Reversal of the growth inhibitory effect of α-difluoromethylornithine by putrescine but not by other divalent cations

Summary Treatment with -difluoromethylornithine (DFMO), an enzyme-activated irreversible inhibitor of ornithine decarboxylase (ODC), depletes the putrescine and spermidine content, and reduces the growth rate of Ehrlich ascites tumor cells.The addition of putrescine, which is the immediate precursor of spermidine, promptly replenished the intracellular putrescine and spermidine pools and completely reversed the antiproliferative effect of DFMO. A sequential accumulation of spermine, spermidine and putrescine was observed.1,3-diaminopropane, a lower homolog of putrescine, did not reverse the antiproliferative effect of DFMO, despite its structural similarity and identical positive charge. By inhibiting remaining ODC activity, resistant to 5 mM DFMO, and possibly by inhibiting spermine synthase activity, 1,3-diaminopropane produced a further decrease in total polyamine content by reducing the spermine content.Mg²⁺, which can replace putrescine in many in vitro reactions, completely lacked the capacity to reverse the antiproliferative effect of putrescine and spermidine deficiency.Abbreviations DFMO -difluoromethylornithine - ODC ornithine decarbxylase     

Opposite effects of fusicoccin and IAA on putrescine synthesis of rice coleop tiles

Changes in polyamine biosynthesis and elongation of etiolated rice coleoptiles ( Oryza sativa L. cv. Taichung Native 1) in response to fusicoccin (FC) and indoleacetic acid (IAA) were investigated. FC stimulated coleoptile elongation at concentrations higher than 1 μ M but caused a decrease in the levels of free putrescine, spermidine and sper-mine, as well as in the activities of arginine decarboxylase (ADC, EC 4.1.1.19) and S -adenosylmethionine decarboxylase (SAMDC, EC 4.1.1.50). The extent to which FC caused these effects was dependent on its concentration. Treatment with 100 μ M IAA also induced coleoptile elongation and resulted in a decrease in free spermidine/sper-mine and SAMDC activity. However, treatment with IAA resulted in an increase in free putrescine levels and ADC activity. The extent of coleoptile elongation and putrescine accumulation also depended on IAA concentration. α-Difluoromethylarginine (DFMA), an irreversible inhibitor of ADC. but not α-difluoromethylornithine (DFMO). an irreversible inhibitor of ODC (EC 4.1.1.17), inhibited the LAA-stimulated coleoptile elongation and putrescine accumulation. Addition of putrescine could not reverse the effect of DFMA.     

Polyamine metabolism,floral initiation and floral development in Chrysanthemum (Chrysanthemum morifolium Ramat.)

In the short-day plant Chrysanthemum (Chrysanthemum morifolium Ramat. variety Pavo) putrescine and spermidine conjugates appeared in the apical bud before the first observable transformation of the meristem into floral structures. These compounds accumulated on floral initiation and well before floral evocation. Spermidine conjugates were predominant during floral initiation whereas free amines did not accumulate to any significant extent. Different associations of amides were observed during floral initiation as compared with the reproductive phase. 3,4-Dimethoxyphenethylamine conjugates (water-insoluble compounds) were the predominant amine conjugates observed during flower development. These compounds decreased drastically after fertilization. In vegetative buds from plants grown in long days polyamine conjugates were very low and appeared as plants aged. We present evidence that ornithine decarboxylase (ODC) regulates putrescine biosynthesis during floral initiation and floral development. When ODC action was blocked by DFMO (-DL-difluoromethylornithine, a specific, irreversible inhibitor of ODC), flowering was inhibited, and free and conjugated polyamines were not detected. This treatment led to a slight enhancement of ADC activity. When putrescine was added, polyamine titers and flowering were restored. A similar treatment with DFMA (-DL difluoromethylarginine, a specific, irreversible inhibitor of ADC) did not affect flowering and the polyamine titers. The results suggest that ODC and polyamine conjugates are involved in regulating floral initiation in Chrysanthemum.Abbreviations ADC arginine decarboxylase - ODC ornithine decarboxylase - DFMA -DL-difluoromethylarginine - DFMO -DL-difluoromethylornithine     

