Selenomethionine induces polyamine biosynthesis in regenerating rat liver tissue

Summary. Our study was undertaken to elucidate the effects of selenomethionine (SeMet) on polyamine metabolism in regenerating rat liver tissue, as useful model of rapidly growing normal tissue. We have examined the levels of spermine, spermidine and putrescine in liver tissue. At the same time we have evaluated the activities of polyamine oxidase (PAO) and diamine oxidase (DAO), the catabolic enzymes of polyamine metabolism. The obtained results suggest that polyamine levels in regenerating liver tissue, at 7^th day after two-thirds partial hepatectomy, were higher in comparison with control group. The administration of selenomethionine to hepatectomized animals during seven days, in a single daily dose of 2.5 μg/100 g body weight, increases the amount of spermine and spermidine; the level of putrescine does not change under the influence of SeMet in regenerating rat liver tissue. PAO activity is lower in regenerating hepatic tissue than in control group. Supplementation of hepatectomized animals with SeMet significantly decreases the activity of this enzyme. DAO activity was significantly higher in hepatectomized and in operated animals treated with SeMet compared to the sham-operated and control ones. The differential sensitivity observed in our model of highly proliferating normal tissue to SeMet, compared with the reported anticancer activity of this molecule is discussed.     

Altered growth and polyamine catabolism following exposure of the chocolate spot pathogen Botrytis fabae to the essential oil of Ocimum basilicum

Biomass of the fungal pathogen Botrytis fabae in liquid culture amended with two chemotypes of the essential oil of basil, Ocimum basilicum, was reduced significantly at concentrations of 50 ppm or less. The methyl chavicol chemotype oil increased the activity of the polyamine biosynthetic enzyme S-adenosylmethionine decarboxylase (AdoMetDC), but polyamine concentrations were not significantly altered. In contrast, the linalol chemotype oil decreased AdoMetDC activity in B. fabae, although again polyamine concentrations were not altered significantly. However activities of the polyamine catabolic enzymes diamine oxidase (DAO) and polyamine oxidase (PAO) were increased significantly in B. fabae grown in the presence of the essential oil of the two chemotypes. It is suggested that the elevated activities of DAO and PAO may be responsible, in part, for the antifungal effects of the basil oil, possibly via the generation of hydrogen peroxide and the subsequent triggering of programmed cell death.     

γ-氨基丁酸对低氧胁迫下甜瓜幼苗多胺代谢的影响

以‘西域一号’甜瓜为试验材料,采用营养液水培法,研究了低氧胁迫下外源添加γ-氨基丁酸(GABA)对甜瓜幼苗多胺代谢的影响.结果表明:与通气对照相比,低氧胁迫处理的甜瓜幼苗谷氨酸脱羧酶(GAD)活性和GABA含量显著提高,同时多胺合成酶活性提高诱导多胺含量显著增加,但二胺氧化酶(DAO)和多胺氧化酶(PAO)活性也显著提高;根系精氨酸脱羧酶(ADC)活性提高幅度较大,导致根系游离态腐胺含量较高,而叶片乌氨酸脱羧酶(ODC)和S-腺苷甲硫氨酸脱羧酶(SAMDC)活性提高幅度较大,导致叶片游离态亚精胺(Spd)含量较高;根系游离态DAO和PAO活性显著低于叶片,其细胞壁结合态PAO活性显著高于叶片.与低氧胁迫处理相比,低氧胁迫下外源添加GABA处理的甜瓜幼苗叶片和根系中GABA和谷氨酸含量均显著提高,而GAD活性显著降低;精氨酸、鸟氨酸、甲硫氨酸含量的提高促使多胺合成酶活性显著提高,从而诱导多胺含量显著增加,DAO和PAO活性显著降低.     

