in cytostatic growth inhibition. While this response may help to minimize host toxicities, it clearly compromises antitumor activity. An intriguing exception to this generality has recently been found among human lung carcinoma cell lines. Previously, Luk et al. (93, 94) and others (95) reported that, among a spectrum of human lung carcinoma lines, small cell carcinoma was exquisitely sensitive to the ODC inhibitor, DFMO. Not only did these cells display a cessation of growth but also an inability to survive during DFMO-induced polyamine depletion. Studies extending these findings to long term maintenance therapy in human small cell lung carcinoma implants in athymic mice revealed sustained growth inhibition of the tumor for longer than one year (96). Casero et al. (97) now find that human large cell carcinoma, which is otherwise refractory to chemotherapeutic intervention, displays a cytotoxic response in vitro to polyamine depletion induced by BES or BESm but not by DFMO.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenosylmethionine Decarboxylase↗
PubMed1990

Experience affects cortical but not subcortical polyamines.

The effect of brief periods of experience in an enriched environment (7 hours per day for 3 days), and of inhibition of polyamine synthesis was studied in four brain regions: occipital cortex, remaining cortex, subcortex and cerebellum plus medulla. Polyamine synthesis was inhibited by alpha-difluoromethylornithine (DFMO), an irreversible inhibitor of ornithine decarboxylase. DFMO caused a 30-50% decrease in putrescine content in all brain areas, irrespective of the environmental treatment. Spermidine was decreased by the inhibitor in subcortex and in cerebellum plus medulla, while spermine was increased in remaining cortex. The regional differences in inhibitor effect suggest that the regulation of polyamine metabolism varies among the four brain areas. Experience increased the weight and spermidine content of remaining cortex and decreased putrescine content of occipital cortex. Noncortical areas were not affected. The effects of experience on polyamine levels were somewhat increased by DFMO. Therefore, experience did not have a generalized effect on polyamine levels; rather, each polyamine responded in a specific manner. In addition, polyamine levels were affected only in those brain areas which are known from previous studies to respond to environmental stimulation with weight increase. These facts suggest that polyamines might have a role in the regulation of experience-induced plasticity.

Animals↗
PubMed1982

Polyamine dependence of Chinese hamster ovary cells in serum-free culture is due to deficient arginase activity.

We recently isolated a Chinese hamster ovary cell line which grows well without serum but requires the exogenous polyamines putrescine, spermidine or spermine for continuous replication. Here we show that these cells are defective in the arginase-catalyzed synthesis of ornithine, the precursor of polyamines, and that ornithine can replace polyamines in the medium for supporting growth of the cells. The activities of two other key enzymes of polyamine biosynthesis, ornithine decarboxylase and adenosylmethionine decarboxylase, are clearly detectable and show increase during polyamine starvation. In ornithine-and polyamine-free medium cellular putrescine and spermidine are rapidly depleted while the concentration of spermine decreases only moderately. We show further that the cells are able to grow in serum-containing medium without added ornithine or polyamines. This is explained by our finding that serum contains arginase which synthesizes ornithine from arginine in the medium. All the sera from different animal species tested contained arginase activity although in greatly varying amounts. Serum-free medium is therefore essential for expression of arginase deficiency in cells in tissue culture. The eventual importance of polyamines for serum-free cultures in general is discussed.

Adenosylmethionine Decarboxylase↗
PubMed1977

Regulation of thyroid ornithine decarboxylase by the polyamines. Induction of a protein inhibitor of ornithine decarboxylase by the end-products of the reaction.

