Intracellular Levels Of Spermidine Research Articles

Herpesvirus-induced spermidine synthesis and eIF5A hypusination for viral episomal maintenance.

Spermidine is essential for cellular growth and acts as a prerequisite of hypusination, a post-translational modification of eukaryotic initiation factor 5A (eIF5A), allowing the translation of polyproline-containing proteins. Here, we show that oncogenic Kaposi's sarcoma-associated herpesvirus (KSHV) increases spermidine synthesis and eIF5A hypusination to enhance expression of polyproline-containing latency-associated nuclear antigen (LANA) for viral episomal maintenance. KSHV upregulates intracellular spermidine levels by dysregulating polyamine metabolic pathways in three-dimensional (3D) culture and 2D de novo infection conditions. Increased intracellular spermidine leads to increased eIF5A hypusination, ultimately enhancing LANA expression. In contrast, inhibition of spermidine synthesis or eIF5A hypusination alleviates LANA expression, decreasing viral episomal maintenance and KSHV-infected cell proliferation invitro and invivo, which is reversed by spermidine supplement. This demonstrates that KSHV hijacks spermidine synthesis and eIF5A hypusination pathways to enhance LANA expression for viral episomal maintenance, suggesting polyamine metabolism and eIF5A hypusination as therapeutic targets for KSHV-induced tumorigenesis.

Polyamine Biosynthetic Pathway as a Drug Target for Osteosarcoma Therapy.

Osteosarcoma (OS) is the most common bone tumor in children. Polyamines (PAs) are ubiquitous cations involved in many cell processes including tumor development, invasion and metastasis. In other pediatric cancer models, inhibition of the PA biosynthesis pathway with ornithine decarboxylase (ODC) inhibitor alpha-difluoromethylornithine (DFMO) results in decreased cell proliferation and differentiation. In OS, the PA pathway has not been evaluated. DFMO is an attractive, orally administered drug, is well tolerated, can be given for prolonged periods, and is already used in pediatric patients. Three OS cell lines were used to study the cellular effects of PA inhibition with DFMO: MG-63, U-2 OS and Saos-2. Effects on proliferation were analyzed by cell count, flow cytometry-based cell cycle analysis and RealTime-Glo™ MT Cell Viability assays. Intracellular PA levels were measured with high-performance liquid chromatography (HPLC). Western blot analysis was used to evaluate cell differentiation. DFMO exposure resulted in significantly decreased cell proliferation in all cell lines. After treatment, intracellular spermidine levels were drastically decreased. Cell cycle arrest at G2/M was observed in U-2 OS and Saos-2. Cell differentiation was most prominent in MG-63 and U-2 OS as determined by increases in the terminal differentiation markers osteopontin and collagen 1a1. Cell proliferation continued to be suppressed for several days after removal of DFMO. Based on our findings, DFMO is a promising new adjunct to current osteosarcoma therapy in patients at high risk of relapse, such as those with poor necrosis at resection or those with metastatic or recurrent osteosarcoma. It is a well-tolerated oral drug that is currently in phase II clinical trials in pediatric neuroblastoma patients as a maintenance therapy. The same type of regimen may also improve outcomes in osteosarcoma patients in whom there have been essentially no medical advances in the last 30 years.

Alterations of Metabolic and Lipid Profiles in Polymyxin-Resistant Pseudomonas aeruginosa.

Multidrug-resistant Pseudomonas aeruginosa presents a global medical challenge, and polymyxins are a key last-resort therapeutic option. Unfortunately, polymyxin resistance in P. aeruginosa has been increasingly reported. The present study was designed to define metabolic differences between paired polymyxin-susceptible and -resistant P. aeruginosa strains using untargeted metabolomics and lipidomics analyses. The metabolomes of wild-type P. aeruginosa strain K ([PAK] polymyxin B MIC, 1 mg/liter) and its paired pmrB mutant strains, PAKpmrB6 and PAKpmrB12 (polymyxin B MICs of 16 mg/liter and 64 mg/liter, respectively) were characterized using liquid chromatography-mass spectrometry, and metabolic differences were identified through multivariate and univariate statistics. PAKpmrB6 and PAKpmrB12, which displayed lipid A modifications with 4-amino-4-deoxy-l-arabinose, showed significant perturbations in amino acid and carbohydrate metabolism, particularly the intermediate metabolites from 4-amino-4-deoxy-l-arabinose synthesis and the methionine salvage cycle pathways. The genomics result showed a premature termination (Y275stop) in speE (encoding spermidine synthase) in PAKpmrB6, and metabolomics data revealed a decreased intracellular level of spermidine in PAKpmrB6 compared to that in PAKpmrB12 Our results indicate that spermidine may play an important role in high-level polymyxin resistance in P. aeruginosa Interestingly, both pmrB mutants had decreased levels of phospholipids, fatty acids, and acyl-coenzyme A compared to those in the wild-type PAK. Moreover, the more resistant PAKpmrB12 mutant exhibited much lower levels of phospholipids than the PAKpmrB6 mutant, suggesting that the decreased phospholipid level was associated with polymyxin resistance. In summary, this study provides novel mechanistic information on polymyxin resistance in P. aeruginosa and highlights its impacts on bacterial metabolism.

