Inhibitor Of S-adenosylmethionine Decarboxylase Research Articles

The role of indole-3-butyric acid and polyamines on in vitro rooting of microshoots of Rosa damascena Mill. and Gladiolus hybridus Hort.

The effect of indole-3-butyric (IBA) acid and polyamines was investigated on in vitro rooting of microshoots of Rosa damascena Mill. (difficult-to-root) and Gladiolus hybridus Hort. (easy-to-root) plants. Both IBA and putrescine (PUT) individually induced rooting and showed a synergistic effect in the rooting of microshoots of both plant species. Maximum rooting of microshoots (%) was observed on Murashige and Skoog medium (MS medium) supplemented with 2.5 μM IBA and 100 μM PUT. The incorporation of methylglyoxal(bis) guanylhydrazone (MGBG: a competitive inhibitor of s-adenosyl methionine decarboxylase and is known to block spermidine and spermine synthesis) completely inhibited the rooting of microshoots of both plant species. Similarly, the incorporation of l-aminoguanidine (AG: an inhibitor of diamine oxidase) also inhibited the adventitious rooting of microshoots of both species, indicating the role of the oxidation product of PUT in rooting. Rooting was not observed in either species when a medium containing MGBG was supplemented with spermidine. In contrast, when spermine was incorporated in a MS medium with MGBG, the rooting was achieved in both species, thus establishing spermine's clear role in the rooting of microshoots. An increase in peroxidase activity was observed during IBA and PUT-induced rooting. The performance of plants rooted on a medium containing 2.5 μM IBA was significantly better than those rooted on a medium containing 2.5 μM IBA and 100 μM PUT.

Conjugated Polyamines in Root Plasma Membrane Enhanced the Tolerance of Plum Seedling to Osmotic Stress by Stabilizing Membrane Structure and Therefore Elevating H+-ATPase Activity.

Polyamines are small positively charged molecules in plants and play important functions in many biological processes under various environmental stresses. One of the most confounding problems relating to polyamines (PAs) in stresses is the lack of understanding of the mechanisms underlying their function(s). Furthermore, a limited number of studies have addressed this issue at the sub-cellular level, especially in tree plants under drought stress. Therefore, in this research, by simulating natural drought stress with polyethylene glycol (PEG) osmotic stress, the relationship between the levels of conjugated polyamines and the activity of H+-ATPase in the plasma membrane was elucidated with the roots of two plum (Prunus salicina L.) cultivars, which were different in drought tolerance, as experimental materials. Furthermore, free PA levels and the activities of S-adenosylmethionine decarboxylase (SAMDC) and transglutaminase (TGase), which were closely associated with the levels of free and conjugated PAs, were also detected. Results showed that under osmotic stress, the increases of the levels of non-covalently conjugated (non-CC) spermidine (Spd) and spermine (Spm), covalently conjugated (CC) putrescine (Put) and Spd in the plasma membrane of drought-tolerant Ganli No. 5 were more significant than those of drought-sensitive Suli No. 3, indicating that these conjugated PAs might be involved in the tolerance of plum seedlings to stress. Furthermore, the conjugated PAs were closely correlated with plum seedling growth, water retention capacity, plasma membrane damage degree, and hydrogen (H+)-ATPase activity in the plasma membrane. To get more complementary pieces of evidence, we subjected plum seedlings to combined treatments of PEG and exogenous PA (Spd and Spm), and an inhibitor of SAMDC [methylglyoxal-bis (guanylhydrazone), (MGBG)] or TGase (o-phenanthroline). These results collectively suggested that non-CC Spd and Spm, CC Put and Spd in plasma membrane might function in enhancing the tolerance of plum seedlings to osmotic stress by stabilizing membrane structure and therefore elevating H+-ATPase activity.

