Polyamine analogues – an update

Summary. The polyamines are growth factors in both normal and cancer cells. As the intracellular polyamine content correlates positively with the growth potential of that cell, the idea that depletion of polyamine content will result in inhibition of cell growth and, particularly tumour cell growth, has been developed over the last 15 years. The polyamine pathway is therefore a target for development of rationally designed, antiproliferative agents. Following the lessons from the single enzyme inhibitors (α-difluoromethylornithine DFMO), three generations of polyamine analogues have been synthesised and tested in vitro and in vivo. The analogues are multi-site inhibitors affecting multiple reactions in the pathway and thus prevent the up-regulation of compensatory reactions that have been the downfall of DFMO in anticancer chemotherapy. Although the initial concept was that the analogues may provide novel anticancer drugs, it now seems likely that the analogues will have wider applications in diseases involving hyperplasia.     

Concise preparation of <Emphasis Type="Italic">N</Emphasis><Superscript>α</Superscript>-Fmoc-<Emphasis Type="Italic">N</Emphasis><Superscript>ɛ</Superscript>-(Boc,methyl)-lysine and its application in the synthesis of site-specifically lysine monomethylated peptide

Summary. A concise preparation of N ^α-Fmoc-N ^ɛ-(Boc, methyl)-lysine and its application in the synthesis of site-specifically lysine monomethylated peptide is described. N ^α-Fmoc-N ^ɛ-(Boc, methyl)-lysine is obtained, via consecutive reductive benzylation and reductive methylation in a one-pot reaction, followed by debenzylation through catalytic hydrogenolysis and Boc protection in another one-pot reaction. A peptide containing monomethylated lysine is successfully synthesized by incorporating N ^α-Fmoc-N ^ɛ-(Boc, methyl)-lysine as a building block via solid-phase peptide synthesis.     

Posttranslational synthesis of hypusine: evolutionary progression and specificity of the hypusine modification

Summary. A naturally occurring unusual amino acid, hypusine [N ^ɛ-(4-amino-2-hydroxybutyl)-lysine] is a component of a single cellular protein, eukaryotic translation initiation factor 5A (eIF5A). It is a modified lysine with structural contribution from the polyamine spermidine. Hypusine is formed in a novel posttranslational modification that involves two enzymes, deoxyhypusine synthase (DHS) and deoxyhypusine hydroxylase (DOHH). eIF5A and deoxyhypusine/hypusine modification are essential for growth of eukaryotic cells. The hypusine synthetic pathway has evolved in eukaryotes and eIF5A, DHS and DOHH are highly conserved, suggesting maintenance of a fundamental cellular function of eIF5A through evolution. The unique feature of the hypusine modification is the strict specificity of the enzymes toward its substrate protein, eIF5A. Moreover, DHS exhibits a narrow specificity toward spermidine. In view of the extraordinary specificity and the requirement for hypusine-containing eIF5A for mammalian cell proliferation, eIF5A and the hypusine biosynthetic enzymes present new potential targets for intervention in aberrant cell proliferation.     

Polyamines are absorbed through a y+ amino acid carrier in rat intestinal epithelial cells

Summary. Due to the similarity in transport characteristics of polyamines and the y⁺ basic amino acid system, we hypothesized that both substrates could be moving through a common carrier site. Competitive and cross inhibition experiments in intestinal epithelial cells revealed the possibility of a common transport site. N-ethylmalemide (NEM) inhibited both lysine and putrescine transport, confirming that both were carried by a y⁺ transporter. Overexpressing the y⁺ transporter CAT-1 in a polyamine transport-deficient cell line, CHO-MG, did not reconstitute polyamine-transport. Thus, polyamines are not traveling through CAT-1. To determine if lysine is carried by a polyamine transport site, an antizyme-overexpressing cell line was used. Antizyme overexpression decreased polyamine uptake by 50%; in contrast, lysine transport was unaffected. Therefore, lysine is not traveling through a polyamine transport site. It appears that polyamines and lysine are likely traveling through a common unknown y⁺ transport site.     

