Regulation of polyamine transport in Chinese hamster ovary cells

Control Chinese hamster ovary (CHO) cells and mutant CHO cells lacking ornithine decarboxylase activity (CHODC-) were used to study the regulation of polyamine uptake. It was found that the transport system responsible for this uptake was regulated by intracellular polyamine levels and that this regulation was responsible for the maintenance of physiological intracellular levels under extreme conditions such as polyamine deprivation or exposure to exogenous polyamines. Polyamine transport activity was enhanced by decreases in polyamine content produced either by inhibition of ornithine decarboxylase with alpha-difluoromethylornithine in CHO cells or via polyamine starvation of CHODC- cells. The provision of exogenous polyamines resulted in rapid and large increases in intracellular polyamine content followed by decreased polyamine transport activity. Soon after this decrease in uptake activity, intracellular polyamine levels then fell to near control values. Cells grown in the presence of exogenous polyamines maintained intracellular polyamine levels at values similar to those of control cells. Protein synthesis was necessary for the increase in transport in response to polyamine depletion, but appeared to play no role in decreasing polyamine transport. Bis(ethyl) polyamine analogues mimicked polyamines in the regulation of polyamine transport but this process was relatively insensitive to regulation by methylglyoxal bis(guanylhydrazone), a spermidine analogue known to enter cells via this transport system and to accumulate to very high levels.     

Polyamine Oxidase Activity in Sera of Depressed and Schizophrenic Patients after ECT Treatment

High level of polyamine oxidase activity is detected in sera of depressed as well as in schizophrenic patients. ECT treatment of depressed and schizophrenic patients reduced significantly the level of polyamine oxidase activity in their sera. After ECT treatment, clinically improved depressed and schizophrenic subjects were found to have sera polyamine oxidase activity not significantly differ from that of normal subjects. Possible biochemical mechanisms, which link polyamine oxidase activity, schizophrenia, depression and ECT effect are discussed here.     

Bis(benzyl)polyamine analogs as novel substrates for polyamine oxidase

N,N'-Bis(benzyl)polyamine analogs were found to be substrates for highly purified polyamine oxidase. Metabolism of these analogs was apparently dependent on molecular O2 and resulted in the formation of benzaldehyde, H2O2, and a polyamine analog with free terminal amines. The debenzylation reaction was optimal between pH 9 and 10, identical to the pH optimum for polyamine oxidase activity when N1-acetylspermine was used as the substrate. On a molecular sieve column the debenzylating activity co-eluted with N1-acetylspermine oxidizing activity, at an apparent molecular mass of approximately 65 kDa. The purified enzyme also appeared to have a molecular mass of approximately 65 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Debenzylation of the bis(benzyl)polyamines was competitively inhibited by N1-acetylspermine and N1-acetylspermidine. The specific irreversible inhibitor of polyamine oxidase, N1,N4-bis(buta-2,3-dienyl)butanediamine also inhibited the debenzylation, whereas inhibitors of diamine and monoamine oxidases did not. The evolution of benzaldehyde from bis(benzyl)polyamine analogs by polyamine oxidase allowed the development of a simple rapid spectrophotometric assay for use in the measurement of polyamine oxidase activity in partially purified tissue or cell extracts. Further, metabolism of a bis(benzyl)polyamine analog by polyamine oxidase was found to be an important element in the growth inhibitory properties of the compound in a mouse model of malaria.     

Effects of dietary polyamines and clofibrate on metabolism of polyamines in the rat

The activities of catalase, polyamine oxidase, diamine oxidase, ornithine decarboxylase, and peroxisomal β-oxidation were assayed in homogenates from liver and small intestinal mucosa of rats which had been fed either a diet very low in polyamines or a diet containing five times the levels of dietary polyamines (putrescine, spermine, and spermidine) found in a standard rat diet. In rats fed the high polyamine diet, hepatic activities of catalase and polyamine oxidase were significantly decreased. Levels of the other activities were unchanged, except that intestinal ornithine decarboxylase was decreased. In rats treated simultaneously with clofibrate, the high polyamine diet restored activities of catalase, ornithine decarboxylase, and polyamine oxidase back to levels found in rats fed the low polyamine diet. The expected increase in activity of peroxisomal β-oxidation was observed, although this was somewhat diminished in rats fed the high polyamine diet. Intestinal diamine oxidase activity was stimulated by clofibrate, particularly in rats fed the high polyamine diet. For the duration of the experiment (20 days), levels of putrescine, spermine, and spermidine in blood remained remarkably constant irrespective of treatment, suggesting that polyamine homeostasis is essentially independent of dietary supply of polyamines. It is suggested that intestinal absorption/metabolism of polyamines is of significance in this respect. Treatment with clofibrate appeared to alter polyamine homeostasis.     

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.     

Changes in polyamine-oxidizing capacity of peroxisomes under various physiological conditions in rats

Rat liver peroxisomal polyamine oxidase activity was determined under various physiological conditions by using the peroxidase method with phenol and 4-aminoantipyrine. N1-Acetylpolyamines such as N1-acetylspermine and N1-acetylspermidine were better substrates than the free polyamines. The polyamine oxidase activity in rat peroxisomes increased significantly when cell proliferation was high. The activity began to appear in fetal liver at the 16th approximately 18th day of pregnancy and peaked in neonatal liver on the first day (approx. 1.7-times higher than in adult liver). In regenerating rat liver, only polyamine oxidase activity among the peroxisomal enzymes tested was increased considerably 12 h after partial hepatectomy (approx. 2.8-fold over the control liver). Finally, the enzyme activity was significantly increased by administration of clofibrate, a peroxisome proliferator, which also causes hepatomegaly. In all cases, the increase in polyamine oxidase activity was not more than 3-fold. Since the level of polyamine oxidase activity in the normal liver is more than adequate in relation to the level of the substrates, the slight but significant increase under conditions of cell proliferation may have a role in modulating levels of polyamines in the proliferating liver tissue.     