Relationship between seed quality and changes in conjugated polyamine in plasma membrane purified from wheat embryos during grain ripening

In order to explore the relationship between seed quality and changes in conjugated polyamine in plasma membrane purified from wheat embryos during grain ripening. Plasma membrane (PM) vesicles were isolated from the embryos of ripening wheat (Triticum aestivum L.) grains by the gradient centrifugation method. The contents of polyamines conjugated (covalently and non-covalently) to the PM vesicles were investigated. Results showed that after pollination, from the 22nd to the 32nd day, the embryos of wheat grains underwent dehydration, as judged by the decrease of embryo relative water content (ERWC). During embryo ripening, non-covalently conjugated (NCC)-Spd and NCC-Spm, covalently conjugated (CC)-Put and CC-Spd contents increased markedly, while relative embryo cell vigor (RECV) decreased slightly. The treatment with methylglyoxyl-bis (guanylhydrazone) (MGBG), an inhibitor of S-adenosylmethionine decarboxylase (SAMDC), inhibited the increases of the NCC-Spd and NCC-Spm contents, enhanced the decrease of RECV simultaneously, and decreased mature seed relative germination potential (RGP). The effects of MGBG were reversed by exogenous Spd and Spm. Phenanthrolin (o–Phen), an inhibitor of transglutaminase (TGase), inhibited the increases of CC-Put and CC-Spd contents, enhanced the decrease of RECV simultaneously, and decreased mature seed RGP. These results suggest that during embryo ripening, the levels of NCC-Spd, NCC-Spm, CC-Put, and CC-Spd increase, and these increases might affect embryo cell vigor and seed germination potential.

Discovery of Covalent Ligands via Noncovalent Docking by Dissecting Covalent Docking Based on a "Steric-Clashes Alleviating Receptor (SCAR)" Strategy.

Covalent ligands modulating protein activities/signals have attracted unprecedented attention in recent years, but the insufficient understanding of their advantages in the early days of drug discovery has hindered their rational discovery and development. This also left us inadequate knowledge on the rational design of covalent ligands, e.g., how to balance the contribution from the covalent group and the noncovalent group, respectively. In this work, we dissected the noncovalent docking from covalent docking by creating SCARs (steric-clashes alleviating receptors). We showed that the SCAR method outperformed those specifically developed but more complicated covalent docking protocols. We furthermore provided a "proof-of-principle" example by implementing this method in the first high-throughput screening and discovery of novel covalent inhibitors of S-adenosylmethionine decarboxylase. This work demonstrated that noncovalent groups play a predeterminate role in the design of covalent ligands, and would be of great value in accelerating the discovery and development of covalent ligands.

Drought-inhibited ribulose-1,5-bisphosphate carboxylase activity is mediated through increased release of ethylene and changes in the ratio of polyamines in pakchoi

To study the mechanisms of drought inhibiting photosynthesis and the role of PAs and ethylene, the photosynthetic rate (Pn), the maximal photochemical efficiency of PSII (Fv/Fm), the intercellular CO2 concentration (Ci), photorespiratory rate (Pr), the amount of chlorophyll (chl), antioxidant enzyme activity, ethylene levels, RuBPC (ribulose-1,5-bisphosphate carboxylase) activity and endogenous polyamine levels of pakchoi were examined, and an inhibitor of S-adenosylmethionine decarboxylase (SAMDC) and an inhibitor of ethylene synthesis and spermidine (Spd) were used to induce the change of endogenous polyamine levels. The results show that drought induced a decrease in Pn and RuBPC activity, an increase in the intercellular CO2 concentration (Ci), but no change in the actual photochemical efficiency of PSII (ΦPSII), and chlorophyll content. In addition, drought caused an increase in the free putrescine (fPut), the ethylene levels, a decrease in the Spd and spermine (Spm) levels, and the PAs/fPut ratio in the leaves. The exogenous application of Spd and amino oxiacetic acid (AOAA, an inhibitor of ethylene synthesis) markedly reversed these drought-induced effects on polyamine, ethylene, Pn, the PAs/fPut ratio and RuBPCase activity in leaves. Methylglyoxal-bis(guanylhydrazone) (MGBG), an inhibitor of SAMDC resulting in the inability of activated cells to synthesize Spd and Spm, exacerbates the negative effects induced by drought. These results suggest that the decrease in Pn is at least partially attributed to the decrease of RuBPC activity under drought stress and that drought inhibits RuBPC activity by decreasing the ratio of PAs/fPut and increasing the release of ethylene.