Synergistic drug combination GC7/DFMO suppresses hypusine/spermidine-dependent eIF5A activation and induces apoptotic cell death in neuroblastoma.

The eukaryotic initiation factor 5A (eIF5A), which contributes to several crucial processes during protein translation, is the only protein that requires activation by a unique post-translational hypusine modification. eIF5A hypusination controls cell proliferation and has been linked to cancer. eIF5A hypusination requires the enzymes deoxyhypusine synthase (DHPS) and deoxyhypusine hydroxylase and uniquely depends on the polyamine (PA) spermidine as the sole substrate. Ornithine decarboxylase (ODC) is the rate-limiting enzyme in PA biosynthesis. Both ODC and PAs control cell proliferation and are frequently dysregulated in cancer. Since only spermidine can activate eIF5A, we chose the hypusine-PA nexus as a rational target to identify new drug combinations with synergistic antiproliferative effects. We show that elevated mRNA levels of the two target enzymes DHPS and ODC correlate with poor prognosis in a large cohort of neuroblastoma (NB) tumors. The DHPS inhibitor GC7 (N1-guanyl-1,7-diaminoheptane) and the ODC inhibitor α-difluoromethylornithine (DFMO) are target-specific and in combination induced synergistic effects in NB at concentrations that were not individually cytotoxic. Strikingly, while each drug alone at higher concentrations is known to induce p21/Rb- or p27/Rb-mediated G1 cell cycle arrest, we found that the drug combination induced caspase 3/7/9, but not caspase 8-mediated apoptosis, in NB cells. Hypusinated eIF5A levels and intracellular spermidine levels correlated directly with drug treatments, signifying specific drug targeting effects. This two-pronged GC7/DFMO combination approach specifically inhibits both spermidine biosynthesis and post-translational, spermidine-dependent hypusine-eIF5A activation, offering an exciting clue for improved NB drug therapy.

Hemeoxygenase-1 Mediates an Adaptive Response to Spermidine-Induced Cell Death in Human Endothelial Cells

Spermidine (SPD) is a ubiquitous polycation that is commonly distributed in living organisms. Intracellular levels of SPD are tightly regulated, and SPD controls cell proliferation and death. However, SPD undergoes oxidation in the presence of serum, producing aldehydes, hydrogen peroxide, and ammonia, which exert cytotoxic effect on cells. Hemeoxygenase-1 (HO-1) is thought to have a protective effect against oxidative stress. Upregulation of HO-1 in endothelial cells is considered to be beneficial in the cardiovascular disease. In the present study, we demonstrate that the ubiquitous polyamine, SPD, induces HO-1 in human umbilical vein endothelial cells (HUVECs). SPD-induced HO-1 expression was examined by Western blot and reverse transcription-polymerase chain reaction (RT-PCR). Involvement of reactive oxygen species, serum amine oxidase, PI3K/Akt signaling pathway, and transcription factor Nrf2 in the induction of HO-1 by SPD was also investigated. Furthermore, small interfering RNA knockdown of Nrf2 or HO-1 and treatment with the specific HO-1 inhibitor ZnPP exhibited a noteworthy increase of death of SPD-stimulated HUVECs. In conclusion, these results suggest that SPD induces PI3K/Akt-Nrf2-mediated HO-1 expression in human endothelial cells, which may have a role in cytoprotection of the cells against oxidative stress-induced death.