Stimulation of root and somatic embryo production in Euonymus europaeus L. by an inhibitor of polyamine biosynthesis

In vitro formation of roots and somatic embryos is obtained from cotyledon explants of a Spindle tree (Euonymus europaeus L.) cultured on two different media: a medium inducing callus formation and the production of roots, and a medium inducing callus formation, root and somatic embryo production. We studied the effects of -difluoromethylornithine (DFMO), a specific, irreversible inhibitor of ornithine decarboxylase (ODC) on root and somatic embryo production, growth and titers of putrescine in Euonymus explants and explant-derived calli. Early changes in putrescine levels were detected in both cultures before the visible emergence of roots or somatic embryos. DFMO rapidly inhibited putrescine accumulation and growth in non-embryogenic calli and highly stimulated rooting activity. DFMO partially inhibited putrescine accumulation in embryogenic calli. This inhibition had no effects on callus growth but significantly reduced the time of emergence of roots and highly stimulated somatic embryo production. The relationship among putrescine, putrescine metabolism, growth, root and somatic embryo formation is discussed.     

Polyamine metabolism and in vitro cell multiplication and differentiation in leaf explants of Chrysanthemum morifolium Ramat

Arginine decarboxylase (ADC), ornithine decarboxylase (ODC), diamine oxydase (DAO) free amine and conjugated amine titers were estimated in leaf explants of Chrysanthemum morifolium Ramat. var. Spinder cultivated in vitro in relation to hormone treatment. Addition of benzyladenine (BA) to a basal medium caused the formation of buds on the explants. BA plus 2,4 dichlorophenoxyacetic acid (2,4 D) caused callus formation and proliferation. Formation of roots was obtained by addition of indolylacetic acid (IAA). Arginine decarboxylase (ADC) ornithine decarboxylase (ODC) and diamine oxidase (DAO) activities increased during the first days of culture when cell multiplication was rapid, followed by a sharp decline as the rate of cell division decreased and differentiation took place. DAO activities increased rapidly in proliferating and growing organs and decreased during maturity. This increase was concomitant with ADC and ODC activities and polyamine content (free and conjugated polyamines). The biosynthesis and oxidation of polyamines which occurred simultaneously in physiological states of intense metabolism such as cell division or organ formation were directly correlated. In callus cultures DAO activity was blocked throughout development and regulated neither the cellular levels of polyamines nor polyamine conjugates. Levels of polyamine conjugates were high in callus cultures throughout development. In foliar explants cultivated on a medium promoting callus, inhibition of ODC activity by DFMO (-DL-difluoromethylornithine, a specific enzyme-activated ODC inhibitor) resulting in an amide deficiency facilated the expression of differentiated cell function; substantial activation of DAO was observed until the emergence of the buds. On a medium promoting bud formation, -OH ethylhydrazine (DAO inhibitor) promoted callus formation without differentiation. In this system DAO activity was blocked and there were high levels of polyamines, especially polyamine conjugates, throughout the culture period. The relationship among free and conjugated polyamines related biosynthetic enzyme activities, DAO activities, cell division and organ formation is discussed.Abbreviations ADC = arginine decarboxylase - ODC = ornithine decarboxylase - DOA = diamine oxidase - DFMA = -DL-difluoromethylarginine - DFMO = -DL-difluoromethylornithine - Put = putrescine     

Regulation of DNA synthesis and cell division by polyamines in Catharanthus roseus suspension cultures

Summary Various inhibitors of polyamine biosynthesis were used to study the role of polyamines in DNA synthesis and cell division in suspension cultures of Catharanthus roseus (L.) G. Don. Arginine decarboxylase (ADC; EC 4.1.1.19) was the major enzyme responsible for putrescine production. DL -difluoromethylarginine inhibited ADC activity, cellular putrescine content, DNA synthesis, and cell division. The effect was reversible by exogenous putrescine. Ornithine decarboxylase (ODC; EC 4.1.1.17) activity was always less than 10% of the ADC activity. Addition of DL -difluoromethylornithine had no effect on ODC activity, cellular polyamine levels, DNA synthesis, and cell division within the first 24 h but by 48 to 72 h it did inhibit these activities. Methylglyoxal bis(guanyl-hydrazone) inhibited S-adenosylmethionine decarboxylase (EC 4.1.1.50) activity without affecting DNA synthesis and cell division.Abbreviations ADC arginine decarboxylase - ODC ornithine decarboxylase - SAMDC S-adenosylmethionine decarboxylase - DFMA DL -difluoro-methylarginine - DFMO DL -difluoromethylornithine - MGBG methylglyoxal bis(guanylhydrazone)     