Changes in H+-ATPase activity and conjugated polyamine contents in plasma membrane purified from developing wheat embryos under short-time drought stress

Plasma membrane (PM) vesicles were isolated from developing embryos of wheat (Triticum aestivum L.) with different drought-tolerance under drought stress by the gradient centrifugation method. The activity of the PM H+-ATPase (EC 3.6.1.35) and contents of polyamine conjugated (covalently and noncovalently) to the PM vesicles were investigated. Results showed that after drought treatment for 3 d, embryo relative water content (ERWC), embryo relative dry weight increase rate (ERDWIR) of drought-sensitive Yumai No. 48 cultivar decreased more significantly than those of drought-tolerant Luomai No. 22 cultivar, while PM H+-ATPase activity, noncovalently conjugated (NCC) spermidine (Spd) and NCC spermine (Spm), the covalently conjugated (CC) putrescine (Put) and CC Spd of PM from Luomai No. 22 cultivar increased more obviously than those from Yumai No. 48 cultivar. As judged by increases in ERWC and ERDWIR, treatment with exogenous Spd alleviated markedly drought injuries to Yumai No. 48, coupled with significant increases in NCC Spd and NCC Spm levels and H+-ATPase activity in the embryo PM vesicle. Under drought stress, the treatment of drought-tolerant Luomai No. 22 cultivar with methylglyoxyl-bis (guanylhydrazone) (MGBG), an inhibitor of S-adenosylmethionine decarboxylase (SAMDC), and phenanthrolin (o−Phen), an inhibitor of transglutaminase (TGase) respectively, caused a decrease of the NCC Spd, NCC Spm, CC Put and CC Spd. Those decreases were associated with decreased PM-H+-ATPase activity and the tolerance of developing wheat embryos to osmotic stress, as judged by decreases in ERWC and ERDWIR. These results suggest that tolerance of the developing wheat embryos to drought stress is associated with the embryo PM H+-ATPase and the levels of NCC Spd, NCC Spm, CC Put and CC Spd in embryo PM vesicles.

Role of polyamines at the G1/S boundary and G2/M phase of the cell cycle

The role of polyamines at the G1/S boundary and in the G2/M phase of the cell cycle was studied using synchronized HeLa cells treated with thymidine or with thymidine and aphidicolin. Synchronized cells were cultured in the absence or presence of α-difluoromethylornithine (DFMO), an inhibitor of ornithine decarboxylase, plus ethylglyoxal bis(guanylhydrazone) (EGBG), an inhibitor of S-adenosylmethionine decarboxylase. When polyamine content was reduced by treatment with DFMO and EGBG, the transition from G1 to S phase was delayed. In parallel, the level of p27Kip1 was greatly increased, so its mechanism was studied in detail. Synthesis of p27Kip1 was stimulated at the level of translation by a decrease in polyamine levels, because of the existence of long 5′-untranslated region (5′-UTR) in p27Kip1 mRNA. Similarly, the transition from the G2/M to the G1 phase was delayed by a reduction in polyamine levels. In parallel, the number of multinucleate cells increased by 3-fold. This was parallel with the inhibition of cytokinesis due to an unusual distribution of actin and α-tubulin at the M phase. Since an association of polyamines with chromosomes was not observed by immunofluorescence microscopy at the M phase, polyamines may have only a minor role in structural changes of chromosomes at the M phase. In general, the involvement of polyamines at the G2/M phase was smaller than that at the G1/S boundary.

Polyamine biosynthesis and control of the development of functional pollen in kiwifruit