Antizyme and antizyme inhibitor activities influence cellular responses to polyamine analogs

Summary. Close structural analogs of spermidine and spermine, polyamine mimetics, are potential chemotheraputic agents as they depress cellular polyamines required for tumor growth. Specific mimetic analogs stimulate synthesis of the regulatory protein antizyme (AZ), which not only inactivates the initial enzyme in polyamine biosynthesis but also inhibits cellular uptake of polyamines. The role of AZ induction in influencing cellular uptake of representative analogs was investigated using three analogs produced by Cellgate Inc., CGC-11047, CGC-11102, and CGC-11144, which exhibit markedly distinct AZ-inducing potential. An inverse correlation was noted between the AZ-inducing activity of a compound and the steady-state levels accumulated in cells. As some tumor cells over express AZI as a means of enhancing the polyamines required for aggressive growth, analog sensitivity was examined in transgenic CHO cells expressing exogenous antizyme inhibitor protein (AZI). Although AZI over expression increased cell sensitivity to analogs, the degree of this affect varied with the analog used.     

Unique polyamines produced by an extreme thermophile, <Emphasis Type="Italic">Thermus thermophilus</Emphasis>

Summary. Recent research progress on polyamines in extreme thermophiles is reviewed. Extreme thermophiles produce two types of unique polyamines; one is longer polyamines such as caldopentamine and caldohexamine, and the other is branched polyamines such as tetrakis(3-aminopropyl)ammonium. The protein synthesis catalyzed by a cell-free extract of Thermus thermophilus, an extreme thermophile, required the presence of a polyamine and the highest activity was found in the presence of tetrakis(3-aminopropyl)ammonium. In vitro experiments, longer polyamines efficiently stabilized double stranded nucleic acids and a branched polyamine, tetrakis(3-aminropyl)ammonium, stabilized stem-and-loop structures. In T. thermophilus, polyamines are synthesized from arginine by a new metabolic pathway; arginine is converted to agmatine and then agmatine is aminopropylated to N¹-aminopropylagmatine which is converted to spermidine by an enzyme coded by a gene homologous to speB (a gene for agmatinase). In this new pathway spermidine is not synthesized from putrescine. Reverse genetic studies indicated that the unique polyamines are synthesized from spermidine.     

Anticancer drugs and hyperthermia enhance cytotoxicity induced by polyamine enzymatic oxidation products

Summary. A correlation between regulation of cell proliferation and polyamine metabolism is described. The latter can enter protein synthesis through the modification of eukaryotic initiation factor 5A (eIF5A) and the formation of the peculiar amino acid hypusine. Specific inhibitors of hypusine formation induce apoptosis that can be potentiated by the combination with cytokines such as interferonα (IFNα) that itself decreases hypusine synthesis. We have also demonstrated that the concomitant treatment of cancer cells with IFNα and the protein synthesis inhibitor fusion protein TGFα/Pseudomonas Aeruginosa toxin synergize in inducing cancer cell growth inhibition. Another way used by polyamines to induce apoptosis is the generation of intracellular oxidative stress through the interaction with bovine serum amine oxidase (BSAO). This enzyme used simultaneously to spermine induces apoptosis, necrosis, inhibition of cell proliferation and inhibition of DNA and protein synthesis in several cell types. The enzymatic oxidation products of polyamine, H₂O₂ and aldehyde(s) cause these effects. We have recently found that the cytotoxicity of anti-cancer agents, either etoposide or docetaxel, in cancer cells is potentiated in the presence of BSAO/Spermine. In conclusion, polyamine metabolites could be useful in the design of new therapeutic strategies.     

Inhibitors of the carrier-mediated influx of auxin in suspension-cultured tobacco cells

