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PubMed2003

Overview of the brain polyamine-stress-response: regulation, development, and modulation by lithium and role in cell survival.

An early transient increase in brain polyamine (PA) metabolism, termed the PA-stress-response (PSR), is a common reaction to stressful stimuli, including physical, emotional, and hormonal stressors, with a magnitude related to the stress intensity. In the extreme, traumatic injury can result in an incomplete PSR, with persistent accumulation of putrescine and eventual reduction in the concentrations of the higher polyamines (PAs), spermidine and spermine. Chronic intermittent application of stressors causes a recurrence of the brain PSR, but, in contrast, it leads to habituation of the response in the periphery (liver). Severe continuous stress, however, may lead to accumulation of brain PAs. Long-term inhibition of PA synthesis depletes brain PAs and can result in altered emotional reactivity to stressors. Furthermore, the brain PSR, in contrast to the periphery, can be blocked by a long-term, but not by short-term, treatment with lithium, the most efficacious treatment of manic-depressive illness. The brain PSR is developmentally regulated, and the switch to the mature pattern coincides with the cessation of the "stress hyporesponsive period" in the hypothalamic-pituitary-adrenocortical (HPA) system. In contrast to the brain and liver, the PSR in the adrenal and thymus is down-regulated by acute stressors. Transient up-regulation of the PSR, as in the brain and liver, is implicated in cell survival while its down-regulation is implicated in cell death. Taken together, the findings indicate that the PSR is a dynamic process that varies with the type, intensity, and duration of stressors, and implicate this response as an adaptive mechanism in the reaction to stressful events. Under persistent stressful conditions, however, the PSR may be maladaptive as may be reflected by PA accumulation. This raises the hypothesis that proper regulation of brain PSR may be critical for neuronal function and for an appropriate behavioral response to stressors.

Adaptation, Physiological↗

PubMed2004

Effects of polyamines on the muscarinic receptor-operated cation current in guinea-pig ileal smooth muscle myocytes.

The effects of extracellular and intracellular polyamines (PAs), spermine and putrescine, on the cation current (mI(CAT)) evoked either by activating muscarinic receptors with carbachol or by intracellularly applied GTPgammaS (in the absence of carbachol) were studied using patch-clamp recording techniques in single guinea-pig ileal myocytes. Extracellular spermine and putrescine rapidly and reversibly inhibited mI(CAT) in a concentration- and voltage-dependent manner with the IC(50) values at -40 mV of about 1 and 5 mM, respectively. Membrane depolarization relieved the blocking action of PAs although cation conductance activation curve remained N-shaped. The inhibition was similar for both carbachol- and GTPgammaS-evoked currents, suggesting that the cation channel rather than the muscarinic receptor was the primary site of the PA action. In outside-out membrane patches, both cation channel unitary conductance and open probability were reduced. In perforated-patch experiments used to retain cytoplasmic PAs sustained 100 microM carbachol-induced mI(CAT) was significantly smaller (478 +/- 76 pA, n = 7) compared to that recorded using conventional whole-cell configuration with nominally PA-free pipette solution (1314 +/- 76 pA, n = 12), but comparable in size to mI(CAT) with 0.3 mM spermine in the pipette solution (509 +/- 41 pA, n = 19). Intracellular putrescine inhibited mI(CAT) less potently compared to spermine. In conclusion, these results show a novel role of intestinal PAs in mI(CAT) inhibition, which can contribute to their well-known suppressing effect on the gastrointestinal smooth muscle excitability and contractility.

Animals↗

PubMed1992

Synchronization of breast cancer cell proliferation in vivo by combined hormonal and polyamine manipulation.