Polyamine Biosynthetic Enzymes and the Effect of their Inhibition on the Growth of Some Phytopathogenic Fungi

Studies were conducted on the distribution of two polyaminebiosynthetic enzymes, or-nithine decarboxylase (ODC) and argininedecarboxylase (ADC), and the effect of their inhibitors on growthand polyamine biosynthesis in four phytopathogenic fungi, namely,Helminthosporium maydis, H. carbonum, Fusarium oxysporum f.sp. lycopersici and Ceratocystis ulmi. Three species had highlevel of ODC as compared to ADC activity; in C. ulmi on theother hand, ADC was predominant with very little or no ODC activity.DL--difluoromethylornithine (DFMO) significantly inhibited ODCactivity in all species in vitro with little effect on ADC activity.ADC in all cases was inhibited by DL--difluoromethylarginine(DFMA) but not by DFMO. Mycelial growth of all fungi was inhibitedby 1 to 5 mM concentrations of either DFMO or DFMA within twodays except in H. maydis which remained unaffected even by thehighest concentration (5 mM) of DFMA. In general, the inhibitionwas more pronounced with DFMO as compared to DFMA. Putrescinecompletely reversed the inhibitory effects of DFMO and DFMAin all species. Among the polyamines, spermidine was predominantin all fungi. The cellular concentrations of putrescine andspermidine were considerably lower in the presence of eitherof the inhibitors while spermine levels were higher than thecontrol. ¹Scientific contribution number 1529 from the New HampshireAgricultural Experiment Station. (Received November 25, 1988; Accepted April 11, 1989)     

Ornithine decarboxylase activities of a Shewanella putrefaciens which produces only diamines

Ornithine decarboxylase (ODC) activity in cell extracts of Shewanella putrefaciens was surveyed. The pH dependency of the ODC activity revealed that the bacterium has two different ODC having optimum pH at 8·25 and 6·50. They were considered to be biosynthetic and biodegradative enzymes, respectively. Their activity ratio varied when the bacterium was cultured at pH 7·0 and 6·0. Both ODC activities were inhibited by α-difluoromethylornithine but cell growth was not affected.     

An essential role for putrescine biosynthesis during meiotic maturation of amphibian oocytes

The objective of this study was to investigate the role of polyamines during meiotic maturation of Xenopus oocytes. The results indicate a rapid and significant increase in the activity of ornithine decarboxylase (ODC), the rate-limiting enzyme in the polyamine biosynthetic pathway, during the meiotic maturation induced by either progesterone or human chorionic gonadotropin (HCG). This increase in the enzyme activity was followed by an accumulation of putrescine without any effect on the levels of spermidine or spermine. The inhibition of ODC activity and the accumulation of putrescine levels by α-difluoromethyl ornithine (DFMO), a catalytic irreversible inhibitor of ODC, also resulted in the inhibition of maturation mediated by progesterone in Xenopus oocytes. DFMO caused an inhibition of both maturation and ovulation induced by HCG in ovarian fragments. This inhibition was readily reversible by exogenous supply of putrescine to the medium. These observations suggest that putrescine plays an important role during the meiotic maturation of amphibian oocytes.     