 Active auxin transport in plant cells is catalyzed by two carriers working in opposite directions at the plasma membrane, the influx and efflux carriers. A role for the efflux carrier in polar auxin transport (PAT) in plants has been shown from studies using phytotropins. Phytotropins have been invaluable in demonstrating that PAT is essential to ensure polarized and coordinated growth and to provide plants with the capacity to respond to environmental stimuli. However, the function of the influx carrier at the whole-plant level is unknown. Our work aims to identify new auxin-transport inhibitors which could be employed to investigate its function. Thirty-five aryl and aryloxyalkylcarboxylic acids were assayed for their ability to perturb the accumulation of 2,4-dichlorophenoxyacetic acid (2,4-D) and naphthalene-1-acetic acid (1-NAA) in suspension-cultured tobacco (Nicotiana tabacum L.) cells. As 2,4-D and 1-NAA are preferentially transported by the influx and efflux carriers, respectively, accumulation experiments utilizing synthetic auxins provide independant information on the activities of both carriers. The majority (60%) of compounds half-inhibited the carrier-mediated influx of [¹⁴C]2,4-D at concentrations of less than 10 μM. Most failed to interfere with [³H]NAA efflux, at least in the short term. Even though they increasingly perturbed auxin efflux when given a prolonged treatment, several compounds were much better at discriminating between influx and efflux carrier activities than naphthalene-2-acetic acid which is commonly employed to investigate influx-carrier properties. Structure-activity relationships and factors influencing ligand specificity with regard to auxin carriers are discussed. Received: 28 June 1999 / Accepted: 28 August 1999     

Enhancement of transglutaminase activity and polyamine depletion in B16-F10 melanoma cells by flavonoids naringenin and hesperitin correlate to reduction of the <Emphasis Type="Italic">in vivo</Emphasis> metastatic potential

Summary. The in vitro and in vivo effects of two flavonons, naringenin (NG) and hesperitin (HP) on the proliferation rate of highly metastatic murine B16-F10 melanoma cell were investigated. NG or HP treatment of melanoma cells produced a remarkable reduction of cell proliferation, paralleled with both the lowering of the intracellular levels of polyamine, spermidine and spermine and the enhancement of transglutaminase (TGase, EC 2.3.2.13) activity. Orally administered NG or HP in C57BL6/N mice inoculated with B16-F10 cells affected the pulmonary invasion of melanoma cells in an in vivo metastatic assay. The number of lung metastases detected by a computerized image analyzer was reduced, compared to untreated animals, by about 69% in NG-treated mice and by about 36% in HP-treated mice. Survival studies showed that 50% of the NG-treated animals died 38 ± 3.1 days after tumor cell injection (control group: 18 ± 1.5 days) and HP-treated mice died 27 ± 2.3 days after cell inoculation. Taken together, these findings provide further evidences for the potential anticancer properties of dietary flavonoids as chemopreventive agents against malignant melanoma.     

Activation of the Human FPRL-1 Receptor Promotes Ca<Superscript>2+</Superscript> Mobilization in U87 Astrocytoma Cells

The human formyl peptide receptor like 1 (FPRL-1) is a variant of the G_i-coupled formyl-peptide receptor. Functional FPRL-1 is endogenously expressed in the U87 astrocytoma cell line and there is accumulating evidence to suggest that FPRL-1 may be involved in neuroinflammation associated with the pathogenesis of Alzheimer’s disease. In this study, we examined the ability of FPRL-1 to mobilize intracellular Ca²⁺ in U87 astrocytoma cells, as well as in Chinese hamster ovary (CHO) cells stably expressing FPRL-1. We showed that Trp–Lys–Tyr–Met–Val–Met–NH₂ (WKYMVM), a specific agonist for FPRL-1, stimulated Ca²⁺ influx in both U87 and FPRL-1/CHO cells. These effects can be inhibited by the FPRL-1 selective antagonist, WRW⁴. Involvement of G_i proteins was demonstrated with the use of pertussis toxin, while inhibitors of store-operated channels (SOC) including 1-[2-(4-methoxyphenyl)]-2-[3-(4-methpxyphenyl)propoxy]ethyl-1H-imidazole hydrochloride (SKF96365) and 2-aminoethoxydiphenyl borate (2-APB) were found to abolish the WKYMVM-induced Ca²⁺ increase. However, intracellular Ca²⁺ mobilization in both cell lines were unaffected by the phospholipase Cβ inhibitor U73122 or selective ryanodine receptor inhibitors. Our data demonstrated that activation of G_i-coupled FPRL-1 can lead to Ca²⁺ influx possibly via SOCs in U87 and FPRL-1/CHO cells.     