When spermidine, putrescine or 1,3-diaminopropane was injected (12.5 mumol/100 g body weight) into rats 1 h before thyrotropin, ornithine decarboxylase activity was increased by 75--150% over control levels. However, when greater than or equal to 75 mumol polyamine/100 g body weight was injected, thyrotropin-activated activity was inhibited by 70--95%. Multiple polyamine injections inhibited goitrogen-induced activity and gland weight increase by approx 35%. The polyamines also inhibited thyrotropin-activated rat thyroid ornithine decarboxylase in vitro in a dose-related fashion, with 50% inhibition occurring at 2--5 . 10(-4)M. The inhibition was not due to a direct effect on the enzyme. No stimulation was seen with low concentrations of polyamine. The polyamines had no effect on in vitro thyroid protein/RNA synthesis or glucose oxidation but had a biphasic effect on plasma membrane adenylate cyclase activity. A protein inhibitor to thyroid ornithine decarboxylase was generated in vivo by multiple injections of the polyamines into rats and in vitro by incubating bovine thyroid slices with 2--10 mM polyamine. The inhibitor was non-dialyzable, destroyed by boiling, and its formation was blocked in a dose-related fashion by cycloheximide. We conclude that: (1) thyroid ornithine decarboxylase is subject not only to positive control, but is also negatively regulated by its end-products, the polyamines, which induce a protein inhibitor to ornithine decarboxylase; (2) since gland growth is also inhibited under these conditions, the polyamine effect on thyroid ornithine decarboxylase may be biologically significant.

Animals↗
PubMed1979

Stimulation by polyamines of carbamylphosphate:glucose phosphotransferase and glucose-6-phosphate phosphohydrolase activities of multifunctional glucose-6-phosphatase.

The effects of added polyamines on carbamylphosphate (carbamyl-P):glucose phosphotransferase and glucose-6-phosphate (Glc-6-P) phosphohydrolase activities of rat hepatic D-Glc-6-P phosphohydrolase (EC 3.1.3.9) of intact and detergent-treated microsomes have been investigated. With the former preparation, in the presence of 1.4 mM phosphate substrate and 90 mM D-glucose (phosphotransferase), 1 mM spermine, spermidine, and putrescine activated Glc-6-P phosphohydrolase 67%, 57%, and 35%, respectively. Carbamyl-P:glucose phosphotransferase, under comparable conditions, was activated 57%, 34%, and 18%. NH+4 (0.25--5.0 mM) produced at best but a minor activation (0--14%), while poly(L-lysine) (Mr = 3400; degree of polymerization 16) equimolar relative to other polyamines with respect to ionized free amino groups activated the hydrolase 358% and the transferase 222%. Treatment of microsomes with the detergent deoxycholate reduced, but did not abolish, polyamine-induced activation. The stimulatory effects of polyamines persisted in the presence of excess catalase, indicating their independence from H2O2 formation; and were eliminated in the presence of Ca2+. Kinetic analysis revealed that all tested polyamines decreased the apparent Michaelis constant values for carbamyl-P and Glc-6-P, but had no effect on the Km for glucose. Poly(L-lysine) increased the V value for both Glc-6-P phosphohydrolase and apparent V values for phosphotransferase extrapolated to infinite concentrations of either carbamyl-P or glucose. The other tested polyamines elevated only this last velocity parameter. It is proposed that a major mechanism by which polyamines activate glucose-6-phosphatase-phosphotransferase is through their electrostatic interactions with phospholipids of the membrane of the endoplasmic reticulum of which this enzyme is a part. Conformational alterations thus induced may in turn affect catalytic behavior. It is suggested that polyamines, or similar positively charged peptides, might participate in the cellular regulation of synthetic and hydrolytic activities of glucose-6-phosphatase.

Ammonium Chloride↗
PubMed1981

Uptake and excretion of polyamines from baby hamster kidney cells (BHK-21/C13). The effect of serum on confluent cell cultures.

In confluent cultures of BHK-21/C13 cells there was little uptake of exogenous polyamines and only a low level of polyamine biosynthesis. These cultures continuously excreted polyamines into the extracellular medium. Spermidine, in both the free and bound form, was the predominant excretion product, whereas the major intracellular polyamine was spermine implying that excretion of polyamines was specific. Reinitiation of growth by the addition of fresh serum immediately increased the uptake of exogenous putrescine, increased the biosynthesis of polyamines and decreased the excretion of polyamines. Thus, polyamine transport into and out of the cell appears to be regulated by the growth status of that cell.

Animals↗
PubMed1996

Polyamine transport in mammalian cells. An update.