Optimal synchronization of breast cancer cell proliferation by hormonal means may be limited by cellular heterogeneity in sensitivity to the multistep activation of growth following initial hormone binding to the receptor. We hypothesized that induced synchronous growth may be improved by combined manipulation of the polyamine (PA) pathway since we have previously shown that PAs are distal effectors of hormonal action on proliferation in breast cancer. To test our hypothesis, we induced an initial phase of hormone and PA depletion (castration plus administration of the PA synthesis inhibitor alpha-difluoromethylornithine) in rats bearing N-nitrosomethylurea induced mammary tumors. This was followed by transition phase of hormone repletion in the presence of alpha-difluoromethylornithine (to push the cells into the proliferative cascade up to the distal step controlled by PA) and finally a phase of hormone and PA repletion. Simultaneously, groups of rats were subjected to hormone/PA depletion/repletion individually. The effects of these manipulations on the labeling indices (LIs) of glandular, myoepithelial, and nonepithelial cells were estimated by autoradiography. The combined hormone/PA manipulation yielded the highest degree of synchronization with LIs of the glandular and myoepithelial cells being approximately 2-fold over intact control after only 2 or 3 days of combined repletion. In contrast, hormone treatment alone restored the LIs of glandular cells only to control levels and minimally influenced those of myoepithelial cells. PA manipulation alone failed to affect the LIs of any cell type. Although the rate of tumor regrowth was highest with the combination treatment, the absolute tumor volumes did not differ significantly at the end of the repletion phase between the three regimens. These results indicate that combined hormone/PA manipulation provides the best "therapeutic window" (LI/tumor volume) for implementation of kinetically based cytotoxic chemotherapy.

Animals↗

PubMedDate not supplied

Polyamine levels in testes and seminal vesicles from adult golden hamsters during gonadal regression-recrudescence.

The exposure of golden hamsters to short days results in early regression of the reproductive organs and subsequent spontaneous recrudescence characterized by active cellular regeneration and differentiation. Thus, adult male hamsters were subjected to short photoperiod (SP, 6L:18D) for 9, 12, 14, 16, 18, and 22 weeks or maintained under long photoperiod (LP, 14L:10D) for 22 weeks, to assess photoperiodic-related changes in testicular and seminal vesicle (SV) levels of polyamines (PA) that are involved in cell growth and differentiation. During the regression phase, the weights of the organs and the circulating levels of luteinizing hormone (LH), follicle-stimulating hormone (FSH), prolactin, testosterone, dihydrotestosterone, and 5 alpha-androstane-3 alpha, 17 beta-diol were significantly diminished and, thereafter, during the recrudescence phase, they recovered total or partially their control values. In both tissues, the exposure to SP for 14-16 weeks resulted in an increase of PA concentrations, followed by a return to control levels in the recrudescence period. At the time of maximal tissue involution, the ornithine decarboxylase (ODC) activity (key regulatory enzyme of PA biosynthesis) showed a significant increase in testis, preceding the sharp peak of PA concentration. However, a marked decrease in ODC activity was detected in SV. The concentration of N-acetyl PA in SV showed an increment at 16 weeks of SP, while no modifications were detected in testicular concentration. When PA, N-acetyl PA, and ODC activity were expressed per testis and per SV, values fell significantly during the involution period, but in the recrudescence phase levels were recovered concomitantly with the restoration of the organ weight and function. In conclusion, the photoperiodic-related changes in PA and their N-acetyl derivatives might play a crucial role in regrowth and differentiation of the male sexual organs during the spontaneous recrudescence phase. Additionally, organ-specific regulation of the PA biosynthesis pathway could also take place.

Androgens↗

PubMed2002

Influence of polyamines on in vitro and in vivo features of aggressive and metastatic behavior by human breast cancer cells.

Increased cellular activity of ornithine decarboxylase (ODC), the first and rate-limiting enzyme in polyamine (PA) synthesis, is an independent adverse prognostic factor for overall survival in human breast cancer, thus suggesting an important role for PA in tumor progression. The experiments presented here were designed to investigate the role of PA in invasion and metastasis, using the highly aggressive MDA-MB-435 and MDA-MB-231 human breast cancer cell lines. Administration of alpha-difluoromethylornithine (DFMO), an irreversible inhibitor of ODC, significantly reduced, in a dose-dependent manner, the invasiveness in matrigel of both MDA-MB-435 and MDA-MB-231 cells by approximately 70%. DFMO treatment also inhibited (P < 0.0001) 'stellate' colony formation (an indicator of aggressive phenotype) by MDA-MB-435 cells plated in the matrigel outgrowth assay. Administration of DFMO (2% in drinking water) reduced the growth rate of both cell lines implanted orthotopically in nude mice. To evaluate metastasis while minimizing effects on proliferation, DFMO-treated mice were sacrificed later to allow their tumors to reach the same size of the tumors in the control mice. The most striking finding was that DFMO, while ineffective in reducing local invasion, nearly totally abolished (P = 0.0152) pulmonary metastasis in mice bearing MDA-MB-435 xenografts. These results support a role of PA in promoting breast cancer aggressiveness, particularly with regard to the development of distant metastasis. Furthermore, the data suggest that PA involvement is distal to local invasion in the metastatic cascade.