Flow cytometric analysis of in vivo polyamine deprivation in Lewis lung carcinoma (3LL) cells using the monoclonal antibody SPM8-2

It has previously been shown that the monoclonal antibody SPM8-2 recognizes free spermine and spermidine as well as polyamines bound by an amide bond. In the present work it is demonstrated that this antibody also interacts with spermidine, spermine, and to a lesser extent N1- and N8-acetyl spermidine in an ELISA test where the polyamines are bound by reaction with formaldehyde. 3LL Lewis lung carcinoma cells from tumor-grafted mice were labeled with fluorescein-conjugated monoclonal antibody SPM8-2 and analyzed by flow cytometry. Both viable cells and formaldehyde-fixed and subsequently permeabilized cells showed fluorescent staining. However, most polyamines present in the cells are not directly available for antibody binding. Treatment of fixed cells with DNase or RNase greatly increased fluorescent staining, suggesting that some polyamines are co-localized with DNA and RNA. Antibody labeling of the cells was prevented by addition of free spermine. 3LL cells from tumors of mice treated by a polyamine depleting regimen had decreased intracellular spermidine levels and bound less antibody when compared to untreated controls. After digestion with RNase, the cells from treated mice bound considerably less fluorescent antibody than tumor cells from untreated mice, while their RNA content was similar. In contrast, fluorescent staining after DNase digestion was only slightly affected by the treatment with a polyamine depleting regimen. This suggests that the pools of polyamines which are co-localized with RNA are depleted more readily than those associated with DNA. Since only a small proportion of the intracellular polyamines is accessible to the bulky antibodies, treatment with hydrolytic enzymes (DNase, RNase) is necessary to reveal specific compartments of the polyamines and to demonstrate qualitative and semi-quantitative differences of their distribution within cells.

Inhibition of cell growth by accumulated spermine is associated with a transient alteration of cell cycle progression

Exposure of HL-60 cells to millimolar levels of spermine resulted in the inhibition of cell growth. Flow cytometry revealed that the addition of exogenous spermine prevented the accumulation of cells in the S and G 2/M phases of the cell cycle as observed in the control cells. High intracellular levels of spermine completely suppressed the early onset of ornithine decarboxylase activity and, consequently, the intracellular increase in spermidine and putrescine. On the other hand, the addition of exogenous spermidine or putrescine also abolished ornithine decarboxylase activity, but in this case neither the growth of spermidine- or putrescine-treated cells nor the cell cycle phase distribution was affected. In the latter cells, intracellular levels of spermidine were not significantly different from control ones. These results suggest that the addition of exogenous spermine inhibits cell proliferation by hindering the increase in cellular spermidine needed to accelerate the G 1 to S phase transition.

Influence of polyamine inhibitors on light-independent and light-dependent chlorophyll biosynthesis and on the photosynthetic rate

The mutant C-2A′ of the unicellular green alga Scenedesmus obliquus behaves at 32°C like higher plants in that it accumulates protochlorophyllide in darkness and converts it to chlorophyll when exposed to light. By lowering the growth temperature from 32 to 22°C, protochlorophyllide can be reduced in darkness. Thus this organism allowed us to study the effects of the inhibition of several enzymes of polyamine biosynthesis on light-dependent and light-independent chlorophyll biosynthesis. Reduction of the intracellular level of putrescine by the use of 1,4-diamino-2-butanone inhibitor blocked the light-independent chlorophyll biosynthesis (dramatically increasing the photochlorophyllide levels with parallel reduction of chlorophyll). The influence of the inhibitor in question is even more dramatic in light-dependent chlorophyll biosynthesis, where small concentrations of 1,4-diamino-2-butanone resulted in considerable reduction of chlorophyll during illumination. The above reduction of chlorophyll was accompanied by an increase in photosynthetic activity and respiration rate, which suggests the formation of a photosynthetic apparatus that behaves similarly to one adapted to high light intensities. Decreases in the intracellular levels of spermidine and spermine by the inhibitors cyclohexylamine and 1,3-diaminopropane respectively, although not showing changes in light-independent chlorophyll biosynthesis, do exhibit a regular reduction of light-dependent chlorophyll biosynthesis and a decrease in the photosynthetic activity as well as in the respiration rate. These characteristics are typical for a photosynthetic apparatus adapted to low light intensities with a higher antenna composition per reaction centre. The possible effects of the reduction of putrescine, spermidine and spermine levels on the stabilization-destabilization of photosynthetic subcomplexes are discussed.