The uptake and release of the natural polyamines putrescine, spermidine and spermine by mammalian cells are integral parts of the systems that regulate the intracellular concentrations of these biogenic amines according to needs. Although a general feature of all tissues, polyamine uptake into intestinal mucosa cells is perhaps the most obvious polyamine transport pathway of physiological and pathophysiological importance. Mutant cell lines lacking the ability to take up polyamines from the environment are capable of releasing polyamines. This indicates that uptake and release are functions of two different transport systems. The isolation of a transporter gene from a mammalian cell line is still lacking. Overaccumulation of polyamines is controlled by release and by a feedback regulation system that involves de novo synthesis of antizyme, a well known protein that also regulates the activity of ornithine decarboxylase. Recent work has demonstrated that Ca(2+)-signalling pathways are also involved. Although there is consensus about the importance of polyamine uptake inhibitors in the treatment of neoplastic disorders, a practically useful uptake inhibitor is still missing. However, the attempts to target tumours, and to increase the selectivity of cytotoxic agents by combining them with the polyamine structure, are promising. New, less toxic and more selective anticancer drugs can be expected from this approach.

Animals↗
PubMed2004

Folding of the Tetrahymena ribozyme by polyamines: importance of counterion valence and size.

Polyamines are abundant metabolites that directly influence gene expression. Although the role of polyamines in DNA condensation is well known, their role in RNA folding is less understood. Non-denaturing gel electrophoresis was used to monitor the equilibrium folding transitions of the Tetrahymena ribozyme in the presence of polyamines. All of the polyamines tested induce near-native structures that readily convert to the native conformation in Mg(2+). The stability of the folded structure increases with the charge of the polyamine and decreases with the size of the polyamine. When the counterion excluded volume becomes large, the transition to the native state does not go to completion even under favorable folding conditions. Brownian dynamics simulations of a model polyelectrolyte suggest that the kinetics of counterion-mediated collapse and the dimensions of the collapsed RNA chains depend on the structure of the counterion. The results are consistent with delocalized condensation of polyamines around the RNA. However, the effective charge of the counterions is lowered by their excluded volume. The stability of the folded RNA is enhanced when the spacing between amino groups matches the distance between adjacent phosphate groups. These results show how changes in intracellular polyamine concentrations could alter RNA folding pathways.

Animals↗
PubMed2002

A streamlined method for the isolation and quantitation of nanomole levels of exported polyamines in cell culture media.

A number of years ago, our laboratory published a method for the isolation of small amounts of polyamines from cell culture media using the ion-exchange resin Bio-Rex 70. We have used this technique extensively to study the export of putrescine and cadaverine from cultured mammalian cells. Unfortunately, this method was highly inefficient in isolating the polyamines spermidine and spermine and was incapable of recovering the acetylated polyamine N(1)-acetylspermidine. In response to these shortcomings, we modified our previous protocol to quantitatively isolate the polyamines N(1)-acetylspermidine, putrescine, cadaverine, N(1)-acetylspermine, spermidine, and spermine. The new method, which is much faster to perform and more efficient than the one previously described, employs the use of disposable minicolumns and a single resin washing step using a weak solution of sodium carbonate at pH 9.3. This new protocol also eliminates the column elution step in favor of directly derivatizing the polyamines with dansyl chloride on the ion-exchange resin. High-performance liquid chromatography analysis of the dansylated polyamines isolated by this procedure showed that 75% of N(1)-acetylspermidine and nearly 100% of the other polyamines present in nanomolar levels were recovered from small amounts of cell culture medium. This new protocol is a valuable new tool for the study of the intracellular/extracellular dynamics of polyamine pools in cultured cells. [A detailed laboratory protocol for this procedure (containing all of the information in this paper but in a condensed form) can be requested by e-mailing the authors.]

Biogenic Polyamines↗
PubMed1992

Feedback repression of polyamine uptake into mammalian cells requires active protein synthesis.

Two mammalian cell lines, rat hepatoma (HTC) and Chinese hamster ovary (CHO), were fed 10 to 50 microM spermidine while changes were monitored in intracellular polyamine levels and spermidine uptake activity. Normal feedback control preventing excessive polyamine uptake was found to be completely blocked by the addition of inhibitors of protein synthesis at the time of polyamine exposure. Under these conditions the cells accumulated abnormally high, toxic concentrations of spermidine. Further, continuous protein synthesis was needed to maintain repression of polyamine transporter proteins that had been inhibited previously by normal or elevated intracellular polyamines. These results suggest that a major factor in the regulation of polyamine uptake is the rapid, reversible inactivation of existing polyamine carrier molecules by an unstable protein whose synthesis is stimulated by intracellular polyamines.