Animals↗

PubMed1989

Polyamine involvement in the growth of hormone-responsive and -resistant human breast cancer cells in culture.

Recent evidence indicates that hormone-responsive but not -resistant human breast cancer cells in culture are sensitive to the antiproliferative effect of the polyamine (PA) biosynthetic inhibitor alpha-difluoromethylornithine (DFMO). The present experiments were designed to investigate the potential differential involvement of the PA pathway in the growth of these different biological subtypes of human breast cancer. Thus, we evaluated the effect of DFMO on proliferation, ornithine decarboxylase (ODC) activity, and PA levels of the hormone-dependent MCF-7 and -independent MDA-MB-231 breast cancer cell lines. When tested at comparable cell density, the two cell lines had similar levels of ODC activity and PA. Administration of DFMO (0.01, 0.1, 1, 4 mM) for 6 days caused a similar dose-dependent inhibition of proliferation (up to approximately 15% of control) associated with suppression of ODC activity to undetectable levels at the highest dose. In both cell lines, putrescine and spermidine levels were maximally suppressed by doses of DFMO greater than 0.1 mM. Higher doses of DFMO (1 and 4 mM) also suppressed spermine levels to approximately 60% of control. In detailed time-course studies, DFMO administration (0.1 mM) similarly suppressed by 80% the rise in ODC observed in both cell lines following a medium change. At all time points, putrescine and spermidine levels were likewise suppressed to a similar extent. Addition of putrescine (0.1-2.5 mM) to DFMO-treated cells repleted cellular PA levels and restored growth to approximately 80% of control in both cell lines. We conclude that, under these experimental conditions, PA are similarly involved in the growth of the hormone-responsive MCF-7 and -resistant MDA-MB-231 human breast cancer cell lines.

Biogenic Polyamines↗

PubMedDate not supplied

Central effects of SL 82.0715, an antagonist of polyamine site of the NMDA receptor complex.

Central effects of SL 82.0715, an antagonist of polyamine site of the NMDA receptor complex, was studied in male Albino-Swiss mice. SL 82.0715, in doses which given alone were inactive in electroshock-induced seizures, potentiated the anticonvulsant effects of CGP 37849. In normal mice, SL 82.0715 decreased the spontaneous locomotor activity, did not affect the locomotor hyperactivity induced by MK-801 and attenuated CGP 37849-induced locomotor hyperactivity. The D-amphetamine locomotor hyperactivity was also antagonized by SL 82.0715. SL 82.0715 did not significantly change the locomotor activity in monoamine-depleted mice (treated with reserpine + alpha-methyl-p-tyrosine). When administered together with clonidine, SL 82.0715 increased the locomotor activity in monoamine-depleted mice but this effect did not reach the level of statistical significance. SL 82.0715 did not change the locomotor activity induced by joint administration of clonidine and MK-801 or clonidine and CGP 37849, in monoamine-depleted mice. The locomotor hyperactivity evoked by L-DOPA (given jointly with benserazide) was not changed by SL 82.0715. SL 82.0715 had no effect on fluphenazine- and haloperidol-induced catalepsy in mice. CGP 37849 attenuated catalepsy induced by haloperidol; that effect was not changed by SL 82.075. The studied compound did not affect the immobility time and had no effect on the action of MK-801 or CGP 37849 in the forced swimming test. The obtained results indicate that SL 82.0715 has a different pharmacological profile than other NMDA antagonists (at least MK-801 and CGP 37849). SL 82.0715 does not increase behavioral actions of MK-801 and CGP 37849, potentiating anticonvulsant effect of CGP 37849 only.

2-Amino-5-phosphonovalerate↗

PubMed2006

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.

Biogenic Polyamines↗

PubMed2006

Peroxisome proliferator-activated receptor gamma as a molecular target of resveratrol-induced modulation of polyamine metabolism.

