Approval of DFMO for high-risk neuroblastoma patients demonstrates a step of success to target MYC pathway.

The "undruggable" MYC oncoproteins are deregulated in 70% human cancers. The approval of DFMO, an irreversible inhibitor of ornithine oxidase (ODC1) that is a direct transcriptional target of MYC, demonstrates that patients can benefit from targeting MYC activity via an indirect approach. However, the mechanism of action of DFMO needs further studies to understand how it works in post-immunotherapy neuroblastomas. Efforts to develop a more potent and safer drug to block MYC function will continue despite challenges.

Effects of putrescine on the quality and epigenetic modification of mouse oocytes during in vitro maturation.

CONTEXT: Low ovarian putrescine levels and decreased peak values following luteinising hormone peaks are related to poor oocyte quantity and quality in ageing women. AIMS: To investigate the effects of putrescine supplementation in in vitro maturation (IVM) medium on oocyte quality and epigenetic modification. METHODS: Germinal vesicle oocytes retrieved from the ovaries of 8-week-old and 9-month-old mice were divided into four groups (the young, young+difluoromethylornithine (DFMO), ageing and ageing+putrescine groups) and cultured in IVM medium with or without 1mM putrescine or DFMO for 16h. The first polar body extrusion (PBE), cleavage and embryonic development were evaluated. Spindles, chromosomes, mitochondria and reactive oxygen species (ROS) were measured. The expression levels of SIRT1, H3K9ac, H3K9me2, H3K9me3, and 5mC levels were evaluated. Sirt1 and imprinted genes were detected. RESULTS: The PBE was higher in the ageing+putrescine group than in the ageing group. Putrescine increased the total and inner cell mass cell numbers of blastocysts in ageing oocytes. Putrescine decreased aberrant spindles and chromosome aneuploidy, increased the mitochondrial membrane potential and decreased ROS levels. Putrescine increased SIRT1 expression and attenuated the upregulation of H3K9ac levels in ageing oocytes. Putrescine did not affect 5mC, H3K9me2 or H3K9me3 levels or imprinted gene expression. CONCLUSIONS: Putrescine supplementation during IVM improved the maturation and quality of ageing oocytes and promoted embryonic development by decreasing ROS generation, maintaining mitochondrial and spindle function and correcting aberrant epigenetic modification. IMPLICATIONS: Putrescine shows application potential for human-assisted reproduction, especially for IVM of oocytes from ageing women.

Polyamines drive myeloid cell survival by buffering intracellular pH to promote immunosuppression in glioblastoma.

Glioblastoma is characterized by the robust infiltration of immunosuppressive tumor-associated myeloid cells (TAMCs). It is not fully understood how TAMCs survive in the acidic tumor microenvironment to cause immunosuppression in glioblastoma. Metabolic and RNA-seq analysis of TAMCs revealed that the arginine-ornithine-polyamine axis is up-regulated in glioblastoma TAMCs but not in tumor-infiltrating CD8+ T cells. Active de novo synthesis of highly basic polyamines within TAMCs efficiently buffered low intracellular pH to support the survival of these immunosuppressive cells in the harsh acidic environment of solid tumors. Administration of difluoromethylornithine (DFMO), a clinically approved inhibitor of polyamine generation, enhanced animal survival in immunocompetent mice by causing a tumor-specific reduction of polyamines and decreased intracellular pH in TAMCs. DFMO combination with immunotherapy or radiotherapy further enhanced animal survival. These findings indicate that polyamines are used by glioblastoma TAMCs to maintain normal intracellular pH and cell survival and thus promote immunosuppression during tumor evolution.

Improving Eflornithine Oral Bioavailability and Brain Uptake by Modulating Intercellular Junctions With an E-cadherin Peptide.