γ-氨基丁酸对低氧胁迫下甜瓜幼苗多胺代谢的影响

以‘西域一号’甜瓜为试验材料,采用营养液水培法,研究了低氧胁迫下外源添加γ-氨基丁酸(GABA)对甜瓜幼苗多胺代谢的影响.结果表明：与通气对照相比,低氧胁迫处理的甜瓜幼苗谷氨酸脱羧酶(GAD)活性和GABA含量显著提高,同时多胺合成酶活性提高诱导多胺含量显著增加,但二胺氧化酶(DAO)和多胺氧化酶(PAO)活性也显著提高;根系精氨酸脱羧酶(ADC)活性提高幅度较大,导致根系游离态腐胺含量较高,而叶片鸟氨酸脱羧酶(ODC)和S-腺苷甲硫氨酸脱羧酶(SAMDC)活性提高幅度较大,导致叶片游离态亚精胺(Spd)含量较高;根系游离态DAO和PAO活性显著低于叶片,其细胞壁结合态PAO活性显著高于叶片.与低氧胁迫处理相比,低氧胁迫下外源添加GABA处理的甜瓜幼苗叶片和根系中GABA和谷氨酸含量均显著提高,而GAD活性显著降低;精氨酸、鸟氨酸、甲硫氨酸含量的提高促使多胺合成酶活性显著提高,从而诱导多胺含量显著增加,DAO和PAO活性显著降低.     

Spermine oxidase SMO(PAOh1), Not N1-acetylpolyamine oxidase PAO, is the primary source of cytotoxic H2O2 in polyamine analogue-treated human breast cancer cell lines

The induction of polyamine catabolism and its production of H2O2 have been implicated in the response to specific antitumor polyamine analogues. The original hypothesis was that analogue induction of the rate-limiting spermidine/spermine N1-acetyltransferase (SSAT) provided substrate for the peroxisomal acetylpolyamine oxidase (PAO), resulting in a decrease in polyamine pools through catabolism, oxidation, and excretion of acetylated polyamines and the production of toxic aldehydes and H2O2. However, the recent discovery of the inducible spermine oxidase SMO(PAOh1) suggested the possibility that the original hypothesis may be incomplete. To examine the role of the catabolic enzymes in the response of breast cancer cells to the polyamine analogue N1,N1-bis(ethyl)norspermine (BENSpm), a stable knockdown small interfering RNA strategy was used. BENSpm differentially induced SSAT and SMO(PAOh1) mRNA and activity in several breast cancer cell lines, whereas no N1-acetylpolyamine oxidase PAO mRNA or activity was detected. BENSpm treatment inhibited cell growth, decreased intracellular polyamine levels, and decreased ornithine decarboxylase activity in all cell lines examined. The stable knockdown of either SSAT or SMO(PAOh1) reduced the sensitivity of MDA-MB-231 cells to BENSpm, whereas double knockdown MDA-MB-231 cells were almost entirely resistant to the growth inhibitory effects of the analogue. Furthermore, the H2O2 produced through BENSpm-induced polyamine catabolism was found to be derived exclusively from SMO(PAOh1) activity and not through PAO activity on acetylated polyamines. These data suggested that SSAT and SMO(PAOh1) activities are the major mediators of the cellular response of breast tumor cells to BENSpm and that PAO plays little or no role in this response.     

Diamine oxidase is involved in H(2)O(2) production in the chalazal cells during barley grain filling

The localization and activities of diamine oxidase (DAO, EC 1.4.3.6) and polyamine oxidase (PAO, EC 1.4.3.4) together with polyamine levels have been investigated in developing grains of barley (Hordeum vulgare L.). DAO (pH 7.5) is present mainly in vascular tissue and its neighbouring cells, namely chalazal cells and nucellar projection, while PAO (pH 6.0) is mainly localized in the chlorenchymatous cells of the crease and at the base of the vascular tissue. Activities of both these enzymes appear to be independently-regulated, as DAO activity increased steadily throughout grain development while PAO activity was higher during the early stages of grain filling, declined thereafter and again increased towards maturity. The maximum activities of DAO coincided with the maximum content of putrescine while the levels of PAO did not seem to be directly correlated with spermidine or spermine contents. Isoelectric focusing (IEF) of DAO and PAO activities revealed the presence of bands at 30 and 45 DPA. The possible involvement of DAO and PAO in the supply of H(2)O(2) to peroxidase-catalysed reactions in the chalazal cells during grain filling is discussed.     