Previous results indicate that the polyphenol resveratrol inhibits cell growth of colon carcinoma cells via modulation of polyamine metabolic key enzymes. The aim of this work was to specify the underlying molecular mechanisms and to identify a possible role of transcription factor peroxisome proliferator-activated receptor gamma (PPARgamma). Cell growth was determined by bromodeoxyuridine incorporation and crystal violet staining. Protein levels were examined by Western blot analysis. Spermine/spermidine acetyltransferase (SSAT) activity was determined by a radiochemical assay. PPARgamma ligand-dependent transcriptional activity was measured by a luciferase assay. A dominant-negative PPARgamma mutant was transfected in Caco-2 cells to suppress PPARgamma-mediated functions. Resveratrol inhibits cell growth of both Caco-2 and HCT-116 cells in a dose- and time-dependent manner (P < 0.001). In contrast to Caco-2-wild type cells (P < 0.05), resveratrol failed to increase SSAT activity in dominant-negative PPARgamma cells. PPARgamma involvement was further confirmed via ligand-dependent activation (P < 0.01) as well as by induction of cytokeratin 20 (P < 0.001) after resveratrol treatment. Coincubation with SB203580 abolished SSAT activation significantly in Caco-2 (P < 0.05) and HCT-116 (P < 0.01) cells. The involvement of p38 mitogen-activated protein kinase (MAPK) was further confirmed by a resveratrol-mediated phosphorylation of p38 protein in both cell lines. Resveratrol further increased the expression of PPARgamma coactivator PGC-1alpha (P < 0.05) as well as SIRT1 (P < 0.01) in a dose-dependent manner after 24 hours of incubation. Based on our findings, p38 MAPK and transcription factor PPARgamma can be considered as molecular targets of resveratrol in the regulation of cell proliferation and SSAT activity, respectively, in a cell culture model of colon cancer.

Acetyltransferases↗

PubMed2000

Pancreatic exocrine secretions as a source of luminal polyamines in pigs.

The goal of the present study was twofold: (1) to detect the possible storage of dietary polyamines (PAs) in various tissues and (2) to investigate the role of dietary PAs in the differentiation of the pig intestinal epithelium. A first experimental series was designed to assess the accumulation of either milk PAs (mostly spermidine) or orally administered spermine (SPM) in piglet red blood cells (RBCs) and plasma, a preliminary stage in their distribution to growing and storage organs. Though PA concentrations of piglet RBCs and plasma were generally significantly higher than their sow counterparts, our experimental conditions failed to demonstrate that this increase could stem from ingested PAs. A second experimental series dealt with the determination of disaccharidase specific activities in proximal and distal parts of piglet gut on the 26th and 29th days after birth (preweaning time). In agreement with observations made previously on rat pups, we observed an increase in maltase specific activity (SA) at the end of the suckling period (the observed increase in sucrase SA was not significant). However, orally administered SPM did not affect this activity. Compared to the constant protein concentrations observed in both parts of the gut, the pancreatic protein content decreased sharply between the 26th and 29th postnatal days. At the same time pancreatic concentrations of spermidine (SPD) also decreased, suggesting that some pancreatic PAs were released as the organ secreted its proteins. In accordance with this hypothesis, we recorded SPM and SPD in pancreatic juice. The increases in PA concentrations seemed to follow the protein secretion pattern (i.e. PA concentrations reached a maximal value when the protein concentration was highest). The presence of PAs in pancreatic juice could be indicative of a control mechanism exerted by the pancreas on PA-induced growth and differentiation of porcine intestinal epithelium.

Animals↗

PubMed1990

Effect of alpha-difluoromethylornithine on 1,3-bis(2-chloroethyl)-1-nitrosourea and cis-diamminedichloroplatinum(II) cytotoxicity, DNA interstrand cross-linking, and growth in human brain tumor cell lines in vitro.

The polyamine biosynthesis inhibitor alpha-difluoromethylornithine (DFMO) has been shown to potentiate the cytotoxicity of 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) in 9L rat brain tumor cells and in non-central nervous system human cancer cells in vitro, but the effects on a human brain tumor cell line have not been reported. Because BCNU is one of the main chemotherapeutic agents used clinically for the treatment of brain tumors, the effect of DFMO treatment on cell growth and potentiation of cytotoxicity was studied in vitro in U-251 MG and SF-126 cells, human tumor cell lines derived from malignant glioma tissue. Pretreatment of U-251 MG with 1 mM DFMO depleted cells of putrescine and spermidine within 48 h but did not sensitize cells to BCNU treatment even after a pretreatment of 72 h. DFMO treatment had no effect on the number of interstrand cross-links formed in BCNU-treated cells. Even treatment with 5 mM DFMO for 72 h caused only the suggestion of potentiation of BCNU cell kill. In contrast, a 72-h pretreatment with 1 mM DFMO decreased the cytotoxic effect of cis-diammine-dichloroplatinum(II) and caused a 38% decrease in the number of DNA interstrand cross-links formed. The glutathione content and cell cycle distribution of U-251 MG cells were not affected by DFMO pretreatment. Because Phase II clinical trials with DFMO and BCNU have shown promise for the treatment of anaplastic astrocytomas in humans, a second brain tumor cell line, SF-126, was studied. In this cell line a consistent potentiation of BCNU cytotoxicity (dose enhancement of 1.2 at the 10% survival level) was observed in cells pretreated with 1 mM DFMO for 72 h.