Eflornithine has been used to treat second-stage human African trypanosomiasis. However, it has inadequate oral bioavailability and low blood-brain barrier permeation, thus requiring a lengthy and complicated intravenous infusion schedule. Here, we investigated the feasibility of using an intercellular junction-modulating E-cadherin peptide HAV6 to enhance the oral bioavailability and blood-brain barrier permeation of eflornithine. Eflornithine was not metabolized in liver microsomes, nor was it a substrate for the human efflux transporter P-glycoprotein. Furthermore, HAV6 and HAV6scr (sequence scrambled HAV6) were stable in simulated gastric fluid with pepsin and rat intestinal mucosal scrapings. Both peptides were stable in human plasma, albeit less stable in rat and mouse plasma. HAV6 increased eflornithine permeability across Madin-Darby canine kidney and Caco-2 cell monolayers (5- and up to 8.5-fold), whereas HAV6scr had little effect. Using an in situ rat brain perfusion model, HAV6, but not HAV6scr, significantly increased eflornithine concentrations in different brain regions up to 4.9-fold. In rats, coadministration of HAV6 increased eflornithine oral bioavailability from 38% to 54%, brain concentrations by up to 83%, and cerebrospinal fluid concentrations by 40%. In conclusion, coadministration of HAV6, either during intravenous infusion or as an oral formulation, has the potential to improve eflornithine-based treatment for second-stage human African trypanosomiasis.

Extracellular Spermine Activates DNA Methyltransferase 3A and 3B.

We first demonstrated that long-term increased polyamine (spermine, spermidine, putrescine) intake elevated blood spermine levels in mice and humans, and lifelong consumption of polyamine-rich chow inhibited aging-associated increase in aberrant DNA methylation, inhibited aging-associated pathological changes, and extend lifespan of mouse. Because gene methylation status is closely associated with aging-associated conditions and polyamine metabolism is closely associated with regulation of gene methylation, we investigated the effects of extracellular spermine supplementation on substrate concentrations and enzyme activities involved in gene methylation. Jurkat cells and human mammary epithelial cells were cultured with spermine and/or D,L-alpha-difluoromethylornithine (DFMO), an inhibitor of ornithine decarboxylase. Spermine supplementation inhibited enzymatic activities of adenosylmethionine decarboxylase in both cells. The ratio of decarboxylated S-adenosylmethionine to S-adenosyl-L-methionine increased by DFMO and decreased by spermine. In Jurkat cells cultured with DFMO, the protein levels of DNA methyltransferases (DNMTs) 1, 3A and 3B were not changed, however the activity of the three enzymes markedly decreased. The protein levels of these enzymes were not changed by addition of spermine, DNMT 3A and especially 3B were activated. We show that changes in polyamine metabolism dramatically affect substrate concentrations and activities of enzymes involved in gene methylation.

A Label-Free Continuous Fluorescence-Based Assay for Monitoring Ornithine Decarboxylase Activity with a Synthetic Putrescine Receptor.

Polyamines play an important role in cell growth, differentiation, and cancer development, and the biosynthetic pathway of polyamines is established as a drug target for the treatment of parasitic diseases, neoplasia, and cancer chemoprevention. The key enzyme in polyamine biosynthesis is ornithine decarboxylase (ODC). We report herein an analytical method for the continuous fluorescence monitoring of ODC activity based on the supramolecular receptor cucurbit[6]uril (CB6) and the fluorescent dye trans-4-[4-(dimethylamino)styryl]-1-methylpyridinium iodide (DSMI). CB6 has a significantly higher binding constant to the ODC product putrescine (>107 M-1) than to the substrate L-ornithine (340 M-1). This enables real-time monitoring of the enzymatic reaction through a continuous fluorescence change caused by dye displacement from the macrocycle by the formed product, which allowed a straightforward determination of enzyme kinetic parameters ( kcat = 0.12 s-1 and KM = 24 µM) and inhibition constants of the two ODC inhibitors α-difluoromethylornithine (DFMO) and epigallocatechin gallate (EGCG). The potential for high-throughput screening (HTS) was demonstrated by excellent Z' factors (>0.9) in a microplate reader format, and the sensitivity of the assay is comparable to or better than most established complementary methods, which invariably have the disadvantage of not being compatible with direct implementation and upscaling to HTS format in the drug discovery process.

Triethylenetetramine modulates polyamine and energy metabolism and inhibits cancer cell proliferation.