Cell kinetics and biochemical parameters in breast cancer.

The study analyzes biochemical and cell kinetic parameters to characterize solid tumor growth in humans. The concentrations of polyamines, CEA, the thymidine labeling index (T.L.I.) and the mitotic index (M.I.) were determined on fragments of neoplastic tissue from 18 patients with breast carcinoma. Urinary polyamines were evaluated in the same patients. Two groups of patients were distinguished according to the median value of the with high T.L.I., M.I. and tissue polyamines were significantly higher than in the group with low T.L.I., whereas tissue CEA was lower, though in a not statistically significant way. Urinary polyamines showed no variations between groups. These preliminary results showed that T.L.I. levels were higher in patients who relapsed during a 4-year follow-up than in patients achieving complete remission and remaining disease free. Results concerning polyamine concentration showed that the tissue polyamine level in breast carcinoma indicated proliferative activity, but this does not seem to be valuable for current prognostic purposes.     

Polyamine metabolism in barley reacting hypersensitively to the powdery mildew fungus Blumeria graminis f. sp. hordei

Polyamine levels and activities of enzymes of polyamine biosynthesis and catabolism were examined in the barley cultivar Delibes (Ml1al + Ml(Ab)) reacting hypersensitively to the powdery mildew fungus, Blumeria graminis f. sp. hordei (race CC220). Levels of free putrescine and spermine and of conjugated forms of putrescine, spermidine and spermine were greatly increased 1–4 d following inoculation of barley with the powdery mildew. These changes in polyamine levels were accompanied by elevated activities of the polyamine biosynthetic enzymes ornithine decarboxylase (ODC), arginine decarboxylase (ADC) and S‐adenosylmethionine decarboxylase (AdoMetDC) and the polyamine catabolic enzymes diamine oxidase (DAO) and polyamine oxidase (PAO). Activities of two enzymes involved in conjugating polyamines to hydroxycinnamic acids, putrescine hydroxycinnamoyl transferase (PHT) and tyramine feruloyl‐CoA transferase (TFT) were also examined and were found to increase significantly 1–4 d after inoculation. The possibility that the increased levels of free spermine, increased polyamine conjugates, and increased DAO and PAO activities are involved in development of the hypersensitive response of Delibes to powdery mildew infection is discussed.     

Inhibition of polyamine oxidase enhances the cytotoxicity of polyamine oxidase substrates. A model study with N1-(n-octanesulfonyl)spermine and human colon cancer cells

N(1)-(n-octanesulfonyl)spermine (N(1) OSSpm) is a substrate of polyamine oxidase. It shares several properties with spermine, such as antagonism of NMDA-type glutamate receptors, calmodulin antagonism, and cytotoxicity, but it is more potent by orders of magnitude in these regards than spermine. The human colon carcinoma-derived cell line CaCo-2 was used as a model to study the toxicity of N(1) OSSpm as a function of polyamine oxidase (PAO) activity and differentiation. If the formation of hydrogen peroxide and aminoaldehyde by the PAO-catalysed reactions was prevented by selective inactivation of the enzyme with MDL 72527, cytotoxicity of N(1)OSSpm was not diminished, but on the contrary, enhanced. Exponentially growing CaCo-2 cells were considerably more sensitive to N(1)OSSpm than differentiating cells. The results suggest that cytotoxic substrates of PAO exhibit enhanced cytotoxicity in cells, if PAO activity is inhibited. Since tumour cells are known to have lower polyamine oxidase activities than their normal counterparts, it will be interesting to explore whether cytotoxic substrates of polyamine oxidase, for which N(1)OSSpm is an example, are suited to preferentially kill tumour cells.     