Effects of inhibitors of polyamine biosynthesis and of polyamines on strawberry microcutting growth and development

The primary free polyamines identified during growth and development of strawberry (Fragaria × ananassa Duch.) microcuttings cultivated in vitro were putrescine, spermidine and spermine. Polyamine composition differed according to tissue and stages of development; putrescine was predominant in aerial green tissues and roots. -DL-difluoromethylarginine (DFMA), a specific and irreversible inhibitor of the putrescine-synthesizing enzyme, arginine decarboxylase (ADC), strongly inhibited growth and development. Application of agmatine or putrescine to the inhibited system resulted in a reversal of inhibition, indicating that polyamines are involved in regulating the growth and development of strawberry microcuttings. -DL-difluoromethylornithine (DFMO), a specific and irreversible inhibitor of putrescine biosynthesis by ornithine decarboxylase, promoted growth and development. We propose that ADC regulates putrescine biosynthesis during microcutting development. The application of exogenous polyamines (agmatine, putrescine, spermidine) stimulated development and growth of microcuttings, suggesting that the endogenous concentrations of these polyamines can be growth limiting.Abbreviations ADC arginine decarboxylase - ODC ornithine decarboxylase - DFMA -difluoromethylarginine - DFMO -difluoromethylornithine - Put putrescine - Spd spermidine - Sp spermine - DW dry weight - PA polyamine - PPF photosynthetic photon flux     

Increased ornithine decarboxylase activity and polyamine biosynthesis are required for optimal cytolytic T lymphocyte induction

The objective of the present investigation was to evaluate the requirement for increased ornithine decarboxylase (ODC) activity and polyamine biosynthesis in the induction of cytolytic T lymphocytes (CTL). In this regard, we have utilized alpha-difluoromethylornithine (DFMO), an irreversible inhibitor of ODC. DFMO treatment completely abrogated Con A-induced NW T-cell ODC activity. Similarly, DFMO treatment reduced putrescine and spermidine biosynthesis 100 and 87% respectively by the end of a 48-hr incubation period. Polyamine depletion reduced the Con A-mediated polyclonal induction of CTL by 52 and 81% at 24 and 48 hr of culture, respectively. The effect of DFMO on CTL induction could be reversed by the addition of exogenous putrescine. These data indicate that the observed effects of DFMO on CTL induction were mediated through inhibition of polyamine biosynthesis. Therefore, increased ODC activity and polyamine biosynthesis are required for optimal CTL induction. Furthermore, polyamine depletion did not impair IL-2 production; however, IL-2-dependent proliferation was reduced. These data are the first to discriminate between the requirement for polyamines with regard to IL-2 responsiveness, rather than IL-2 production, during a primary T-cell mitogenic response.     

Polyamines,floral induction and floral development of strawberry (Fragaria ananassa Duch.)

In the short-day plant, strawberry (Fragaria ananassa Duch.), polyamines (putrescine, spermidine and spermine), conjugated spermidine (water-insoluble compounds) and bound amines (putrescine, spermidine, phenylethylamine, 3-hydroxy, 4-methoxyphenylethylamine) accumulated in the shoot tips during floral induction and before floral emergence. Different associations of free amines and conjugated amines were observed during floral induction, as compared with the reproductive phase. During the whole period of floral development, phenylethylamine (an aromatic amine) was the predominant amine, representing 80 to 90% of the total free amine pool. Phenylethylamine conjugates (water-insoluble compounds) were the predominant amides observed prior to fertilization. These substances decreased drastically after fertilization. In vegetative shoot tips from plants grown continously under long days, free polyamines (putrescine, spermidine) and bound polyamines (putrescine, spermidine) were low and no change was observed. Free amines (spermine and phenylethylamine), bound aromatic amines (phenylethylamine, 3-hydroxy, 4-methoxyphenylethylamine), conjugated spermidine and phenylethylamine did not appear. Male-sterile flowers were distinguished by their lack of conjugated spermidine and phenylethyalamine and by a decrease in free phenylethylamine. In normal and sterile strawberry plants -DL-difluoromethylornithine (DFMO), a specific irreversible inhibitor of ornithine decarboxylase (ODC), caused inhibition of flowering and free and polyamine conjugates. When putrescine was added, polyamine titers and flowering were restored. A similar treatment with -DL-difluoromethylarginine (DFMA), a specific, irreversible inhibitor of arginine decarboxylase (ADC), did not affect flowering and polyamine titers. These results suggest that ornithine decarboxylase (ODC) and polyamines are involved in regulating floral initiation in strawberry. The relationship between polyamines, aromatic amines, conjugates, floral initiation and male sterility is discussed.Abbreviations ADC arginine decarboxylase - ODC ornithine decarboxylase - DFMA -DL-difluoromethylarginine - DFMO -DL-difluoromethylornithine - Put putrescine - Spd spermidine - Spm spermine - Phen phenylethylamine - 3H4M Phen 3-hydroxy, 4-methoxyphenylethylamine     