Polyamine metabolism is an attractive anticancer drug target, since polyamines are absolutely required for cellular proliferation, and increased levels of polyamines and their biosynthetic enzyme ornithine decarboxylase (ODC) are associated with cancer. Triethylenetetramine (TETA) is a charge-deficient isosteric analogue of the polyamine spermidine (Spd) and a Cu(II)-chelating compound used for the treatment of Wilson's disease, and it has been implicated as a potential anticancer therapeutic drug. In the present study, we studied the effects of TETA in comparison with two other Cu(II)-chelators, D-penicillamine (PA) and tetrathiomolybdate (TTM), on polyamine metabolism in DU145 prostate carcinoma, MCF-7 breast carcinoma and JEG-3 choriocarcinoma cells. TETA induced antizyme, down-regulated ODC and inhibited [(14)C] Spd uptake. Moreover, it completely prevented α-difluoromethylornithine (DFMO)-induced increase in [(14)C] Spd uptake, and inhibited [(14)C] putrescine (Put) uptake and ODC activity in vivo Seven-day treatment of DU145 cells with TETA caused growth cessation by reducing intracellular polyamine levels and suppressing the formation of hypusinated eukaryotic translation initiation factor 5A (eIF5A). TETA or its N-acetylated metabolites also inhibited spermine (Spm), diamine and semicarbazide-sensitive amine oxidases and decreased the level of intracellular reactive oxygen species. Moreover, TETA inhibited the utilization of Put as energy source via the tricarboxylic acid (TCA) cycle, as indicated by decreased production of (14)CO2 from [(14)C] Put. These results indicate that TETA attacks multiple proven anticancer drug targets not attributed to copper chelation, which warrants further studies to reveal its potential in cancer chemoprevention and cure.

A method to improve the efficacy of topical eflornithine hydrochloride cream.

CONTEXT: Facial hirsutism is a cosmetic concern for women and can lead to significant anxiety and lack of self-esteem. Eflornithine cream is indicated for the treatment of facial hirsutism. However, limited success rate and overall patient's satisfaction, even with a long-term and high-frequency application, leave room for improvement. OBJECTIVE: The objective of this study is to test the effect of microneedle treatment on the in vitro skin permeation and the in vivo efficacy of eflornithine cream in a mouse model. MATERIALS AND METHOD: In vitro permeation study of eflornithine was performed using Franz diffusion cell. In vivo efficacy study was performed in a mouse model by monitoring the re-growth of hair in the lower dorsal skin of mice after the eflornithine cream was applied onto an area pretreated with microneedles. The skin and the hair follicles in the treated area were also examined histologically. RESULTS AND DISCUSSION: The hair growth inhibitory activity of eflornithine was significantly enhanced when the eflornithine cream was applied onto a mouse skin area pretreated with microneedles, most likely because the micropores created by microneedles allowed the permeation of eflornithine into the skin, as confirmed in an in vitro permeation study. Immunohistochemistry data revealed that cell proliferation in the skin and hair follicles was also significantly inhibited when the eflornithine cream was applied onto a skin area pretreated with microneedles. CONCLUSION: The integration of microneedle treatment into topical eflornithine therapy represents a potentially viable approach to increase eflornithine's ability to inhibit hair growth.

Hessian fly larval feeding triggers enhanced polyamine levels in susceptible but not resistant wheat.