百合小鳞茎离体发生过程中内源多胺水平和多胺氧化酶活性的变化

以百合小鳞茎离体发生为实验系统,研究了鳞片(外植体)内多胺和多胺氧化酶活性的变化与小鳞茎发生之间的相关性。随着细胞脱分化、愈伤组织形成、小鳞茎发生等过程,内源腐胺、亚精胺和精胺的含量均失上升后下降,变化曲线为“钟形”,其含量达最高峰的时间为肉眼可见小鳞茎出现之前。多胺氧化酶活性在小鳞茎发生初期逐渐下降,6d后开始上升,并在小鳞茎突起时达最大值。由于内源多胺水平和多胺氧化酶活性的变化与小鳞茎发生有密切关系,可作为这一形态发生过程的生化指示。     

Developmental aspects of polyamine-oxidizing enzyme activities in the mouse kidney. Effects of testosterone

Summary In the present study developmental patterns of renal polyamineoxidizing enzymes polyamine oxidase (PAO) and diamine oxidase (DAO) in male and female ICR mice were demonstrated. The effects of testosterone (10g/100g body weight) on renal PAO and DAO activities were also studied. The differences between sexes in both PAO and DAO activities were most clearly expressed in the immature kidney. At the age of 20 days PAO and DAO activities were 1.52 fold (p < 0.01) and 1.75 (p < 0.02) respectively higher in male mouse kidney than in female. Maturational processes reflected in significant increases in polyamine- oxidizing enzyme activities mainly in female mouse kidney, comparable with the gain in the kidney wet weight. Our data show that testosterone is able to influence renal PAO and DAO activities in addition to the well-known stimulation of polyamine biosynthesis. The hormonal effects were sex and age dependent. The influence of testosterone on renal PAO activity was mainly age dependent. The slight stimulation of renal PAO activity observed in 20- and 50-day old mice, 24h after testosterone administration, change with a decrease in the enzyme activity at the age of 70 days. The effects of testosterone on renal DAO activity were mainly sex dependent. Testosterone caused stimulation of DAO activity with a very close magnitude (nearly twice) in female mouse kidney, independently of the age of mice. In contrast, in male mice the hormone treatment resulted in a statistically significant increase in renal DAO activity at the age of 70 days (.1.3 fold, p < 0.05) only. It could be suggested that our data indicate the different contribution of renal PAO and DAO in androgen regulation of polyamine levels, depending on sex and the stage of the postnatal development.     

Endogenous enzyme activities and polyamine levels in diverse rice cultivars depend on the genetic background and are not affected by the presence of the hygromycin phosphotransferase selectable marker

We used the polyamine biosynthetic pathway and rice as a relevant model to understand the genetic basis of variation in endogenous levels of metabolites and key enzymes involved in the pathway. Wild-type tissues and also tissues containing a commonly used selectable marker gene were employed. We detected a wide variation in levels of arginine decarboxylase activity and in the three polyamines, putrescine, spermidine and spermine, in different tissues and varieties, but this was not dependent on the presence of the selectable marker. A more-extensive profile of enzyme activities (ADC, ODC, SAMDC, DAO and PAO) and polyamine levels in different tissues was generated in two different varieties. Our results indicate that genetic background is important in terms of the basal levels of metabolites and enzyme activity, particularly in situations in which we aim to engineer metabolic pathways that are also encoded by homologous endogenous genes. We did not find any evidence that the presence of a selectable marker in any way influences enzyme activity or metabolite levels.     

Rat colon ornithine and arginine metabolism: coordinated effects after proliferative stimuli

Ornithine decarboxylase (ODC) catalyzes the first step in the polyamine biosynthetic pathway, a highly regulated pathway in which activity increases during rapid growth. Other enzymes also metabolize ornithine, and in hepatomas, rate of growth correlates with decreased activity of these other enzymes, which thus channels more ornithine to polyamine biosynthesis. Ornithine is produced from arginase cleavage of arginine, which also serves as the precursor for nitric oxide production. To study whether short-term coordination of ornithine and arginine metabolism exists in rat colon, ODC, ornithine aminotransferase (OAT), arginase, ornithine, arginine, and polyamine levels were measured after two stimuli (refeeding and/or deoxycholate exposure) known to synergistically induce ODC activity. Increased ODC activity was accompanied by increased putrescine levels, whereas OAT and arginase activity were reduced by either treatment, accompanied by an increase in both arginine and ornithine levels. These results indicate a rapid reciprocal change in ODC, OAT, and arginase activity in response to refeeding or deoxycholate. The accompanying increases in ornithine and arginine concentration are likely to contribute to increased flux through the polyamine and nitric oxide biosynthetic pathways in vivo.     