Biogenic Polyamines↗

PubMed2005

Expression of an antisense Datura stramonium S-adenosylmethionine decarboxylase cDNA in tobacco: changes in enzyme activity, putrescine-spermidine ratio, rhizogenic potential, and response to methyl jasmonate.

S-adenosylmethionine decarboxylase activity (SAMDC; EC 4.1.1.21) leads to spermidine and spermine synthesis through specific synthases which use putrescine, spermidine and decarboxylated S-adenosylmethionine as substrates. In order to better understand the regulation of polyamine (PA), namely spermidine and spermine, biosynthesis, a SAMDC cDNA of Datura stramonium was introduced in tobacco (Nicotiana tabacum L. cv. Xanthi) in antisense orientation under the CaMV 35S promoter, by means of Agrobacterium tumefaciens and leaf disc transformation. The effect of the genetic manipulation on PA metabolism, ethylene production and plant morphology was analysed in primary transformants (R0), and in the transgenic progeny (second generation, R1) of self-fertilised primary transformants, relative to empty vector-transformed (pBin19) and wild-type (WT) controls. All were maintained in vitro by micropropagation. Primary transformants, which were confirmed by Southern and northern analyses, efficiently transcribed the antisense SAMDC gene, but SAMDC activity and PA titres did not change. By contrast, in most transgenic R1 shoots, SAMDC activity was remarkably lower than in controls, and the putrescine-to-spermidine ratio was altered, mainly due to increased putrescine, even though putrescine oxidising activity (diamine oxidase, EC 1.4.3.6) did not change relative to controls. Despite the reduction in SAMDC activity, the production of ethylene, which shares with PAs the common precursor SAM, was not influenced by the foreign gene. Some plants were transferred to pots and acclimatised in a growth chamber. In these in vivo-grown second generation transgenic plants, at the vegetative stage, SAMDC activity was scarcely reduced, and PA titres did not change. Finally, the rhizogenic potential of in vitro-cultured leaf explants excised from antisense plants was significantly diminished as compared with WT ones, and the response to methyl jasmonate, a stress-mimicking compound, in terms of PA conjugation, was higher and differentially affected in transgenic leaf discs relative to WT ones. The effects of SAMDC manipulation are discussed in relation to plant generation, culture conditions and response to stress.

Acetates↗

PubMed1993

Effects of polyamine synthesis blockade on neuronal loss and astroglial reaction after transient forebrain ischemia.

Polyamines and ornithine decarboxylase, the polyamine biosynthetic enzyme, have been demonstrated to increase in the early phase of several types of brain lesion. However, their role in the pathogenesis of tissue damage is still debated. In the present paper the effects of treatments with alpha-difluoromethylornithine, a suicide inhibitor of ornithine decarboxylase, have been investigated in a model of transient forebrain ischemia. Three treatment schedules were used: alpha-difluoromethylornithine treatment was either started 3 hr before and repeated 1 hr after the insult, or started at the time of the insult and continued for 3 or 7 days after post-ischemic reperfusion. The rats were sacrificed 4 hr, 7 or 40 days after reperfusion, respectively. The acute experiment demonstrated that alpha-difluoromethylornithine can reduce the increase of glial fibrillary acid protein immunoreactivity, an early marker of astroglial reaction, in ischemic striatum. Subchronic and chronic alpha-difluoromethylornithine treatments induced a worsening of the morphological outcome of the ischemic lesion. In caudate-putamen a trend for an increase of the area of neuronal loss was present after both treatments. In the hippocampal formation, a significant increase in the severity of neuronal lesion was observed in the mildly lesioned CA3 field. In addition, other alterations of lesioned tissue were observed in alpha-difluoromethylornithine-treated animals, including increases of non-neuronal cells at 7 and especially 40 days post-lesion in striatum and CA3 hippocampal field. In conclusion, present data indicate that ornithine decarboxylase activation after ischemic lesion is a crucial factor for survival of mildly lesioned neurons and proper tissue reaction to the ischemic lesion. The experiment on acute alpha-difluoromethylornithine treatment suggests that these effects may be, at least in part, related to putrescine-induced activation of astroglial cells in the early post-lesion period.