Apparent ornithine decarboxylase activity, measured by 14CO2 trapping, after frozen storage of rat tissue and rat tissue supernatants

Ornithine decarboxylase (ODC) activity of rat tissues was measured by the standard 14CO2 trapping method after frozen storage (-60 or -70 degrees C) of the tissues or their 105,000g supernatants. True ODC activity was determined by two methods: (a) addition of the inhibitors alpha-difluoromethylornithine (DFMO), a specific irreversible inhibitor of ODC, or aminooxyacetate (AOA), an inhibitor that blocks the decarboxylation of ornithine by mitochondrial enzymes; and (b) chromatographic analysis of the reaction products. In the frozen supernatants of liver and spleen, ODC activity changed only slightly after 1 day but increased 29 and 14%, respectively, by 30 days; activity in kidney supernatant decreased 17% after 1 day and remained near that level at 30 days. Kidney and spleen ODC activity was inhibited 90-100% by DFMO, but apparent liver ODC activity was inhibited only 60-75%. In the supernatant prepared from tissue stored frozen for 1 day, apparent ODC activity in liver increased 500% over that activity in the freshly prepared supernatant; at 23 days, apparent activity increased 755% for liver and 121% for kidney. After 23 days, DFMO did not inhibit apparent ODC activity in supernatants from frozen liver and inhibited ODC in frozen kidney by only 49%. With AOA, the ODC activities of the fresh and frozen supernatants were similar, indicating that the large increase in apparent ODC activity in frozen tissue was due to artifacts from the metabolism of ornithine via the mitochondrial pathway. HPLC analysis of the reaction products resulting from the incubation of uniformly labeled [14C]ornithine with the fresh and frozen preparations indicated no increase in putrescine with the frozen preparation.(ABSTRACT TRUNCATED AT 250 WORDS)     

An evaluation of the involvement of polyamines in modulating MCF-7 human breast cancer cell proliferation and progesterone receptor levels by estrogen and antiestrogen

The present studies were undertaken to determine the importance of the polyamine biosynthetic pathway in cellular proliferation and hormone-regulated progesterone receptor synthesis in estrogen receptor-containing breast cancer cells. Treatment of MCF-7 cells with difluoromethylornithine (DFMO), the irreversible inhibitor of the enzyme ornithine decarboxylase (ODC), prevented estradiol-induced cell proliferation in a dose-dependent fashion. DFMO inhibition of estradiol-induced cell proliferation was completely recoverable by the addition of exogenous putrescine while putrescine alone did not stimulate proliferation of control cells. ODC activity was 4-fold greater in estrogen-treated cells and DFMO (5 mM) fully inhibited ODC activity. DFMO was able to suppress only slightly further the proliferation of antiestrogen (tamoxifen) treated cells and putrescine was able to recover this DFMO inhibition. In contrast to the suppressive effect of DFMO on cell proliferation, DFMO had no effect on the ability of estrogen to stimulate increased (4-fold elevated) levels of progesterone receptor. Hence, while ODC activity appears important for estrogen-induced cell proliferation, inhibition of the activity of this enzyme has no effect on the ability of estradiol to increase cellular progesterone receptor content.     