Growth,morphological and biochemical changes in oxa-spermine derivative-treated MCF-7 human breast cancer cells

The growth inhibitory properties of two oxa-spermine derivatives named compound 1 and compound 2, representatives of a novel type of polyamine derivatives, were studied. Dose-response growth inhibitory curves obtained after 48h drug exposure demonstrated the much higher cytotoxic activity of compound 1 towards MCF-7 human breast cancer cells. Further experiments with compound 1 showed that this oxa-spermine derivative exhibited considerable cytotoxicity with IC(50) values of 3.74 microM and 2.93 microM after 24h and 48h drug exposure respectively. In MCF-7 cells, after 8h drug (10 microM) exposure it caused shrinkage, chromatin condensation and nuclear fragmentation. However, no clear DNA laddering was detected in treated cells. Drug treatment provoked an increase in polyamine oxidase (PAO) activity. This enzyme is able to produce cytotoxic H(2)O(2) and 3-acetamidopropanal, catalyzing the oxidative deamination of N(1)-acetylated derivatives of spermine and spermidine to spermidine and putrescine respectively. Taken together these data demonstrate that the novel oxa-polyamine derivative compound 1 has considerable cytotoxic activity towards MCF-7 cells and indicate that an induction of PAO may be involved in its cytotoxic and apoptotic effects.     

Activation of polyamine catabolic enzymes involved in diverse responses against epibrassinolide-induced apoptosis in LNCaP and DU145 prostate cancer cell lines

Epibrassinolide (EBR) is a biologically active compound of the brassinosteroids, steroid-derived plant growth regulator family. Generally, brassinosteroids are known for their cell expansion and cell division-promoting roles. Recently, EBR was shown as a potential apoptotic inducer in various cancer cells without affecting the non-tumor cell growth. Androgen signaling controls cell proliferation through the interaction with the androgen receptor (AR) in the prostate gland. Initially, the development of prostate cancer is driven by androgens. However, in later stages, a progress to the androgen-independent stage is observed, resulting in metastatic prostate cancer. The androgen-responsive or -irresponsive cells are responsible for tumor heterogeneity, which is an obstacle to effective anti-cancer therapy. Polyamines are amine-derived organic compounds, known for their role in abnormal cell proliferation as well as during malignant transformation. Polyamine catabolism-targeting agents are being investigated against human cancers. Many chemotherapeutic agents including polyamine analogs have been demonstrated to induce polyamine catabolism that depletes polyamine levels and causes apoptosis in tumor models. In our study, we aimed to investigate the mechanism of apoptotic cell death induced by EBR, related with polyamine biosynthetic and catabolic pathways in LNCaP (AR+), DU145 (AR?) prostate cancer cell lines and PNT1a normal prostate epithelial cell line. Induction of apoptotic cell death was observed in prostate cancer cell lines after EBR treatment. In addition, EBR induced the decrease of intracellular polyamine levels, accompanied by a significant ornithine decarboxylase (ODC) down-regulation in each prostate cancer cell and also modulated ODC antizyme and antizyme inhibitor expression levels only in LNCaP cells. Catabolic enzymes SSAT and PAO expression levels were up-regulated in both cell lines; however, the specific SSAT and PAO siRNA treatments prevented the EBR-induced apoptosis only in LNCaP (AR+) cells. In a similar way, MDL 72,527, the specific PAO and SMO inhibitor, co-treatment with EBR during 24 h, reduced the formation of cleaved fragments of PARP in LNCaP (AR+) cells.     