The role of polyamines (PAs) in plant reproduction, especially pollen development and germination has been demonstrated in several higher plants. The aim of the present research was to investigate PA involvement in pollen development and germination in dioecious kiwifruit (Actinidia deliciosa). Differences in PA content, level and gene expression for PA biosynthetic enzymes, and the effect of PA biosynthetic inhibitors were found during pollen development (or abortion in female flowers). Whereas PAs, especially spermidine (Spd), remained high throughout the development of functional pollen, the levels collapsed by the last stage of development of sterile pollen. Mature and functional pollen from male-fertile anthers showed S-adenosyl methionine decarboxylase activity (SAMDC; involved in Spd biosynthesis) throughout microgametogenesis, with high levels of soluble SAMDC found starting from the late uninucleate microspore stage. Soluble SAMDC was absent in male-sterile anthers. Arginine decarboxylase [ADC; for putrescine (Put) biosynthesis] showed little difference in functional vs sterile pollen; ornithine decarboxylase [ODC; also for putrescine (Put) biosynthesis] was present only in sterile pollen. Ultrastructural studies of aborted pollen grains in male-sterile flowers showed that cytoplasmic residues near the intine contain vesicles, extruding towards the pollen wall. Very high SAMDC activity was found in the wall residues of the aborted pollen. The combined application in planta of competitive inhibitors of S-adenosylmethionine decarboxylase (MGBG) and of spermidine synthase (CHA), or of D-arginine (inhibitor of Put synthesis), to male-fertile plants led to abnormal pollen grains with reduced viability. The importance of PAs during male-fertile pollen germination was also found. In fact, PA biosynthetic enzymes (ADC and, mainly, SAMDC) were active early during pollen hydration and germination in vitro. Two different SAMDC gene transcripts were expressed in germinating pollen together with a lower level of ADC transcript. Gene expression preceded PA enzyme activity. The application of PA inhibitors in planta drastically reduced pollen germination. Thus, low free Spd can lead either to degeneration or loss of functionality of kiwifruit pollen grains.

Cinnamic acid-inhibited ribulose-1,5-bisphosphate carboxylase activity is mediated through decreased spermine and changes in the ratio of polyamines in cowpea

This study investigated the effects of cinnamic acid (CA) on ribulose-1,5-bisphosphate carboxylase (RuBPC) activity and the endogenous polyamine levels of cowpea leaves. The results show that 0.1 mM CA treatment decreased photosynthetic rate ( P n) and RuBPC activity, but it did not affect the maximal photochemical efficiency of PSII ( F v/ F m), the actual photochemical efficiency of PSII ( ΦPSII), intercellular CO 2 concentration ( C i), and relative chlorophyll content. These suggest that the decrease in P n is at least partially attributed to a lowered RuBPC activity. In addition, 0.1 mM CA treatment increased the putrescine (Put) level, but decreased spermidine (Spd) and spermine (Spm) levels, thereby reducing the (Spd+Spm)/Put (PAs) ratio in the leaves. The exogenous application of 1 mM Spd markedly reversed these CA-induced effects for polyamine and partially restored the PAs ratio and RuBPC activity in leaves. Methylglyoxal-bis (guanylhydrazone) (MGBG), which is an inhibitor of S-adenosylmethionine decarboxylase (SAMDC), results in the inability of activated cells to synthesize Spd and exacerbates the negative effects induced by CA. The exogenous application of 1 mM d-arginine ( d-Arg), which is an inhibitor of Put biosynthesis, decreased the levels of Put, but increased the PAs ratio and RuBPC activity in leaves. These results suggest that 0.1 mM CA inhibits RuBPC activity by decreasing the levels of endogenous free and perchloric acid soluble (PS) conjugated Spm, as well as the PAs ratio.

RNA Interference-Mediated Silencing of Ornithine Decarboxylase and Spermidine Synthase Genes in Trypanosoma brucei Provides Insight into Regulation of Polyamine Biosynthesis

Polyamine biosynthesis is a drug target for the treatment of African sleeping sickness; however, mechanisms regulating the pathway in Trypanosoma brucei are not well understood. Recently, we showed that RNA interference (RNAi)-mediated gene silencing or the inhibition of S-adenosylmethionine decarboxylase (AdoMetDC) led to the upregulation of the AdoMetDC activator, prozyme, and ornithine decarboxylase (ODC) proteins. To determine if this regulatory response is specific to AdoMetDC, we studied the effects of the RNAi-induced silencing of the spermidine synthase (SpdSyn) and ODC genes in bloodstream form T. brucei. The knockdown of either gene product led to the depletion of the polyamine and trypanothione pools and to cell death. Decarboxylated AdoMet levels were elevated, while AdoMet was not affected. There was no significant effect on the protein levels of other polyamine pathway enzymes. The treatment of parasites with the ODC inhibitor alpha-difluoromethylornithine gave similar results to those observed for ODC knockdown. Thus, the cellular response to the loss of AdoMetDC activity is distinctive, suggesting that AdoMetDC activity controls the expression levels of the other spermidine biosynthetic enzymes. RNAi-mediated cell death occurred more rapidly for ODC than for SpdSyn. Further, the ODC RNAi cells were rescued by putrescine, but not spermidine, suggesting that the depletion of both putrescine and spermidine is more detrimental than the depletion of spermidine alone. This finding may contribute to the effectiveness of ODC as a target for the treatment of African sleeping sickness, thus providing important insight into the mechanism of action of a key antitrypanosomal agent.