Polyamine metabolism during seedling development in rice

The main free amines identified during growth and development of rice seedlings were agmatine, putrescine, spermidine, diaminopropane and tyramine. Amine composition differed according to tissue and stages of development. Conjugated amines were only found in roots. We present evidence that arginine decarboxylase (ADC) regulates putrescine during the development of rice seedlings. When ADC action was blocked by DFMA (-DL-difluoromethylarginine, a specific irreversible inhibitor of ADC), polyamine titers and seedling development were diminished; when agmatine or putrescine was added, normal polyamine titers and growth were restored. The effects of DFMA were concentration dependent. DFMO (-DL-difluoromethylornithine, a specific irreversible inhibitor of ornithine decarboxylase or ODC) promoted growth and development at concentrations below 2 mM. This effect was probably related to its unexplained, but consistently observed slight enhancement of rice ADC. When the increase in the concentration of spermidine was prevented by CHA (cyclohexylammonium sulfate), the number of roots increased and the increase in length of leaves and roots was strongly inhibited. The addition of exogenous spermidine at the time of treatment with CHA reversed the inhibition by CHA.Abbreviations ADC arginine decarboxylase - ODC ornithine decarboxylase - DFMA -DL-difluoromethylarginine - DFMO -DL-difluoromethylornithine - CHA cyclohexylammonium sulfate     

Ornithine decarboxylase induction and polyamine biosynthesis are required for the growth of interleukin-2- and interleukin-3-dependent cell lines

The objective of this study was to evaluate induction of ornithine decarboxylase (ODC), the rate-limiting enzyme in polyamine biosynthesis, and subsequent polyamine accumulation in interleukin-2 (IL-2)- and interleukin-3 (IL-3)-dependent growth. The CTLL-20 and FDC-P1 cell lines, which have been shown to be absolutely dependent on IL-2 and IL-3, respectively, were used in these studies. The CTLL-20 and FDC-P1 cells each had different temporal patterns of ODC induction following lymphokine stimulation. ODC levels increased rapidly in the FDC-P1 cells, peaking 4 hr after stimulation with IL-3. In contrast, peak ODC activity in the CTLL-20 cells occurred 18 hr following stimulation with IL-2 and reached eightfold higher levels than those observed in the FDC-P1 cells. Treatment with D,L-alpha-difluoromethylornithine X HCl X H2O (DFMO), a specific irreversible inhibitor of ODC activity, completely abrogated lymphokine-dependent ODC induction in both the CTLL-20 and FDC-P1 cell lines. Similarly, intracellular levels of the polyamines putrescine and spermidine were reduced in both cell lines following DFMO treatment. DFMO treatment reduced both IL-2- and IL-3-dependent proliferation in a dose-dependent manner. However, this inhibition could be reversed by the addition of exogenous putrescine. DFMO treatment had no effect on cell viability. Polyamine-depleted CTLL-20 and FDC-P1 cells showed decreased absorption of IL-2 and IL-3 activity, respectively. However, the addition of exogenous putrescine restored the ability of the cells to absorb the appropriate lymphokine. These data are the first to demonstrate that ODC induction and polyamine biosynthesis are required in lymphokine dependent growth.     

Regulation of N-Methyl-d-aspartate receptor-mediated calcium transport and norepinephrine release in rat hippocampus synaptosomes by polyamines

The role of polyamines (PA) synthesis in NMDA receptor-mediated⁴⁵Ca²⁺ fluxes and norepinephrine release was studied in rat hippocampal synaptosomes. NMDA (50M) caused a sharp (>2-fold) transient increase in PA synthesis regulating enzyme, ornithine decarboxylase (ODC) activity with concomitant elevation in PA levels in the order putrescine>spermidine>spermine. ODC inhibitor, -difluoromethylornithine (DFMO), and NMDA antagonist, 2-amino-5-phosphonovaleric acid (D-AP5), both blocked increases in ODC activity and PA levels. Activation of NMDA receptors induced a sharp (3 to 4-fold) and quick (15 seconds) increase in⁴⁵Ca²⁺ uptake by synaptosomes within 15 seconds of exposure at 37°C. The efflux of⁴⁵Ca²⁺ and³H-norepinephrine (NE) release at 22°C from pre-loaded synaptosomes was also significantly (2 to 4-fold) enhanced by NMDA within 15 seconds. These NMDA receptor-mediated effects on calcium fluxes and NE release were blocked by NMDA receptor-antagonists (DAP-5 and MK-801) and PA synthesis inhibitor, DFMO and the DFMO inhibition nullified by exogenous putrescine. These observations establish that ODC/PA cascade play an important role in transduction of excitatory amino acid mediated signals at NMDA receptors.Special issue dedicated to Dr. Sidney Ochs.     