Alternative spermidine biosynthetic route is critical for growth of Campylobacter jejuni and is the dominant polyamine pathway in human gut microbiota

The availability of fully sequenced bacterial genomes has revealed that many species known to synthesize the polyamine spermidine lack the spermidine biosynthetic enzymes S-adenosylmethionine decarboxylase and spermidine synthase. We found that such species possess orthologues of the sym-norspermidine biosynthetic enzymes carboxynorspermidine dehydrogenase and carboxynorspermidine decarboxylase. By deleting these genes in the food-borne pathogen Campylobacter jejuni, we found that the carboxynorspermidine decarboxylase orthologue is responsible for synthesizing spermidine and not sym-norspermidine in vivo. In polyamine auxotrophic gene deletion strains of C. jejuni, growth is highly compromised but can be restored by exogenous sym-homospermidine and to a lesser extent by sym-norspermidine. The alternative spermidine biosynthetic pathway is present in many bacterial phyla and is the dominant spermidine route in the human gut, stomach, and oral microbiomes, and it appears to have supplanted the S-adenosylmethionine decarboxylase/spermidine synthase pathway in the gut microbiota. Approximately half of the gut Firmicutes species appear to be polyamine auxotrophs, but all encode the potABCD spermidine/putrescine transporter. Orthologues encoding carboxyspermidine dehydrogenase and carboxyspermidine decarboxylase are found clustered with an array of diverse putrescine biosynthetic genes in different bacterial genomes, consistent with a role in spermidine, rather than sym-norspermidine biosynthesis. Due to the pervasiveness of ε-proteobacteria in deep sea hydrothermal vents and to the ubiquity of the alternative spermidine biosynthetic pathway in that phylum, the carboxyspermidine route is also dominant in deep sea hydrothermal vents. The carboxyspermidine pathway for polyamine biosynthesis is found in diverse human pathogens, and this alternative spermidine biosynthetic route presents an attractive target for developing novel antimicrobial compounds.     

Polyamine biosynthesis in the developing rabbit palate

Levels of putrescine, spermidine, and spermine and their biosynthetic enzymes, ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (SAMDC) were measured in the developing rabbit palate between day 14 and day 18 of gestation. DNA, RNA, and protein synthesis were also measured during this time period to determine if a correlation exists between polyamine biogenesis and macromolecular synthesis. ODC activity was found to be twice as high on day 14 as on the succeeding days of gestation, while SAMDC activity did not change significantly. Levels of putrescine and spermine were higher on day 14 by 22% and 30%, respectively, than levels on day 18. Spermidine concentration did not change. DNA synthesis remained relatively constant between days 14 and 18 of gestation, suggesting that there is no peak in cell proliferation during this period. RNA synthesis was elevated significantly on day 14 and protein synthesis was significantly higher on both days 14 and 16. This data indicates that there is no correlation between polyamine synthesis and cell proliferation during this period of palatal development, but polyamines could play a regulatory role in RNA and/or protein synthesis.     

Age and tissue-dependent changes of ornithine decarboxylase and s-adenosylmethionine decarboxylase activities in neural tissue and fat body of adult crickets

The activities of ornithine decarboxylase and S-adenosylmethionine decarboxylase, two key enzymes in polyamine metabolism, were determined during the first 10 days of imaginal life in the nervous tissue and the fat body of the adult cricket Acheta domesticus. The kinetic constants of the two enzymes were also determined in both tissues. Both decarboxylases presented a higher activity in fat body than in nervous tissue. In nervous tissue, the activity of the two enzymes peaked at 16 h postemergence, then slowly decreased up to day 3–4. By contrast, the enzymatic activities in fat body, low at emergence, strongly increased on day 2. Thereafter, whereas ornithine decarboxylase activity remained rather high. S-adenosyl-methionine decarboxylase activity dropped back to emergence levels by day 10. These results, examined in light of the temporal alterations of polyamine levels observed in the two tissues, demonstrate synchronous variations between polyamine contents and the enzymes involved in their biosynthesis. © 1993 Wiley-Liss, Inc.