Animals↗

PubMed1990

Alpha-difluoromethylornithine resistance in Leishmania donovani is associated with increased ornithine decarboxylase activity.

The promastigote form of Leishmania donovani is sensitive to growth inhibition by DL-alpha-difluoromethylornithine (DFMO), an inhibitor of ornithine decarboxylase (ODC), the first enzyme of the polyamine biosynthetic pathway, with an EC50 value of approximately 30 microM. Exposure of a wild type (DI700) cell population to gradually increasing concentrations of DFMO resulted in the selection of a strain of Leishmania, DFMO-10, which was capable of proliferating in 10 mM DFMO. DFMO-10 cells possessed an EC50 value for DFMO greater than 4 mM, and were cross-resistant to alpha-methylornithine, alpha-monofluoromethyl-3,4-dehydroornithine methyl ester, and delta-methyl-acetylenic putrescine, three other inhibitors of ODC activity. DI700 and DFMO-10 cells accumulated and/or transported [3H]DFMO and a spectrum of basic, neutral, and acidic amino acids at comparative rates. However, the DFMO-resistant Leishmania, if suspended in culture medium in the absence of DFMO for several days, expressed up to 15-fold greater levels of ODC activity than did wild-type cells. The overexpressed ODC in mutant cells appeared kinetically normal, since the ODC activities from DI700 and DFMO-10 cells possessed similar apparent Km values for ornithine and were equally sensitive to inactivation by DFMO. Incubation of extracts of DFMO-10 cells, but not of wild-type parental cells, with [3H]DFMO for 1 h resulted in the labeling of a polypeptide, presumably ODC, which migrated with a molecular weight of 76,000 +/- 4000 on SDS-gel electrophoretograms. As a consequence of the elevated ODC activities, the levels of putrescine in mutant cells released from DFMO exposure were also elevated by about 15-fold over those of wild-type cells, although spermidine levels in DI700 and DFMO-10 cells were similar. In the absence of prolonged selective pressure, the resistance to DFMO, the ODC activity, and the putrescine levels of DFMO-10 cells all returned to those of wild type cells, indicating that the mutant phenotype of DFMO-selected L. donovani was unstable.

Affinity Labels↗

PubMed1998

Alpha-difluoromethylornithine inhibits N-nitrosomethylbenzylamine-induced esophageal carcinogenesis in zinc-deficient rats: effects on esophageal cell proliferation and apoptosis.

Sustained, increased cell proliferation induced by dietary zinc deficiency in rats plays a critical role in esophageal carcinogenesis. It is the determining factor that converts an otherwise nontumorigenic dose of N-nitrosomethylbenzylamine (NMBA) into a highly tumorigenic one. We studied whether the increased esophageal cell proliferation and susceptibility to NMBA-induced carcinogenesis induced by zinc deficiency can be inhibited by alpha-difluoromethylornithine (DFMO), an enzyme-activated, irreversible inhibitor of ornithine decarboxylase (the first enzyme in polyamine synthesis). Weanling rats were divided into four groups: Zn+/DFMO-, Zn+/DFMO+, Zn-/DFMO-, and Zn-/DFMO+. They were fed ad libitum either a zinc-sufficient (Zn+, 75 ppm zinc) or a zinc-deficient (Zn-, 4 ppm zinc) diet and given either deionized water (DFMO-) or 1% DFMO in deionized water (DFMO+). After 5 weeks, 5-19 animals from each group were sacrificed after in vivo 5-bromo-2'-deoxyuridine labeling to detect cells in S phase. The remaining animals in each group were given a single intragastric dose of NMBA at 2 mg/kg and sacrificed 12 weeks later for tumor incidence analysis. At week 5, DFMO treatment greatly decreased (by 48-82%) the levels of putrescine and spermidine in rat esophagus, colon, and liver, irrespective of dietary zinc intake. The increased esophageal cell proliferation induced by dietary zinc deficiency, as measured by the labeling index, the number of labeled cells, and the total number of cells, was substantially reduced by DFMO. This was accompanied by an increase in the rate of apoptosis. In addition, the expression of bax protein, an apoptosis accelerator, was markedly stronger in esophagi from Zn-/DFMO+ animals that showed increased apoptosis, whereas increased expression of bcl-2, an inhibitor of apoptosis, was only seen in the highly proliferative, zinc-deficient esophagus (Zn-/DFMO-). At week 12 after NMBA dosing, DFMO reduced the incidence of esophageal tumors from 80 to 4% in zinc-deficient rats. Our data showed that DFMO effectively inhibited the increased esophageal cell proliferation induced by dietary zinc deficiency and reduced the incidence of esophageal tumors induced by a single dose of NMBA in zinc-deficient animals. Our results also indicate a role for increased apoptosis in the mechanism(s) whereby DFMO brings about the inhibition of cell proliferation and tumor induction. These findings support a role for DFMO as a chemopreventive agent.