BACKGROUND: Hessian fly (Mayetiola destructor), a member of the gall midge family, is one of the most destructive pests of wheat (Triticum aestivum) worldwide. Probing of wheat plants by the larvae results in either an incompatible (avirulent larvae, resistant plant) or a compatible (virulent larvae, susceptible plant) interaction. Virulent larvae induce the formation of a nutritive tissue, resembling the inside surface of a gall, in susceptible wheat. These nutritive cells are a rich source of proteins and sugars that sustain the developing virulent Hessian fly larvae. In addition, on susceptible wheat, larvae trigger a significant increase in levels of amino acids including proline and glutamic acid, which are precursors for the biosynthesis of ornithine and arginine that in turn enter the pathway for polyamine biosynthesis. RESULTS: Following Hessian fly larval attack, transcript abundance in susceptible wheat increased for several genes involved in polyamine biosynthesis, leading to higher levels of the free polyamines, putrescine, spermidine and spermine. A concurrent increase in polyamine levels occurred in the virulent larvae despite a decrease in abundance of Mdes-odc (ornithine decarboxylase) transcript encoding a key enzyme in insect putrescine biosynthesis. In contrast, resistant wheat and avirulent Hessian fly larvae did not exhibit significant changes in transcript abundance of genes involved in polyamine biosynthesis or in free polyamine levels. CONCLUSIONS: The major findings from this study are: (i) although polyamines contribute to defense in some plant-pathogen interactions, their production is induced in susceptible wheat during interactions with Hessian fly larvae without contributing to defense, and (ii) due to low abundance of transcripts encoding the rate-limiting ornithine decarboxylase enzyme in the larval polyamine pathway the source of polyamines found in virulent larvae is plausibly wheat-derived. The activation of the host polyamine biosynthesis pathway during compatible wheat-Hessian fly interactions is consistent with a model wherein the virulent larvae usurp the polyamine biosynthesis machinery of the susceptible plant to acquire nutrients required for their own growth and development.

Untargeted metabolomics reveals a lack of synergy between nifurtimox and eflornithine against Trypanosoma brucei.

A non-targeted metabolomics-based approach is presented that enables the study of pathways in response to drug action with the aim of defining the mode of action of trypanocides. Eflornithine, a polyamine pathway inhibitor, and nifurtimox, whose mode of action involves its metabolic activation, are currently used in combination as first line treatment against stage 2, CNS-involved, human African trypanosomiasis (HAT). Drug action was assessed using an LC-MS based non-targeted metabolomics approach. Eflornithine revealed the expected changes to the polyamine pathway as well as several unexpected changes that point to pathways and metabolites not previously described in bloodstream form trypanosomes, including a lack of arginase activity and N-acetylated ornithine and putrescine. Nifurtimox was shown to be converted to a trinitrile metabolite indicative of metabolic activation, as well as inducing changes in levels of metabolites involved in carbohydrate and nucleotide metabolism. However, eflornithine and nifurtimox failed to synergise anti-trypanosomal activity in vitro, and the metabolomic changes associated with the combination are the sum of those found in each monotherapy with no indication of additional effects. The study reveals how untargeted metabolomics can yield rapid information on drug targets that could be adapted to any pharmacological situation.

Studies on cell migration model in intestinal epithelial restitution for pharmacological research.

OBJECTIVE: To set up a suitable IEC-6 migration model for pharmacological research and observe the effect of complex polysaccharide from Astragalus membranaceus (Fisch.) Bge. var. mongholicus (Bge.) Hsiao to IEC-6 cell migration. METHODS: The main conditions related to the establishment of the model, including the planting density of the cell, the observation time after scratching, the concentration of the auxiliary material Matrigel, the treatment of the serum-starvation, the concentration of alpha-difluoromethylornithine (DFMO), an inhibitor of the cell migration, were investigated respectively; and the effects of the tested medicines on the model were observed. RESULTS: 4 x 10(5) cell/mL was the suitable planting density of the cell in the 6-well plate; at the 24th hour after scratching was the appropriate time to count the migrating cells; and the proper concentration of Matrigel was 5%; the serum-starvation could evidently reduce the migrating cells, so the culture medium should contain the serum; 2.5 - 5 mmol/L DFMO was proper for inhibition of the cell migration. Complex polysaccharide from Astragalus membranaceus and spermidine both can promote cell migration. CONCLUSION: The established model of IEC-6 cell migration was suitable for intestinal epithelial restitution such as the researches on pathophysiological mechanisms is the effects of the medicines on the cell migration.

The change of antizyme inhibitor expression and its possible role during mammalian cell cycle.