The effect of polyamine biosynthesis inhibition on growth and differentiation of the phytopathogenic fungus Sclerotinia sclerotiorum

We studied the effects of several polyamine biosynthesis inhibitors on growth, differentiation, free polyamine levels and in vivo and in vitro activity of polyamine biosynthesis enzymes in Sclerotinia sclerotiorum. -Difluoromethylornithine (DFMO) and -difluoromethylarginine (DFMA) were potent inhibitors of mycelial growth. The effect of DFMO was due to inhibition of ornithine decarboxylase (ODC). No evidence for the existence of an arginine decarboxylase (ADC) pathway was found. The effect of DFMA was partly due to inhibition of ODC, presumably after its conversion into DFMO by mycelial arginase, as suggested by the high activity of this enzyme detected both in intact mycelium and mycelial extracts. In addition, toxic effects of DFMA on cellular processes other than polyamine metabolism might have occurred. Cyclohexylamine (CHA) slightly inhibited mycelial growth and caused an important decrease of free spermidine associated with a drastic increase of free putrescine concentration. Methylglyoxal bis-[guanyl hydrazone] (MGBG) had no effect on mycelial growth. Excepting MGBG, all the inhibitors strongly decreased sclerotial formation. Results demonstrate that sclerotial development is much more sensitive to polyamine biosynthesis inhibition than mycelial growth. Our results suggest that mycelial growth can be supported either by spermidine or putrescine, while spermidine (or the putrescine/spermidine ratio) is important for sclerotial formation to occur. Ascospore germination was completely insensitive to the inhibitors.     

Inhibition of ornithine decarboxylase by retinoic acid and difluoromethylornithine in relation to their effects on differentiation and proliferation

Murine embryonal carcinoma F9 cells can be induced to differentiate by 2-difluoromethylornithine (DFMO), an irreversible inhibitor of ornithine decarboxylase (ODC). The differentiated phenotype is similar to that of retinoic acid (RA)-treated F9 cells. In contrast to F9 cells the differentiated cells secrete plasminogen activator and express keratin intermediate filaments. Both DFMO and RA reduce ornithine decarboxylase activity, polyamine levels and inhibit cell proliferation of F9 cells. These compounds also reduce ODC, polyamine levels and proliferation of mouse BALB/c 3T6 fibroblasts. RA inhibits the induction of ODC by insulin, serum and to a lesser extent that of epidermal growth factor (EGF) and 12-O-tetradecanoylphorbol-13-acetate (TPA). The action of DFMO and RA can be distinguished by their response to putrescine. The induction of differentiation and the inhibition of cell proliferation by DFMO can be totally abolished upon the addition of putrescine, whereas the actions of RA are not affected at all. These results suggest that the inhibition of ODC and reduction of polyamines are not causal in the induction of differentiation and the inhibition of proliferation by RA.     

Effect of polyamine biosynthetic inhibitors on alkaloids and organogenesis in tobacco callus cultures

We studied the effects of inhibitors of ornithine decarboxylase (ODC), arginine decarboxylase (ADC) and spermidine synthase (Spd synthase) on organogenesis and the titers of polyamines (PA) and alkaloids in tobacco calli. DL--difluoromethylarginine (DFMA) and D-arginine (D-Arg), both inhibitors of ADC activity, were more effective than DL--difluoromethylornithine (DFMO), an inhibitor of ODC, in reducing titers of PA and the putrescine (Put)-derived alkaloids (nornicotine and nicotine). Dicyclohexylammonium sulfate (DCHA), an inhibitor of Spd synthase, was also more efficient than DFMO in reducing PA and alkaloid levels. Root organogenesis is inversely related to the titers of Put and alkaloids. Thus, DFMA and D-Arg, which strongly inhibit Put and alkaloid biosynthesis, markedly promote root organogenesis, while control callus with high Put and alkaloid content showed poor root organization. These results suggest that morphological differentiation is not required for activation of secondary metabolic pathways and support the view that ADC has a major role in the generation of Put going to the pyrrolidine ring of tobacco alkaloids.