Abnormal accumulation and toxicity of polyamines in a difluoromethylornithine-resistant HTC cell variant

Mammalian cells possess an inducible, active polyamine transport system that is stringently regulated by feedback controls. This study provides evidence that DH23b cells, which were initially selected from the rat hepatoma HTC line for overproduction of ornithine decar☐ylase, demonstrate an abnormality in the regulation of polyamine transport. Exposure of these cells to micromolar levels of spermidine or spermine resulted in inhibition of protein synthesis and eventual cell lysis. These effects were not due to by-products of polyamine oxidation by serum oxidases as neither inhibition of protein synthesis nor cell lysis was mitigated by aminoguanidine, reduced glutathione, dithiothreitol, or catalase. Although the polyamine transport system in the DH23b cells has the same K m and V max as that in the parental HTC line, the variant cells accumulated abnormally high levels of both spermidine (8-times normal) and spermine (4-times normal). In the HTC line, however, transport of both polyamines as well as putrescine was feedback inhibited within approx. 3 h, while in the variant cells uptake was not diminished by 12 h and terminated only with cell lysis. The DH23b cells appear to lack the normal mechanism responsible for feedback control of active polyamine incorporation. This defect provided the opportunity to manipulate intracellular levels of spermidine from 30 to approx. 800% of normal, allowing the demonstration that cellular protein synthesis is as sensitive to spermidine levels as previous in-vitro studies had suggested.

Mechanisms of heat protection by cycloheximide or puromycin: Involvement of polyamines?

1. 1.|When Chinese hamster ovary cells are treated with cycloheximide (10 μg/ml) or puromycin (100 μg/ml) for 2 h before and during heating at 43°C for 3 h, there is protection from hyperthermic killing; i.e. the plating efficiency increases 2000-fold from 3.7 × 10 −5 to (6–9) × 10 −2. 2. 2.|The total intracellular levels of spermidine and spermine are not altered by the hyperthermic or drug treatments. 3. 3.|The small 30% decrease in intracellular putrescine observed after heating is not altered by drug treatment. 4. 4.|Heat protection by treatment with cycloheximide or puromycin cannot be attributed to changes in levels of total intracellular polyamines.

Control of Leishmania mexicana proliferation by modulation of polyamine intracellular levels

Repeated treatments of Leishmania mexicana promastigote cultures with a-difluoromethylornithine could not block proliferation when the parasite was grown in a rich medium. Although the irreversible inhibitor of ornithine decarboxylase was able to abolish the enzymatic activity under these conditions, polyamine depletion was only partial probably due to the uptake of these substances from the external medium. Conversely, when Leishmania was cultivated in a defined medium essentially free of polyamines, a-difluoromethylornithine was able to decrease the growth rate and proliferation was arrested after several passages in the presence of the drug. Parasite multiplication could be resumed by addition of exogenous polyamines, and a strict correlation between Leishmania promastigote growth and intracellular levels of spermidine was observed.

Modulation of growth gene expression by selective alteration of polyamines in human colon carcinoma cells

The biosynthesis of the polyamines, putrescine, spermidine and spermine, is temporally linked with expression of many growth related genes. Our previous studies have shown that generalized polyamine depletion of the human colon cancer cell line COLO 320 by 2-difluoromethylornithine is associated with decreased transcription of the c-myc, c-fos, and ornithine decarboxylase (ODC) genes. In the current study, the role of individual polyamines was further defined by the use of a specific inhibitor of spermidine synthase, S-adenosyl-1,8, diamino-3-thio-octane (AdoDATO), and a spermine analogue, N 1, N 12 bis(ethyl)spermine. Our data demonstrate that depletion of spermidine results in a 60–90% decrease in c-myc mRNA steady state levels. In contrast, c-fos mRNA levels are decreased only when both spermidine and spermine are diminshed. Furthermore, ODC mRNA levels are increased when all polyamines are decreased by DFMO, but are unaffected by a selective reduction in intracellular spermidine levels by AdoDATO. These studies suggest that individual polyamines may have a selective role in the expression of specific growth related genes.