Animals↗

PubMed2002

Effect of immobilization stress on brain polyamine levels in spontaneously hypertensive and Wistar-Kyoto rats.

The present study aimed to compare the basal brain polyamine levels and stress-induced brain polyamine level changes in spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY) rats. For immobilization stress, both strains underwent acute (3 h per day immobilization for 2 days), chronic (3 h per day immobilization for 15 consecutive days), or no immobilization stress (control group). Basal putrescine (PU) levels in frontal cortex and hippocampus of SHR (11.03 +/- 0.81 and 11.36 +/- 0.33 nmol/g tissue, respectively) were significantly higher than WKY rats (6.90 +/- 1.44 and 7.82 +/- 0.71 nmol/g tissue, respectively). However, there were no strain differences in basal spermidine and spermine levels between the two. After acute stress, the PU levels in frontal cortex and hippocampus (15.99 +/- 0.45 and 14.10 +/- 0.95 nmol/g tissue, respectively) were significantly increased in SHR as compared to the non-stressed SHR (11.03 +/- 0.81 and 11.36 +/- 0.33 nmol/g tissue, respectively). In WKY rats, the PU level was significantly increased by acute stress in frontal cortex (11.68 +/- 1.12 nmol/g tissue) as compared to the non-stressed WKY (6.90 +/- 1.44 nmol/g tissue). After chronic stress, the PU levels in frontal cortex and hippocampus of SHR (12.44 +/- 0.54 and 11.34 +/- 0.66 nmol/g tissue, respectively) significantly decreased as compared to acute-stressed groups (15.99 +/- 0.45 and 14.01 +/- 0.95 nmol/g tissue, respectively). In WKY rats, after chronic stress, the PU level was significantly decreased in frontal cortex (5.73 +/- 0.36 nmol/g tissue) as compared to acute-stressed groups (11.68 +/- 1.12 nmol/g tissue). The PU levels in frontal cortex and hippocampus of acute-stressed (15.99 +/- 0.45 nmol/g tissue and 14.10 +/- 0.95 nmol/g tissue, respectively) and chronic-stressed (12.44 +/- 0.54 and 11.34 +/- 0.66 nmol/g tissue, respectively) SHR were significantly higher than acute-stressed (11.68 +/- 1.12 and 9.76 +/- 0.45 nmol/g tissue, respectively) and chronic-stressed (5.73 +/- 0.36 and 8.44 +/- 0.71 nmol/g tissue, respectively) WKY rats. The present study provides the higher basal PU level and stress-induced PU response in SHR as compared to WKY rats may be related to enhanced response of hypothalamic-pituitary-adrenocortical axis and sympathetic influence that may significantly contribute to the development of hypertension in SHR.

Acute Disease↗

PubMed2001

Ornithine decarboxylase and thymidine kinase activities and polyamine levels from selected organs of adult miniature swine receiving three concentrations of dietary menhaden oil.