Animals↗
PubMed2002

The crystal structure of N(1)-[2-(2-amino-ethylamino)-ethyl]-ethane-1,2-diamine (polyamines) binding to the minor groove of d(CGCGCG)(2), hexamer at room temperature.

The crystal structure of a left-handed Z-DNA hexamer, d(CG)(3) in complex with a synthetic polyamine, N(1)-[2-(2-amino-ethylamino)-ethyl]-ethane-1,2-diamine, NH(3)(+)-(CH(2))(2)-NH(2)(+)-(CH(2))(2)-NH(2)(+)-(CH(2))(2)-NH(3)(+) [PA(222)], has been determined by the X-ray diffraction method at 1.0 A resolution. In an orthorhombic crystal, the d(CG)(3) duplex binds two PA(222) molecules, and this synthetic polyamine exhibits dual conformational properties. One of the two PA(222) molecules resides on the floor of the minor groove of a Z-DNA duplex and imino groups bridge the two phosphate chains across a double helix, while the terminal amino groups link the oxygen atoms O2 of four cytosine bases. This PA(222) molecule makes a U-turn like a fishhook at one of its ends to provide a micro-environmental network previously unseen in complexes of DNA with polyamines. The width of the minor groove does not become considerably greater with the looped end of the polyamine, indicating conformational rigidity of the Z-DNA backbone imposed by the high stacking energy of the GC base pairs. While polyamine binding to the minor groove has been postulated by theoretical studies for stabilizing the Z-DNA double helical conformation, the finding in the crystal of the looped polyamine chain binding the minor groove of Z-DNA is observed for the first time from the data collected at 10 degrees C (so-called room temperature data). Another PA(222) molecule binds on the convex outer surface of the major groove of the Z-DNA duplex and links three d(CG)(3) duplexes which are symmetrically related to each other. The structure of this PA(222) presents the previously reported zig-zag type conformation [Egli et al., Biochemistry 30 (1991) 11388-11402]. Comparison of this structure with other polyamine-DNA cocrystals reveals structural themes and differences that may relate to the length of the polyamine.

Binding Sites↗
PubMed1998

Effects of L-valine on growth and polyamine metabolism in human colon carcinoma cells.

HT-29 cells, originating from a human colon carcinoma, can proliferate in standard culture conditions with an absolute requirement for polyamines. The major precursor provided in the culture medium for polyamine biosynthesis is L-arginine. L-Arginine conversion to L-ornithine by arginase is followed by stepwise conversion of this latter amino acid to putrescine, spermidine and spermine. The aim of the present work was to document the consequences of a total inhibition of L-arginine flux through arginase, resulting in a decreased L-ornithine availability, on HT-29 cell proliferation and polyamine metabolism. L-Valine, a known arginase inhibitor, when used at a high concentration, i.e., 100 mM, inhibits L-arginine flux through arginase almost totally. The addition in the culture medium of 100 mM L-valine or 50 mM NaCl used to mimic the L-valine induced increase in medium osmolality both reduced equally cellular growth. Cell viability, protein synthesis or oxidative metabolism measured in isolated cells were unaffected by the L-valine treatment, suggesting that decreased proliferation was not associated with an acute toxic effect of this aminoacid, but was rather due to the increase in the medium osmolality. L-Valine treated cells displayed an altered polyamine metabolism when compared with control cells grown in the absence of the amino acid. After 4 days of treatment with 100 mM L-valine, L-ornithine flux through ornithine decarboxylase was significantly higher as well as putrescine and spermidine cellular uptakes in treated cells. However, the changes in polyamine metabolism led to similar polyamine cell contents in untreated and L-valine treated cells. In conclusion, we propose that the observed alterations of polyamine metabolism may reflect an adaptative response of HT-29 cells to the presence of L-valine which contribute together with the low amount of L-ornithine present in the culture medium to polyamine homeostasis.

Arginine↗
PubMed1998

Differential effect on polyamine metabolism in mitogen- and superantigen-activated human T-cells.