NovelS-Adenosylmethionine Decarboxylase Inhibitors for the Treatment of Human African Trypanosomiasis

Trypanosomiasis remains a significant disease across the sub-Saharan African continent, with 50,000 to 70,000 individuals infected. The utility of current therapies is limited by issues of toxicity and the need to administer compounds intravenously. We have begun a program to pursue lead optimization around MDL 73811, an irreversible inhibitor of S-adenosylmethionine decarboxylase (AdoMetDC). This compound is potent but in previous studies cleared rapidly from the blood of rats (T. L. Byers, T. L. Bush, P. P. McCann, and A. J. Bitonti, Biochem. J. 274:527-533). One of the analogs synthesized (Genz-644131) was shown to be highly active against Trypanosoma brucei rhodesiense in vitro (50% inhibitory concentration, 400 pg/ml). Enzyme kinetic studies showed Genz-644131 to be approximately fivefold more potent than MDL 73811 against the T. brucei brucei AdoMetDC-prozyme complex. This compound was stable in vitro in rat and human liver microsomal and hepatocyte assays, was stable in rat whole-blood assays, did not significantly inhibit human cytochrome P450 enzymes, had no measurable efflux in CaCo-2 cells, and was only 41% bound by serum proteins. Pharmacokinetic studies of mice following intraperitoneal dosing showed that the half-life of Genz-644131 was threefold greater than that of MDL 73811 (7.4 h versus 2.5 h). Furthermore, brain penetration of Genz-644131 was 4.3-fold higher than that of MDL 73811. Finally, in vivo efficacy studies of T. b. brucei strain STIB 795-infected mice showed that Genz-644131 significantly extended survival (from 6.75 days for controls to >30 days for treated animals) and cured animals infected with T. b. brucei strain LAB 110 EATRO. Taken together, the data strengthen validation of AdoMetDC as an important parasite target, and these studies have shown that analogs of MDL 73811 can be synthesized with improved potency and brain penetration.

Effects of spermidine and spermine levels on salt tolerance associated with tonoplast H+-ATPase and H+-PPase activities in barley roots

The effects of polyamines (Putrescine— Put; Spermidine—Spd; and Spermine—Spm) on␣salt tolerance of seedlings of two barley (Hordeum vulgare L.) cultivars (J4, salt-tolerant; KP7, salt-sensitive) were investigated. The results showed that, the salt-tolerant cultivar J4 seedlings accumulated much higher levels of Spd and Spm and lower Put than the salt-sensitive cultivar KP7␣under salt stress. At the same time, the dry weight of KP7 decreased significantly than that of␣J4. After methylglyoxal bis(guanylhydrazone) [MGBG, an inhibitor of S-adenosylmethionine decarboxylase (SAMDC)] treatment, Spd and Spm levels together with the dry weight of both cultivars were reduced, but the salt-caused dry weight reduction in two cultivars could be reversed by the concomitant treatment with Spd. MGBG decreased the activities of tonoplast H+-ATPase and H+-PPase too, but the experiments in vitro indicated that MGBG was not able to affect the above two enzyme activities. However, the polyamines, especially Spd, promoted their activities obviously. These results suggested that the conversion of Put to Spd and Spm and maintenance of higher levels of Spd and Spm were necessary for plant salt tolerance.