Mature, female swine were randomly assigned to one of seven dietary groups. Swine in groups 1-3 were fed a cholesterol-rich diet for 55 days while the remaining groups remained on a basal swine diet. At the end of the cholesterol(Chol)-preloading period the swine in groups 1-7 were placed on menhaden oil (MO) and/or corn oil (CO) as follows: groups 1 and 4, 15% CO (control); groups 2 and 5, 0.75% MO+14.25% CO; groups 3 and 7, 15% MO; and group 6, 7.5% MO+7.5% CO. Animals were killed at the end of the approximately 6-month feeding period and portions of liver, pancreas and colon mucosa were analyzed for both ornithine decarboxylase (ODC) and thymidine kinase (TK) activity while polyamine levels were measured in the liver and pancreas. Statistical analyses were carried out by one-way and two-way ANOVA and by trend analysis. In the pancreas, the highest MO group (group 7) had significantly higher ODC levels when compared with the CO control (group 4) and the next to highest MO group (group 6) (one-way ANOVA)-all non-cholesterol preloaded groups. Using a two-way ANOVA (Chol-by-MO), liver ODC was significantly lower in the CO control when compared with the lowest and highest MO groups (groups 5 and 7, respectively), again in the non-cholesterol-preloaded animals. In the colon, the swine in the Chol-low MO group (group 2) had significantly lower TK activity than the Chol/CO control group (group 1) and Chol/Hi MO group (group 3) (one-way ANOVA) and also had significantly lower activity than all groups except the CO control (group 4) (two-way ANOVA). Liver acetylputrescine in the lowest and highest MO groups (groups 5 and 7, respectively) was significantly higher than in the CO group (group 4). Liver spermidine in the Chol-Hi MO group (group 3) was significantly higher than the Chol-Lo MO group (group 2), while the highest MO group (group 7) had a statistically higher level than the other non-cholesterol groups (groups 4-6) (one-way ANOVA). Liver spermine was significantly higher in the Chol-Hi MO group (group 3) when compared to the CO control (group 1) and the Chol-Lo MO group (group 2) (one-way ANOVA). Pancreatic putrescine in the CO control (group 4) was significantly higher than all other groups (two-way ANOVA) while spermine from the 2 Chol-MO groups (groups 2 and 3) was higher than the Chol-CO control (group 1) (one-way ANOVA). Using trend analysis, liver TK, putrescine and spermidine increased in the non-cholesterol preloaded groups with increasing dietary MO, similar to the increase seen in ODC. Thus, of the three organs studied, only liver responded to menhaden oil with changes in both ODC itself or some of its metabolic engendered products and thymidine kinase; at least for one of the parameters, ODC, change associated with dietary MO was dependent on whether the swine were preloaded with cholesterol.

Animals↗

PubMed2003

Thirty years of polyamine-related approaches to cancer therapy. Retrospect and prospect. Part 1. Selective enzyme inhibitors.

As soon as the natural polyamines (PAs), putrescine (Put), spermidine (Spd) and spermine (Spm), were recognized as ubiquitous constituents of eukaryotic cells, their involvement in growth-related processes attracted particular interest. The high activities of ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (AdoMetDC) in rapidly growing tissues and cells, particularly in tumour cells, suggested PA biosynthesis as a target for antineoplastic therapy. In the course of the years selective inhibitors have been developed for literally all enzymes of PA metabolism. Some became important as tools in the elucidation of the PA metabolic system, but only few of them were efficient as inhibitors of tumour growth. A major reason for the inefficacy of selective enzyme inhibitors as anticancer drugs is the sophistication of the system, which regulates intracellular PA pools. Selective blockade of a single enzyme induces changes of metabolism and transport, which compensate for the deficit. The selective impairment of tumour growth is in addition hampered by the ubiquitous occurrence of the PAs, their importance in normal functions of nearly all mammalian cells, and the ability or the mammalian organism to utilize exogenous (gastrointestinal) PAs. Among the inhibitors of PA-related enzymes, the ODC inactivator (R, S)-2-(difluoromethyl)ornithine (DFMO) became most famous. Although it was disappointing in most therapeutic attempts to use it as single drug, it has--based on its low toxicity--considerable potential in cancer chemoprevention, and it turned out to be a highly efficient anti-trypanosome agent. Very likely DFMO is suitable to improve the efficacy of some of the current cytotoxic drugs, and it may allow one to create new therapies in combination with other PA-directed drugs. Some of the less selective enzyme inhibitors, particularly those, which inhibit two or more enzymes of PA metabolism, appear to have had a chance to become practically useful, but they have not been developed energetically. Disregarding DFMO, the AdoMetDC inhibitor SAM486A is the only compound for which clinical trials were published. The future of this drug is unclear at present; presumably phase III clinical trials have been discontinued. One of the lessons that had to be learned from the work on selective enzyme inhibitors was that PA metabolism is a much more difficult target, than has been expected on the basis of the simplicity of the PA structures, and the simple reactions involved in their biosynthesis. In order to inhibit tumour growth several reactions or regulatory functions of PA metabolism have to be impaired at the same time. Recent efforts devoted to the development new types of anticancer drugs, which are based on the perturbation of PA metabolism by structural analogues of the natural PAs, take this message into account. These approaches are the topic of the 2nd part of this overview.

Adenosylmethionine Decarboxylase↗