Anthraquinone polyamines: novel channel blockers of <Emphasis Type="Italic">N</Emphasis>-methyl-<Emphasis Type="SmallCaps">d</Emphasis>-aspartate receptors

Summary. Polyamines, in particular spermine, as well as some natural and synthetic polyamine derivatives have been found to be blockers of N-methyl-d-aspartate receptors. We developed novel, polyamine-based channel blockers to analyze the structure of NMDA receptors. Anthraquinone polyamines block NMDA receptors with some selectivity compared to other glutamate receptors. Results using mutant NR1 and NR2 subunits identified amino acid residues that influence blockade by anthraquinone polyamines. The head group (anthraquinone) may be positioned at the selectivity filter/narrowest constriction of the channel and the polyamine tail penetrates this constriction into the inner vestibule below the level of the selectivity filter. The results are consistent with other work showing that NR1 (Asn616) and NR2B (Asn616), but not NR2B (Asn615), make the narrowest constriction of NMDA channel, and that the M3 segments from the two subunits, which form the outer vestibule, are likely staggered relative to each other in the vertical axis of the channel.     

Synthesis and bioevaluation of aryl-guanidino polyamine conjugates targeting the polyamine transporter

Aryl-guanidino polyamine conjugates were prepared to evaluate their recognition for polyamine transporter (PAT) via a-difluoromethylornithine (DFMO) and spermidine (SPD)-treated B16 cell lines. The potent synergistic effects of DFMO on guanidino polyamine conjugates indicated that the presence of DFMO strongly facilitates the transport of conjugates into cells via PAT on cell membrane. The apoptotic mechanisms of triamine conjugates 10 and 1 (with and without guanidine groups) revealed that they induced apoptosis of Hela cells through the mitochondrial pathway associated with lysosomes, while DFMO strongly synergizes the function of 10 without changing the apoptotic route. Taken together, guanidino polyamine conjugates can target PAT for transport as normal polyamine ones, and the presence of guanidine in the polyamine vectors does not seem to alter the cellular targets of the conjugates, which may depend mainly on the pharmacophore.     

1-(N-chloroacetylamino)-alkylphosphonic acids – synthetic precursors of phosphonopeptides

Summary. General procedures of N-chloroacetylation of the representative 1-aminoalkylphosphonic acids (Gly^P, Ala^P, Val^P, Pgly^P and Phe^P) are described. These 1-(N-chloroacetylamino)-alkylphosphonic acids were converted into the corresponding glycylphosphonodipeptides (Gly-AA^P) and/or related N-alkylglycylphosphonodipeptides (Me_nGly-AA^P) in the course of ammonolysis/aminolysis. Physico-chemical properties of synthesized 1-(N-chloroacetylamino)-alkylphosphonic acids and phosphonodipeptides are characterized. Authors’ address: Z. H. Kudzin, Institute of Chemistry, University of Łódź, Narutowicza 68, Łódź 91-136, Poland     

Inhibition of ornithine decarboxylase by α-difluoromethylornithine induces apoptosis of HC11 mouse mammary epithelial cells

Summary. The effect of α-difluoromethylornithine (DFMO) on the apoptosis of HC11 mouse mammary epithelial cells was investigated. The involvement of reactive oxygen species (ROS) and Bcl-2 protein in the mechanism of apoptosis induced by ornithine decarboxylase (ODC) inhibition was also assessed. DFMO (0.1, 1 and 5 mM) induced apoptosis of HC11 cells in dose- and time-dependent manner. Apoptosis manifests itself with morphological features like: cell shrinkage, condensation of chromatin, pyknosis and fragmentation of nucleus, followed by secondary necrosis (putrosis). The decrease in the nuclear DNA contents appearing as the hypodiploidal peak sub-G₁ in the DNA histogram was not dependent on the presence of prolactin (5 μg/ml) in DFMO-treated cultures. Apoptosis induced by ODC inhibition was associated with a rapid increase in ROS concentration in HC11 cells observed within 1 h after DFMO treatment. The down-regulation of Bcl-2 as a decrease in cell number expressing bcl-2 and a lowered Bcl-2 protein content in cells expressing this protooncogene was also noted. The administration of putrescine (50 μM) lowered the number of early-apoptotic, late-apoptotic and necrotic cells. Moreover, it increased the number of cells expressing bcl-2. In conclusion, the disturbance of cellular polyamine homeostasis by inhibition of their synthesis enhances mammary epithelial cell susceptibility to apoptosis. It may occur in the mammary gland at the end of lactation, when the depletion of circulating lactogenic hormones and activation of intra-mammary apoptogenic factors expression take place. Received May 6, 1999; Accepted December 15, 1999     