Antizyme inhibitor (AIn), a homolog of ODC, binds to antizyme and inactivates it. We report here that AIn increased at the G1 phase of the cell cycle, preceding the peak of ODC activity in HTC cells in culture. During interphase AIn was present mainly in the cytoplasm and turned over rapidly with the half-life of 10 to 20 min, while antizyme was localized in the nucleus. The level of AIn increased again at the G2/M phase along with ODC, and the rate of turn-over of AIn in mitotic cells decreased with the half-life of approximately 40 min. AIn was colocalized with antizyme at centrosomes during the period from prophase through late anaphase and at the midzone/midbody during telophase. Thereafter, AIn and antizyme were separated and present at different regions on the midbody at late telophase. AIn disappeared at late cytokinesis, whereas antizyme remained at the cytokinesis remnant. Reduction of AIn by RNA interference caused the increase in the number of binucleated cells in HTC cells in culture. These findings suggested that AIn contributed to a rapid increase in ODC at the G1 phase and also played a role in facilitating cells to complete mitosis during the cell cycle.

Effect of difluoromethylornithine on thyroid function in rats.

BACKGROUND: A variety of stimuli cause a rapid increase in polyamine synthesis by increasing an enzyme ornithine decarboxylase required for the biosynthetic pathway of protein synthesis. Difluoromethyl ornithine is a selective inhibitor of this enzyme and hence arrests cell replication strikingly. Its effects on thyroid gland are studied with respect to change in animal's weight and levels of Triiodothyronine, Thyroxine and Thyroid stimulating hormone. The study was conducted to evaluate the inhibitory effects of Di-fluoromethyl ornithine (DFMO) administration on polyamine metabolism of thyroid gland in rats. METHODS: The study was conducted on rats weighing 248 to 320 grams, divided into control and DFMO treated group. A dose of 50 mg/rat was administered subcutaneously to the treated group for 5 consecutive days and placebo (normal saline) injections to control group. On sixth day, blood was collected by cardiac puncture and serum was separated. Serum T3, T4 and TSH were analyzed with the help of radioimmunoassay in both groups. RESULTS: In treated group there was a fall in T3, T4 concentration with significant rise in TSH concentration as compared to control group. CONCLUSION: DFMO (Difluoro methyl ornithine) decreases cellular proliferation of thyroid gland as is assessed by decrease in thyroid hormone levels. The hypothalamo pituitary thyroid axis however remains intact as is shown by a feedback rise in TSH concentration. DFMO can thus be employed for anti-neoplastic clinical trials on account of interference with activity of ODC (Ornithine Decarboxylase) fundamental for polyamine biosynthesis.

Effect of the polyamine analogue N1,N11-diethylnorspermine on cell survival and susceptibility to apoptosis of human chondrocytes.

Chondrocyte survival is closely linked to cartilage integrity, and forms of chondrocyte apoptotic death can contribute to cartilage degeneration in articular diseases. Since growing evidence also implicates polyamines in the control of cell death, we have been investigating the role of polyamine metabolism in chondrocyte survival and apoptosis. Treatment of human C-28/I2 chondrocytes with N(1),N(11)-diethylnorspermine (DENSPM), a polyamine analogue with clinical relevance as an experimental anticancer agent, inhibited polyamine biosynthesis and induced polyamine catabolism, thus rapidly depleting all main polyamines. DENSPM did not increase significantly caspase activity, but provoked a late cell death associated to DNA fragmentation. A short treatment with DENSPM did not reduce cell viability when given alone, but enhanced caspase-3 and -9 activation in chondrocytes exposed to tumor necrosis factor-alpha (TNF) and cycloheximide (CHX). A longer treatment with DENSPM however reduced caspase response to TNF plus CHX. Depletion of all polyamines obtained by specific inhibitors of polyamine biosynthesis did not cause cell death and contrasted apoptosis by decreasing caspase activities. In conclusion, following DENSPM treatment, C-28/I2 chondrocytes are initially sensitized to caspase 9-dependent apoptosis in the presence of TNF and CHX and may eventually undergo a late and mainly caspase-independent cell death in the absence of other stimuli. Moreover, these results indicate that a reduction of polyamine levels not only leads to inhibition of cell proliferation, but also of caspase-mediated pathways of chondrocyte apoptosis.

Effects of polyamine depletion by alpha-difluoromethylornithine on in vitro and in vivo biological properties of 4T1 murine mammary cancer cells.