Putrescine stimulates DNA synthesis in intestinal epithelial cells

Experiments were designed to examine the effects of exogenously supplied putrescine on the synthesis of DNA, RNA, and protein in cultured epithelial cells (IEC-6). Putrescine increased aphidicolin-sensitive DNA synthesis at concentrations as low as 0.3 microM putrescine with maximal stimulation (267% control) at 10 microM. This response appeared to be an effect of increases in the intracellular concentration of putrescine as the intracellular levels of spermidine and spermine did not change over the time period examined. Furthermore, pulse-chase experiments revealed that putrescine that entered the cell was not metabolized to another polyamine or degraded. In addition, 10 microM putrescine enhanced both cycloheximide-sensitive lysine incorporation and actinomycin D-sensitive uridine incorporation, indexes of protein and RNA synthesis, respectively. Incorporation of both lysine and uridine was maximal 12 h after the addition of putrescine, whereas thymidine incorporation was still increasing at 24 h, the longest time point examined. These data suggest that putrescine synthesis and/or transport during mucosal proliferation is directly involved in the stimulation of epithelial DNA, RNA, and protein synthesis.

Spermidine synthesis by Pseudomonas sp. strain Kim, previously reported to lack this polyamine.

Pseudomonas sp. strain Kim has previously been reported to be the only known naturally occurring organism lacking spermidine. We now show that it synthesizes this polyamine. The apparent lack of intracellular levels of spermidine results from an efficient conversion of spermidine to putrescine and hydroxyputrescine.

Characterization of the inducible polyamine transporter in bovine lymphocytes.

The polyamine uptake system in bovine lymphocytes was activated by concanavalin A. The system was common to putrescine, spermidine and spermine. The Kt values for uptake activities of putrescine, spermidine and spermine were 3.7 microM, 0.38 microM and 0.23 microM in that order. The uptake activity was inhibited by carbonyl cyanide m-chlorophenylhydrazone, gramicidin D or valinomycin in the presence of 20 mM K+ suggesting that polyamine uptake depends on the membrane potential. The uptake activity appeared 10 h after addition of concanavalin A, and the maximum was reached at 28 h indicating that induction of the polyamine transporter precedes the initiation of DNA synthesis. Addition of polyamine antimetabolites, such as alpha-difluoromethylornithine and ethylglyoxal bis(guanylhydrazone), to the medium enhanced at least eightfold the induction of the polyamine transporter. The induction was repressed by addition of 50 microM spermidine or spermine, but not putrescine. We propose here that the induction of the membrane-potential-dependent polyamine transporter is regulated by the intracellular level of spermidine and spermine.

Synthesis of N-chlorosulfonyl dicyclohexylamine as a potent inhibitor for spermidine synthase and its effects on human leukemia molt4B cells

N-Chlorosulfonyl dicyclohexylamine (CSD) was synthesized as a potent inhibitor of spermidine synthase and analyzed for antiproliferative effects on leukemic cells. The compound specifically inhibited spermidine synthase in a competitive mode with the substrate putrescine (Ki, 1.8 X 10(-7) M). When human leukemia Molt4B cells were cultured in the presence of the inhibitor, the intracellular level of spermidine and the rate of cell proliferation were markedly depressed. In these polyamine depleted and growth retarded cells the synthesis of protein, but not of DNA or RNA, was found to be significantly diminished.

Involvement of spermidine in proliferation and differentiation of human promyelocytic leukemia cells

The polyamines putrescine, spermidine, and spermine have been implicated in the regulation of cell proliferation and differentiation. Previous studies, however, have demonstrated that the polyamines are essential for proliferation, but not differentiation, of HL-60 human promyelocytic leukemia cells. We have extended these findings by demonstrating a highly significant relationship between intracellular spermidine levels and HL-60 proliferation. However, in contrast to previous studies, we have also demonstrated that induction of HL-60 differentiation with dimethyl sulfoxide, hexamethylene bisacetamide, butyric acid, or retinoic acid is inhibited by alpha-difluoromethyl ornithine (DFMO) depletion of intracellular putrescine and spermidine. Further, the addition of exogenous spermidine abrogates DFMO inhibition of HL-60 differentiation, thus confirming the involvement of this polyamine in the expression of a differentiated phenotype. The discrepancy between our results and those of previous studies probably stems from the nearly complete, rather than partial, depletion of intracellular spermidine achieved in the present work. The results of the present study thus demonstrate the involvement of spermidine in both proliferation and induction of HL-60 differentiation with certain agents.