Polyamines are important for regulation of lymphocyte differentiation and proliferation. Mitogens induce synthesis of ornithine decarboxylase (ODC), the rate limiting enzyme in polyamine biosynthesis. Since mitogens stimulate T-cells by non-physiological routes, the role of polyamine metabolism in T-cell receptor (TCR)-mediated T-cell activation has not been adequately evaluated. The effect of phytohemagglutinin (PHA) and staphylococcal enterotoxin B (SEB) on T-cell ODC and polyamine synthesis was compared. ODC activity was 6-11-fold higher in PHA compared to SEB stimulated T-cells. These differences were not attributed to differences in the magnitude of T-cell proliferation. Kinetics of ODC and polyamine synthesis were also different in PHA- and SEB-stimulated T-cells. In PHA-stimulated cells ODC levels and the induction of putrescine and spermidine synthesis peaked 6 h prior to peak IL-2 production, while in SEB-stimulated cells, ODC levels and polyamine synthesis peaked 6-12 h after IL-2 production. Differences in the temporal relationship between IL-2 production and polyamine induction in mitogen- versus superantigen-stimulated cells may account for the significant inhibition of the proliferative response by alpha-difluoromethylornithine following PHA but not SEB stimulation. Polyamine metabolism is regulated differently in T-cells stimulated via TCR engagement than with polyclonal mitogens.

Eflornithine↗
PubMed2002

Analysis of polyamines as markers of (patho)physiological conditions.

The aliphatic polyamines, putrescine, spermidine and spermine, are normal cell constituents that play important roles in cell proliferation and differentiation. The equilibrium between cellular uptake and release and the balanced activities of biosynthetic and catabolic enzymes of polyamines are essential for normal homeostasis in the proliferation and functions of cells and tissues. However, the intracellular polyamine content increases in hyperplastic or neoplastic growth. Although the involvement of polyamines in physiological and pathological cell proliferation and differentiation has been well established, the role they play is quite different in relation to cell systems and animal models and is dependent on inducer agents and stimuli. Also, the experimental procedures used to deplete polyamines have been shown to influence the cell responses. In this paper, the assay methods currently in use for polyamines are reviewed and compared with respect to sensitivity, reproducibility and applicability to routine analysis. The relevance of polyamine metabolism and the uptake/release process in many physiological and pathological processes is highlighted, and the cellular polyamine pathways are discussed in relation to the possible diagnostic and therapeutic significance of these mediators.

Biomarkers↗
PubMed2001

DNA condensation by polyamines: a laser light scattering study of structural effects.

Polyamines such as spermidine and spermine are abundant in living cells and are believed to aid in the dense packaging of cellular DNA. DNA condensation is a prerequisite for the transport of gene vectors in living cells. To elucidate the structural features of polyamines governing DNA condensation, we studied the collapse of lambda-DNA by spermine and a series of its homologues, H2N(CH2)3NH(CH2)n=2-12NH(CH2)3NH2 (n = 4 for spermine), using static and dynamic light scattering techniques. All polyamines provoked DNA condensation; however, their efficacy varied with the structural geometry of the polyamine. In 10 mM sodium cacodylate buffer, the EC50 values for DNA condensation were comparable (4 +/- 1 microM) for spermine homologues with n = 4-8, whereas the lower and higher homologues provoked DNA condensation at higher EC50 values. The EC50 values increased with an increase in the monovalent ion (Na+) concentration in the buffer. The slope of a plot of log [EC50(polyamine4+)] against log [Na+] was approximately 1.5 for polyamines with even number values of n, whereas the slope value was approximately 1 for compounds with odd number values of n. Dynamic light scattering measurements showed the presence of compact particles with hydrodynamic radii (Rh) of about 40-50 nm for compounds with n = 3-6. Rh increased with further increase in methylene chain length separating the secondary amino groups of the polyamines (Rh = 60-70 nm for n = 7-10 and >100 nm for n = 11 and 12). Determination of the relative binding affinity of polyamines to DNA using an ethidium bromide displacement assay showed that homologues with n = 2 and 3 as well as those with n > 7 had significantly lower DNA binding affinity compared to spermine and homologues with n = 5 and 6. These data suggest that the chemical structure of isovalent polyamines exerts a profound influence on their ability to recognize and condense DNA, and on the size of the DNA condensates formed in aqueous solution.