Synthesis, tandem MS- and NMR-based characterization, and quantification of the carbon 13-labeled advanced glycation endproduct, 6-N-carboxymethyllysine

Summary. 6-N-carboxymethyllysine (CML), generated by the glycation and/or oxidation of lysine residues, has been measured in biological materials and food products using techniques such as ELISA, HPLC with fluorescence detection and mass spectrometry methods. Only limited information has been reported regarding the preparation of standards labeled with either deuterium, ¹³C or ¹⁵N atoms to be used as internal standards. In the present paper, a synthesis of carbon-13 labeled CML is described using l,2-¹³C₂-glyoxylic acid and 2-N-acetyllysine as starting materials. The resulting labeled 2-N-acetyl-CML was purified by HPLC-UV as a dibutyl ester. After a deprotection step, the yield was evaluated to be 53% when the reaction was conducted 17 h at 37°C. CML was extensively studied by ¹H- and ¹³C-NMR and the fragments observed in the collision induced dissociation (CID) spectrum were also assigned. Finally, the standards of CML and carbon-13 labeled CML were accurately quantified based on ¹H-NMR and tandem MS using lysine as an internal reference.     

Chlamydomonas contains calcium stores that are mobilized when phospholipase C is activated

Mastoparan induces Ca²⁺-dependent deflagellation of the unicellular green alga Chlamydomonas moewusii Gerloff, as well as the activation of phospholipase C and the production of inositol 1,4,5-trisphosphate (InsP₃; T. Munnik et al., 1998, Planta 207: 133–145). Even in the absence of extracellular Ca²⁺, mastoparan still induces deflagellation (L.M. Quarmby and H.C. Hartzell, 1994, J Cell Biol 124: 807–815; J.A.J. van Himbergen et al., 1999, J Exp Bot, in press) suggesting that InsP₃ mediates Ca²⁺ release from intracellular stores. To test this hypothesis, cells were pre-loaded with ⁴⁵Ca²⁺ and their plasma membranes permeabilized by digitonin. Subsequent treatment of the cells with mastoparan (3.5 μM) induced release of intracellular ⁴⁵Ca²⁺. Mastoparan also activated phospholipase C in permeabilized cells, as demonstrated by the breakdown of ³²P-phosphatidylinositol 4,5-bisphosphate and the production of diacylglycerol. The mastoparan analogues mas7 and mas17 were also effective and their efficacy was correlated with their biological activity. X-ray microanalysis showed that electron-dense bodies (EDBs) are a major Ca²⁺ store in  C. moewusii. Analysis of digitonin-permeabilized cells showed that EDBs lost calcium at digitonin concentrations that released radioactivity from ⁴⁵Ca²⁺-labelled cells, suggesting that ⁴⁵Ca²⁺ monitored the content of EDBs. X-ray microanaysis of living cells treated with mastoparan also revealed that calcium was released from EDBs. Received: 30 December 1998 / Accepted: 25 June 1999     

A generalized transport model for biased cell migration in an anisotropic environment

 A generalized transport model is derived for cell migration in an anisotropic environment and is applied to the specific cases of biased cell migration in a gradient of a stimulus (taxis; e.g., chemotaxis or haptotaxis) or along an axis of anisotropy (e.g., contact guidance). The model accounts for spatial or directional dependence of cell speed and cell turning behavior to predict a constitutive cell flux equation with drift velocity and diffusivity tensor (termed random motility tensor) that are explicit functions of the parameters of the underlying random walk model. This model provides the connection between cell locomotion and the resulting persistent random walk behavior to the observed cell migration on longer time scales, thus it provides a framework for interpreting cell migration data in terms of underlying motility mechanisms. Received: 8 April 1999     

Non-sufficient cell cycle control as possible clue for the resistance of human malignant glioma cells to clinically relevant treatment conditions