Relationship between osmotic stress and polyamines conjugated to the deoxyribonucleic acid-protein in wheat seedling roots

The contents of covalently conjugated polyamines (CC-PAs) and noncovalently conjugated polyamines (NCC-PAs) to deoxyribonucleic acid-protein (DNP) isolated from wheat (Triticum aestivum L.) seedling roots under osmotic stress were detected. Results showed thatafter osmotic stress treatment for 7 d, the levels in NCC-spermine (NCC-Spm) and NCC-spermidine (NCC-Spd) of drought-tolerant Yumai No. 18 cv. increased more markedly than that of drought-sensitive Yangmai No. 9 cv., while the NCC-putrescine (NCC-Put) could not be statistically detected in two cultivars. Exogenous Spm treatment alleviated osmotic stress injury to Yangmai No. 9 cv. seedlings, coupled with marked increases of NCC-Spm and NCC-Spd levels of this cultivar. Under PEG osmotic stress, the concomitant treatment of drought-tolerant Yumai No. 18 cv.seedlings with methylglyoxyl-bis (guanylhydrazone) (MGBG), an inhibitor of S-adenosylmethionine decarboxylase (SAMDC), aggravated osmotic stress injury to this cultivar, coupled with market decreases of the NCC-Spm and NCC-Spd levels. The levels in CC-Put and CC-Spd of drought-tolerant Yumai No. 18 cv. increased more markedly than that of drought-sensitive Yangmai No. 9 cv. Under osmotic stress. The treatment of drought-tolerant Yumai No. 18 cv. seedlings with phenanthrolin (o-Phen), an inhibitor of transglutaminase (TGase), aggravated osmotic stress injury to this cultivar, coupled with a reduction of sum contents of CC-Put+CC-Spd. These results suggested that NCC-Spm and NCC-Spd, together with CC-Put and CC-Spd, in DNP of roots could enhance tolerance of the wheat seedlings to osmotic stress.

Multi-centre Phase II trial of the polyamine synthesis inhibitor SAM486A (CGP48664) in patients with metastatic melanoma

To determine the activity and tolerability of SAM496A, an inhibitor of S-adenosylmethionine decarboxylase (SAMDC), in patients with metastatic melanoma who had not received prior chemotherapy. Selected patients were offered participation in two sub-studies examining early changes in tumor metabolism with FDG-PET and changes in tumor polyamine content. Fifteen patients with measurable metastatic melanoma, normal cardiac function, and no known CNS metastases were eligible and received SAM486A by 1-hour IV infusion daily for 5 days every 3 weeks. Response was assessed by SWOG criteria. No patient had a confirmed partial response. Fatigue/lethargy, myalgia and neutropenia were the main toxicities but no febrile neutropenia or grade 4 non-hematological toxicity occurred. Five patients had PET scans pre-treatment and on days 8-12 of cycle 1. No patient had reduction of tumor metabolism. Serial biopsy in one patient showed alterations in polyamines consistent with SAMDC inhibition. Using the present dose and schedule of administration, SAM486A does not have significant therapeutic potential in patients with metastatic melanoma.

Effects of Polyamine Synthesis Inhibitors on Primary Tumor Features and Metastatic Capacity of Human Breast Cancer Cells

We have previously reported that inhibition of polyamine biosynthesis with alpha-difluoromethylornithine (DFMO) reduces pulmonary metastasis from MDA-MB-435 human breast cancer xenografts without affecting the volume of the primary tumors (Manni et al. Clin Exp Mets 20:321, 2003). In these experiments, we show that DFMO treatment (2% in drinking H(2)O) reduced the growth fraction of the primary tumors by 60%. However, this effect was counter-balanced by a similar reduction in non-apoptotic necrosis, thus accounting for the preservation of tumor volume in DFMO-treated mice. DFMO treatment caused a 4-fold increase in cytoplasmic staining for cleaved caspase-3 (as opposed to the nuclear staining observed in control tonsil tissue) in the absence of histologic evidence of apoptosis. DFMO treatment reduced the number of mice with pulmonary metastasis by approximately 80% and the number of metastasis per mouse by >90% in association with a reduction in invasiveness of the primary tumor in the surrounding dermis and muscle by approximately 30%. DFMO treatment increased ERK phosphorylation in the tumors, an effect that has been found by us in vitro to be causally linked to the anti-invasive effect of the drug (Manni et al. Clin Exp Metast 2004; 21: 461]. DFMO also increased tyrosine phosphorylation of STAT-3 and expression of STAT-1 and JNK proteins. Administration of SAM486A (1 mg/kg/i.p. daily), an inhibitor of S-adenosylmethionine decarboxylase, either individually or in combination with DFMO, was not found to exert any biological or biochemical effects, most likely as a result of its failure to suppress tissue polyamine levels under these experimental conditions.