Increased polyamine synthesis has been associated with proliferation and progression of breast cancer, and thus, is a potential target for anticancer therapy. Polyamine depletion by alpha-difluoromethylornithine (DFMO) has been shown to decrease pulmonary and bone metastasis from human breast cancer cell xenografts. Following these observations, this study was designed to test the effects of DFMO on in vitro and in vivo features of the highly invasive and metastatic 4T1 murine mammary cancer cells. DFMO inhibited proliferation, caused G1-S arrest, and suppressed in vitro invasiveness of 4T1 cells. In contrast to our previous findings with MDA-MB-435 cells, DFMO did not affect the activation of signal transducers and activator of transcription 3, c-Jun N-terminal kinase, and extracellular signal-regulated kinase, but decreased phosphorylation of p38. DFMO did not alter the expression of Twist. DFMO delayed the orthotopic growth of 4T1 xenografts in association with suppressed putrescine and spermidine levels but increased levels of spermine. DFMO did not affect pulmonary metastasis when primary tumors of control and DFMO-treated mice were matched for size. Interestingly, DFMO reduced Ki-67 expression only in the primary tumors but did not affect its expression in the metastatic tumors in the lung. Cleaved caspase-3 expression was not affected by DFMO in either the primary tumors or the pulmonary metastasis. In summary, DFMO treatment markedly inhibited in vitro proliferation and invasiveness of 4T1 cells and retarded the growth of orthotopic xenografts in mice. The failure of DFMO to inhibit pulmonary metastasis in this system appears to be due, at least in part, to its lack of antiproliferative effect at the metastatic sites.

Pathways involved in alanyl-glutamine-induced changes in neutrophil amino- and alpha-keto acid homeostasis or immunocompetence.

We examined the effects of DON [glutamine-analogue and inhibitor of glutamine-requiring enzymes], alanyl-glutamine (regarding its role in neutrophil immunonutrition) and alanyl-glutamine combined with L-NAME, SNAP, DON, beta-alanine and DFMO on neutrophil amino and alpha-keto acid concentrations or important neutrophil immune functions in order to establish whether an inhibitor of *NO-synthase [L-NAME], an *NO donor [SNAP], an analogue of taurine and a taurine transport antagonist [beta-alanine], an inhibitor of ornithine-decarboxylase [DFMO] as well as DON could influence any of the alanyl-glutamine-induced effects. In summary, irrespective of which pharmacological, metabolism-inhibiting or receptor-mediated mechanisms were involved, our results showed that impairment of granulocytic glutamine uptake, modulation of intracellular glutamine metabolisation and/or de novo synthesis as well as a blockade of important glutamine-dependent metabolic processes may led to significant modifications of physiological and immunological functions of the affected cells.

Protein phosphatase 2A regulates apoptosis in intestinal epithelial cells.

Polyamine depletion prevents apoptosis by increasing serine/threonine phosphorylation leading to either inactivation or activation of pro- and anti-apoptotic proteins, respectively. Despite evidence that protein kinases are regulators of apoptosis, a specific role for protein phosphatases in regulating cell survival has not been established. In this study, we show that polyamine depletion inhibits serine/threonine phosphatase 2A (PP2A). Inhibition of PP2A in cells depleted of polyamines correlated well with increased phosphorylation of Bad at Ser112. Bad Ser112 phosphorylation in response to tumor necrosis factor (TNF)-alpha treatment decreased with time in cells grown in control as well as those grown in the presence of alpha-difluoromethylornithine plus putrescine. However, a sustained increase in the levels of Bad Ser112 phosphorylation was maintained in response to TNF-alpha treatment in cells grown in the presence of alpha-difluoromethylornithine. Inhibition of PP2A by okadaic acid and fostriecin or PP2A small interfering RNA transfection significantly decreased TNF-alpha-induced apoptosis in control and polyamine-depleted cells. Inhibition of PP2A by okadaic acid: 1) increased Bad and Bcl-2 phosphorylation at Ser112 and Ser70, respectively; 2) increased ERK activity; 3) prevented JNK activation; 4) prevented cytochrome c release, and activation of caspases-9 and -3 in response to TNF-alpha. Inhibition of MEK1 by U0126 prevented phosphorylation of Bad at Ser112. These results indicate that polyamines regulate PP2A activity, and inhibition of PP2A in response to polyamine depletion increases steady state levels of Bad and Bcl-2 proteins and their phosphorylation and thereby prevents cytochrome c release, caspase-9, and caspase-3 activation.