Bacteriophage lambda↗
PubMed2006

A novel protein kinase CK2 substrate indicates CK2 is not directly stimulated by polyamines in vivo.

The activity of the protein kinase (CK2) is enhanced in vitro by the binding of polyamines to the CK2beta regulatory subunit. The overexpression of ornithine decarboxylase (ODC), the rate-limiting enzyme in polyamine biosynthesis, also elevates CK2 kinase activity in primary keratinocytes and tissues of K6/ODC transgenic mice. In an effort to better characterize the mechanisms by which polyamines may affect CK2 in vivo, we constructed a transfectable CK2 substrate cDNA consisting of the enhanced green fluorescence protein appended with a canonical CK2 phosphorylation sequence (EGFP-S). In contrast to unmodified EGFP, the EGFP-S protein was extensively phosphorylated by CK2, and this phosphorylation was stimulated by the polyamine spermine in a dose-dependent manner. The in vivo phosphorylation of EGFP-S was examined in cell lines which inducibly express either wild-type CK2 holoenzyme or a CK2 holoenzyme which contains activating mutations in the polyamine-binding region of its CK2beta regulatory subunit. Neither the overexpression of ODC in either cell line nor the mutation of the CK2beta subunit conferred an increase in CK2 kinase activity as measured by the in vivo phosphorylation of EGFP-S. Rather, our data indicate that polyamines increase total CK2 kinase activity through increases in steady-state levels of both CK2alpha and CK2beta subunits. The overexpression of ODC resulted in a 3-fold increase in steady-state levels of both exogenous and endogenous CK2 transcripts but did not increase the half-life of wild-type or mutated CK2 protein. These data suggest that the regulation of intracellular CK2 by the polyamines may occur through mechanisms distinct from the direct stimulation of CK2 by polyamines in vitro as previously described.

Casein Kinase II↗
PubMed1999

Activation of nuclear factor kappaB by polyamines in breast cancer cells.

Polyamines-putrescine, spermidine, and spermine-are involved in the growth of breast cancer cells. A possible target of polyamine action is at the site of interaction of transcription factors with their response elements. NF-kappaB is a member of the rel family of transcription factors that regulate transcription of genes in the proliferative/anti-apoptotic pathways. We performed electrophoretic mobility shift assays to study the role of polyamines in NF-kappaB binding to NF-kappaB response elements (NREs), the consensus sequence of which is GGGGAATTCCCC. Using cellular extract from MCF-7 breast cancer cells, we found very little binding of NF-kappaB to NRE in the absence of polyamines. Addition of 1 mM spermidine or spermine caused a 4- and 6-fold increase in NF-kappaB-NRE binding, respectively. Putrescine induced a 2-fold increase in the binding at 2 mM concentration. Using antibody supershift assays, we identified the p50 subunit of NF-kappaB to be a major component in NF-kappaB-NRE complex formation in the presence of polyamines. However, the decreased intensity of the band corresponding to NF-kappaB-NRE complex in the presence of anti-p65, c-rel, relB and p52 antibodies suggested the participation of these subunits also. Spermine also stimulated NF-kappaB-NRE binding using cellular extracts from other breast cancer cell lines and a normal breast epithelial cell line. A differential effect of spermine analogues on NF-kappaB-NRE binding was observed, with spermine exerting the maximal effect. CD spectra of NRE containing oligonucleotides was asymmetric and distinct from that of a typical B-DNA CD spectrum. A concentration-dependent increase in T(m) of the duplex NRE was seen in the presence of polyamines. In transient transfection experiments using an NF-kappaB driven secreted alkaline phosphatase (SEAP) reporter, spermine induced NF-kappaB activity by approximately 2-fold as compared to controls. Spermine induced activation of NF-kappaB was also confirmed using an NF-kappaB-EGFP (enhanced green fluorescent protein) vector in transient transfections in which expression of the green fluorescent protein was visualized by fluorescence microscopy. These data show a gene regulatory function of polyamines involving enhanced binding of NF-kappaB to NRE and a possible mechanism for the action of polyamines in breast cancer cell proliferation.

Breast Neoplasms↗