Involvement of Spermidine in Proliferation and Differentiation of Human Promyelocytic Leukemia Cells

The polyamines putrescine, spermidine, and spermine have been implicated in the regulation of cell proliferation and differentiation. Previous studies, however, have demonstrated that the polyamines are essential for proliferation, but not differentiation, of HL-60 human promyelocytic leukemia cells. We have extended these findings by demonstrating a highly significant relationship between intracellular spermidine levels and HL-60 proliferation. However, in contrast to previous studies, we have also demonstrated that induction of HL-60 differentiation with dimethyl sulfoxide, hexamethylene bisacetamide, butyric acid, or retinoic acid is inhibited by alpha-difluoromethyl ornithine (DFMO) depletion of intracellular putrescine and spermidine. Further, the addition of exogenous spermidine abrogates DFMO inhibition of HL-60 differentiation, thus confirming the involvement of this polyamine in the expression of a differentiated phenotype. The discrepancy between our results and those of previous studies probably stems from the nearly complete, rather than partial, depletion of intracellular spermidine achieved in the present work. The results of the present study thus demonstrate the involvement of spermidine in both proliferation and induction of HL-60 differentiation with certain agents.

S-adenosylmethionine levels and protein methylation during morphogenesis of Mucor racemosus.

The intracellular level of S-adenosylmethionine increased as the yeast-phase cells of Mucor racemosus were induced to convert to hyphae. This increase correlated well with time course of the conversion in cell type and was independent of the metabolic changes caused by the shift to aerobic conditions. There was no significant change in the intracellular level of spermidine, a polyamine synthesized from putrescine and the propylamine group of S-adenosylmethionine. Spermine was not detected. An examination of protein methylation revealed an increase in the methylation of total protein during the shift in cell type and possible qualitative as well as quantitative changes in specific base proteins.

Polyamines and the accumulation of ribonucleic acid in some polyauxotrophic strains of Escherichia coli.

The cellular accumulations of polyamines and ribonucleic acid (RNA) were compared in the polyauxotrophic mutants of Escherichia coli strain 15 TAU and E. coli K-12 RC(re1) met(-) leu(-). Putrescine, spermidine, and their monoacetyl derivatives were the main polyamines in both strains, when grown in glucose-mineral medium. No significant degradation of either (14)C-putrescine or (14)C-spermidine was found in growing cultures of strain 15 TAU, which requires thymine, arginine, and uracil for growth. Experiments with this organism showed that in a variety of different incubation conditions, which included normal growth, amino acid starvation, inhibition by chloramphenicol or streptomycin, or thymine deprivation, a close correlation was seen between the intracellular accumulation of unconjugated spermidine and RNA. In the presence of arginine, the antibiotics stimulated the production of putrescine and spermidine per unit of bacterial mass. Deprivation of arginine also resulted in an increase in the production of putrescine per unit of bacterial mass, most of which was excreted into the growth medium. However, in this system the antibiotics reduced the synthesis of putrescine. Furthermore, streptomycin caused a rapid loss of cellular putrescine into the medium. The latter effect was not seen in anaerobic conditions or in a streptomycin-resistant mutant of 15 TAU. Methionine added to the growth medium of growing TAU not only markedly increased the total production of spermidine, but also increased both the intracellular concentration of spermidine and the accumulation of RNA. Exogenous spermidine extensively relaxed RNA synthesis in amino acid-starved cultures of 15 TAU. Analysis in sucrose density gradients showed that the RNA accumulated in the presence of spermidine was ribosomal RNA. Cells of E. coli K-12 RC(rel) met(-) leu(-), grown in a complete medium, had approximately the same ratio of free spermidine to RNA as did strain 15 TAU. However, the relaxed strain showed a much lower ratio of putrescine to spermidine than the stringent 15 TAU. Omission of methionine stopped spermidine synthesis and markedly increased both the intracellular accumulation and the total production of putrescine. It seems that a high intracellular level of spermidine acts as a feedback inhibitor in the biosynthesis of putrescine in this strain. The hypothesis that the intracellular concentration of polyamines may participate in the control of the synthesis of ribosomal RNA in bacteria is discussed.