Inhibitors of polyamine metabolism: review article.

The identification of increased polyamine concentrations in a variety of diseases from cancer and psoriasis to parasitic infections has led to the hypothesis that manipulation of polyamine metabolism is a realistic target for therapeutic or preventative intervention in the treatment of certain diseases. The early development of polyamine biosynthetic single enzyme inhibitors such as alpha-difluoromethylornithine (DFMO) and methylglyoxal bis(guanylhydrazone) showed some interesting early promise as anticancer drugs, but ultimately failed in vivo. Despite this, DFMO is currently in use as an effective anti-parasitic agent and has recently also been shown to have further potential as a chemopreventative agent in colorectal cancer. The initial promise in vitro led to the development and testing of other potential inhibitors of the pathway namely the polyamine analogues. The analogues have met with greater success than the single enzyme inhibitors possibly due to their multiple targets. These include down regulation of polyamine biosynthesis through inhibition of ornithine decarboxylase and S-adenosylmethionine decarboxylase and decreased polyamine uptake. This coupled with increased activity of the catabolic enzymes, polyamine oxidase and spermidine/spermine N1-acetyltransferase, and increased polyamine export has made the analogues more effective in depleting polyamine pools. Recently, the identification of a new oxidase (PAO-h1/SMO) in polyamine catabolism and evidence of induction of both PAO and PAO-h1/SMO in response to polyamine analogue treatment, suggests the analogues may become an important part of future chemotherapeutic and/or chemopreventative regimens.

Inhibition of placental ornithine decarboxylase by DL-alpha-difluoro-methyl ornithine causes fetal growth restriction in rat.

The roles of polyamines in intrauterine growth restriction (IUGR) is studied. The DL-alpha-difluoromethyl ornithine (DFMO), an irreversible inhibitor of ornithine decarboxylase (ODC) which is a rate limiting enzyme of polyamine synthesis was administrated to pregnant rats so that we obtained rat fetuses with IUGR. The changes of maternal nutrition, damage of the placenta, and the direct effect of DFMO on the fetus were examined in this IUGR model. Administration of DFMO did not induced changes of maternal nutrition except for triglyceride and the fetal metabolic state. But the placental weight, ODC activity, and DNA in the placenta were decreased significantly. The ODC activity in the total placenta decreased to less than 10% of that of the control. Depression of ODC activity in the placenta may be the major cause of IUGR induced by DFMO administration, and polyamines play important roles to carry pregnancy.

Changes in polyamines, auxins and peroxidase activity during in vitro rooting of Fraxinus angustifolia shoots: an auxin-independent rooting model.

Among shoots of Fraxinus angustifolia Vahl raised in vitro, 76% rooted after culture on root induction medium for 5 days in darkness followed by culture on root expression medium for 15 days in light. The addition of 20.7 microM indole-butyric acid (IBA) to the root induction medium did not significantly increase the rooting percentage (88%). Putrescine, spermidine, cyclohexylamine (CHA) and aminoguanidine (AG) enhanced rooting up to 100% (98.66% for AG), when applied during root induction in the absence of IBA, otherwise these compounds inhibited rooting, as did spermine and difluoromethylornithine (DFMO) + difluoromethylarginine (DFMA). The root induction phase was characterized by a temporary increase in endogenous free indole-acetic acid (IAA) and putrescine concentrations during root induction, whereas the root expression phase was characterized by increased peroxidase activity and low concentrations of polyamines. These changes were specifically associated with the rooting process and did not depend on the presence of exogenous IBA, because application of exogenous IBA enhanced the amount of IAA in the cuttings but did not affect rooting or the pattern of changes in polyamines and peroxidase. The effects of CHA, AG and DFMO + DFMA on endogenous concentrations of auxins and polyamines highlight the close relationship between the effects of IAA and putrescine in root induction and suggest that polyamine catabolism has an important role in root formation and elongation.