Summary. Objectives. Human gliomas have a catastrophic prognosis with a median survival in the range of one year even after therapeutic treatment. Relatively high resistance towards apoptotic stimuli is the characteristic feature of malignant gliomas. Since cell cycle control has been shown to be the key mechanism controlling both apoptosis and proliferation, this study focuses on DNA damage analysis and protein expression patterns of essential cell cycle regulators P53 and P21^waf1/cip1 in glioma under clinically relevant therapeutic conditions. Material and methods. U87MG cell line, characterised by wild p53-phenotype relevant for the majority of primary malignant glioblastomas, was used. Glioma cells underwent either irradiation or temozolomide treatment alone, or combined radio/chemo treatment. DNA damage was analysed by the “Comet Assay”. Expression rates of target proteins were analysed using “Western-Blot” technique. Results and conclusions. “Comet Assay” demonstrated extensive DNA damage caused by temozolomide treatment alone and in combination with irradiation, correlating well with the low survival rate observed under these treatment conditions. In contrast, irradiation alone resulted in a relatively low DNA damage, correlating well with a high survival rate and indicating a poor therapeutic efficiency of irradiation alone. Unusually low up-regulation of P53 and P21^waf1/cip1 expression patterns was produced by the hereby tested stressful conditions. A deficit in cell cycle control might be the clue to the high resistance of malignant glioma cells to established therapeutic approaches.     

Intracellular chloroplast photorelocation in the moss Physcomitrella patens is mediated by phytochrome as well as by a blue-light receptor

 The light-induced intracellular relocation of chloroplasts was examined in red-light-grown protonemal cells of the moss Physcomitrella patens. When irradiated with polarized red or blue light, chloroplast distribution in the cell depended upon the direction of the electrical vector (E-vector) in both light qualities. When the E-vector was parallel to the cross-wall (i.e. perpendicular to the protonemal axis), chloroplasts accumulated along the cross-wall; however, no accumulation along the cross-wall was observed when the E-vector was perpendicular to it (i.e. parallel to the protonemal axis). When a part of the cell was irradiated with a microbeam of red or blue light, chloroplasts accumulated at or avoided the illumination point depending on the fluence rate used. Red light of 0.1–18 W m⁻² and blue light of 0.01–85.5 W m⁻² induced an accumulation response (low-fluence-rate response; LFR), while an avoidance response (high-fluence-rate response; HFR) was induced by red light of 60 W m⁻² or higher and by blue light of 285 W m⁻². The red-light-induced LFR and HFR were nullified by a simultaneous background irradiation of far-red light, whereas the blue-light-induced LFR and HFR were not affected at all by this treatment. These results show, for the first time, that dichroic phytochrome, as well as the dichroic blue-light receptor, is involved in the chloroplast relocation movement in these bryophyte cells. Further, the phytochrome-mediated responses but not the blue-light responses were revealed to be lost when red-light-grown cells were cultured under white light for 2 d. Received: 7 September 1999 / Accepted: 15 October 1999     

Inducible expression of maize polyamine oxidase in the nucleus of MCF-7 human breast cancer cells confers sensitivity to etoposide

Summary. In this study, polyamine oxidase from maize (MPAO), which is involved in the terminal catabolism of spermidine and spermine to produce an aminoaldehyde, 1,3-diaminopropane and H₂O₂, has been conditionally expressed at high levels in the nucleus of MCF-7 human breast cancer cells, with the aim to interfere with polyamine homeostasis and cell proliferation. Recombinant MPAO expression induced accumulation of a high amount of 1,3-diaminopropane, an increase of putrescine levels and no alteration in the cellular content of spermine and spermidine. Furthermore, recombinant MPAO expression did not interfere with cell growth of MCF-7 cells under normal conditions but it did confer higher growth sensitivity to etoposide, a DNA topoisomerase II inhibitor widely used as antineoplastic drug. These data suggest polyamine oxidases as a potential tool to improve the efficiency of antiproliferative agents despite the difficulty to interfere with cellular homeostasis of spermine and spermidine. Authors’ address: Dr. Paraskevi Tavladoraki, Department of Biology, University ‘Roma Tre’, Viale G. Marconi 446, 00146 Rome, Italy