S‐adenosyl methionine decarboxylase activity is required for the outcome of herpes simplex virus type 1 infection and represents a new potential therapeutic target

All the available antiherpetic drugs are directed against viral proteins. Their extensive clinical use has led to the emergence of resistant viral strains. There is a need for the treatment of herpes infections due to resistant strains, especially for immunocompromised patients. To design new kinds of drugs, we have developed a strategy to identify cellular targets. Herpes simplex virus type 1 (HSV-1) infection is concomitant to a repression of most host protein synthesis. However, some cellular proteins continue to be efficiently synthesized. We speculated that some of them could determine the outcome of infection. Since two polyamines, spermidine and spermine, are components of the HSV-1 virions, we investigated whether enzymes involved in their synthesis could be required for viral infection. We show that inhibition of S-adenosyl methionine decarboxylase, a key enzyme of the polyamine metabolic pathway, prevents HSV-1 infection. Inhibition of polyamine synthesis prevents infection of culture cells with HSV-1 laboratory strains as well as clinical isolates that are resistant to the conventional antiviral drugs acyclovir and foscarnet. Our data provide the opportunity to develop molecules with a novel mechanism of action for the treatment of herpes infection.

Identification and Characterization of a Polyamine Permease from the Protozoan Parasite Leishmania major

The proteins that mediate polyamine translocation into eukaryotic cells have not been identified at the molecular level. To define the polyamine transport pathways in eukaryotic cells we have cloned a gene, LmPOT1, that encodes a polyamine transporter from the protozoan pathogen, Leishmania major. Sequence analysis of LmPOT1 predicted an unusual 803-residue polytopic protein with 9-12 transmembrane domains. Expression of LmPOT1 cRNA in Xenopus laevis oocytes revealed LmPOT1 to be a high affinity transporter for both putrescine and spermidine, whereas expression of LmPOT1 in Trypanosoma brucei stimulated putrescine uptake that was sensitive to inhibition by pentamidine and proton ionophores. Immunoblot analysis established that LmPOT1 was expressed predominantly in the insect vector form of L. major, and immunofluorescence demonstrated that LmPOT1 was localized predominantly to the parasite plasma membrane. To our knowledge this is the first molecular identification and characterization of a cell surface polyamine transporter in eukaryotic cells.

Effect of osmotic stress on the activity of H +-ATPase and the levels of covalently and noncovalently conjugated polyamines in plasma membrane preparation from wheat seedling roots

The influence of osmotic stress on the H +-ATPase (EC 3.6.1.35) activity and the contents of covalently and noncovalently conjugated polyamines were investigated using plasma membrane (PM) vesicles isolated from the roots of wheat ( Triticum aestivum L.) seedlings with different drought-tolerance by the sucrose gradient centrifugation method. Results showed that after polyethylene glycol (PEG) 6000 (−0.55 MPa) treatment for 7 days, leaf relative water content (LRWC), relative dry weight increase rate (RDWIR) of seedlings of drought-sensitive Yangmai No. 9 cv. decreased more markedly than that of drought-tolerant Yumai No. 18 cv., while plasma membrane (PM) H +-ATPase activity, the content ratio of noncovalently conjugated polyamine (NCC-PAs) NCC-Spd + NCC-Spm to NCC-Put and the sum contents of covalently conjugated polyamines (CC-PAs) CC-Put + CC-Spd of PM of Yumai No. 18 cv. increased more markedly than that of Yangmai No. 9 cv. Exogenous Spd treatment alleviated osmotic stress injury to Yangmai No.9 seedlings, coupled with marked increases of NCC-Spd and NCC-Spm levels and H +-ATPase activity in the root PM vesicle. Under PEG osmotic stress, the concomitant treatment of drought-tolerant cv. Yumai No. 18 seedlings with methylglyoxyl-bis (guanylhydrazone) (MGBG), an inhibitor of S-adenosylmethionine decarboxylase (SAMDC), and phenanthrolin (o-Phen), an inhibitor of transglutaminase (TGase), caused a decrease of the NCC-Spd + NCC-Spm: NCC-Put ratio and a reduction of sum contents of CC-Put + CC-Spd, respectively. This was associated with decreased PM-H +-ATPase activity and tolerance of seedlings to osmotic stress. Together, these results suggest that tolerance of the wheat seedlings to osmotic stress is associated with the activity of H +-ATPase and the contents of NCC-Spd and NCC-Spm together with CC-Put and CC-Spd in the plasma membrane of roots.

Biological activity of the S-adenosylmethionine decarboxylase inhibitor SAM486A in human breast cancer cells in vitro and in vivo

The antitumor activity of the S-adenosylmethionine decarboxylase (SAMDC) inhibitor SAM486A in human breast cancer cells was investigated. Our in vitro study focused on testing the effects of SAM486A on the proliferation, clonogenicity, invasiveness, cell signaling and PA levels of hormone-independent MDA-MB-435 human breast cancer cells. We also investigated the antitumor action, effects on polyamine pools and tolerability of SAM486A administered to nude mice carrying MDA-MB-435 xenografts. SAM486A suppressed anchorage-independent and -dependent growth and invasiveness of breast cancer cells and the inhibition of cell growth was associated with suppression of spermine synthesis. Combined administration of SAM486A and alpha-difluoromethylornithine (DFMO), a selective inhibitor of ornithine decarboxylase (ODC), exerted greater antiproliferative and anti-invasive effects and induced an overall greater suppression of cellular PA levels than the individual treatments. Both SAM486A and DFMO increased phosphorylation of STAT-1, -3, ERK1/2 and p38, thus indicating activation of both STAT signaling and the MAPK pathway. SAM486A (1 mg/kg) significantly suppressed the growth and spermine levels of established MDA-MB435 breast tumors in nude mice. SAM486A exerts a potent antitumor action in MDA-MB-435 breast cancer cells both in vitro and in vivo. Inhibition of cellular spermine is consistently observed with SAM486A treatment and may mediate its antitumor action. Combination treatment with DFMO may allow the use of lower and, hence, less toxic doses of each compound with preservation of optimal therapeutic effect. The role of activation of STAT signaling and the MAPK pathway in the antitumor action of SAM486A remains to be determined.

TATA-binding Protein-associated Factor 7 Regulates Polyamine Transport Activity and Polyamine Analog-induced Apoptosis

Identification of the polyamine transporter gene will be useful for modulating polyamine accumulation in cells and should be a good target for controlling cell proliferation. Polyamine transport activity in mammalian cells is critical for accumulation of the polyamine analog methylglyoxal bis(guanylhydrazone) (MGBG) that induces apoptosis, although a gene responsible for transport activity has not been identified. Using a retroviral gene trap screen, we generated MGBG-resistant Chinese hamster ovary (CHO) cells to identify genes involved in polyamine transport activity. One gene identified by the method encodes TATA-binding protein-associated factor 7 (TAF7), which functions not only as one of the TAFs, but also a coactivator for c-Jun. TAF7-deficient cells had decreased capacity for polyamine uptake (20% of CHO cells), decreased AP-1 activation, as well as resistance to MGBG-induced apoptosis. Stable expression of TAF7 in TAF7-deficient cells restored transport activity (55% of CHO cells), AP-1 gene transactivation (100% of CHO cells), and sensitivity to MGBG-induced apoptosis. Overexpression of TAF7 in CHO cells did not increase transport activity, suggesting that TAF7 may be involved in the maintenance of basal activity. c-Jun NH2-terminal kinase inhibitors blocked MGBG-induced apoptosis without alteration of polyamine transport. Decreased TAF7 expression, by RNA interference, in androgen-independent human prostate cancer LN-CaP104-R1 cells resulted in lower polyamine transport activity (25% of control) and resistance to MGBG-induced growth arrest. Taken together, these results reveal a physiological function of TAF7 as a basal regulator for mammalian polyamine transport activity and MGBG-induced apoptosis.