Regulation of ornithine decarboxylase and polyamine import by hypoxia in pulmonary artery endothelial cells

In rat lung and cultured lung vascular cells, hypoxia decreases ornithine decarboxylase (ODC) activity and increases polyamine import. In this study, we used rat cultured pulmonary artery endothelial cells to explore the mechanism of hypoxia-induced reduction in ODC activity and determined whether this event was functionally related to the increase in polyamine import. Two strategies known to suppress proteasome-mediated ODC degradation, lactacystin treatment and use of cells expressing a truncated ODC incapable of interacting with the proteasome, prevented the hypoxia-induced decrease in ODC activity. Interestingly, though, cellular abundance of the 24-kDa antizyme, a known physiological accelerator of ODC degradation, was not increased by hypoxia. These observations suggest that an antizyme-independent ODC degradation pathway contributes to hypoxia-induced reductions of ODC activity. When reductions in ODC activity in hypoxia were prevented by the proteasome inhibitor strategies, hypoxia failed to increase polyamine transport. The induction of polyamine transport in hypoxic pulmonary artery endothelial cells thus seems to require decreased ODC activity as an initiating event.     

Differences in polyamine metabolism between carcinomatous and uninvolved human breast tissues

The polyamine biosynthetic enzymes ODC and SAMDC show higher activity in carcinomatous human breast tissue than in uninvolved tissue of the same breast; the interconversion enzyme PAO shows significantly lower activity in carcinomatous than uninvolved tissue. The polyamine metabolism in carcinomatous human breast tissue thus appears to differ from that in uninvolved tissue. Intracellular polyamine concentrations, particularly spermine, are high in carcinomatous tissue. This increase and that of the biosynthetic enzymes suggest that a higher polyamine concentration is needed for carcinomatous cell growth. If this is the case, the lower PAO activity in carcinomatous tissue may be explained as a mechanism that conserves the high intracellular polyamine concentration.     

Characterization of the inducible polyamine transporter in bovine lymphocytes

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

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.     

TATA-binding protein-associated factor 7 regulates polyamine transport activity and polyamine analog-induced apoptosis

Identification of the polyamine transporter gene will be useful for modulating polyamine accumulation in cells and should be a good target for controlling cell proliferation. Polyamine transport activity in mammalian cells is critical for accumulation of the polyamine analog methylglyoxal bis(guanylhydrazone) (MGBG) that induces apoptosis, although a gene responsible for transport activity has not been identified. Using a retroviral gene trap screen, we generated MGBG-resistant Chinese hamster ovary (CHO) cells to identify genes involved in polyamine transport activity. One gene identified by the method encodes TATA-binding protein-associated factor 7 (TAF7), which functions not only as one of the TAFs, but also a coactivator for c-Jun. TAF7-deficient cells had decreased capacity for polyamine uptake (20% of CHO cells), decreased AP-1 activation, as well as resistance to MGBG-induced apoptosis. Stable expression of TAF7 in TAF7-deficient cells restored transport activity (55% of CHO cells), AP-1 gene transactivation (100% of CHO cells), and sensitivity to MGBG-induced apoptosis. Overexpression of TAF7 in CHO cells did not increase transport activity, suggesting that TAF7 may be involved in the maintenance of basal activity. c-Jun NH2-terminal kinase inhibitors blocked MGBG-induced apoptosis without alteration of polyamine transport. Decreased TAF7 expression, by RNA interference, in androgen-independent human prostate cancer LN-CaP104-R1 cells resulted in lower polyamine transport activity (25% of control) and resistance to MGBG-induced growth arrest. Taken together, these results reveal a physiological function of TAF7 as a basal regulator for mammalian polyamine transport activity and MGBG-induced apoptosis.     

Biosynthesis of long-chain polyamines by crenarchaeal polyamine synthases from Hyperthermus butylicus and Pyrobaculum aerophilum

Polyamines are ubiquitously present in all organisms. In addition to the common polyamines, thermophilic archaea synthesize long-chain polyamines. In the present study polyamine synthases from Hyperthermus butylicus and Pyrobaculum aerophilum were cloned and their substrate specificity was analyzed. The polyamine synthase HbSpeE II from H. butylicus synthesized long-chain polyamines with high activity using the same mechanism that is used by a wide range of organisms to synthesize common polyamines, in which the aminopropyl residue derives from decarboxylated S-adenosylmethionine. This is the first polyamine synthase described that synthesizes a polyamine longer than a tetramine with high activity.     

Feedback repression of polyamine uptake into mammalian cells requires active protein synthesis.

Two mammalian cell lines, rat hepatoma (HTC) and Chinese hamster ovary (CHO), were fed 10 to 50 microM spermidine while changes were monitored in intracellular polyamine levels and spermidine uptake activity. Normal feedback control preventing excessive polyamine uptake was found to be completely blocked by the addition of inhibitors of protein synthesis at the time of polyamine exposure. Under these conditions the cells accumulated abnormally high, toxic concentrations of spermidine. Further, continuous protein synthesis was needed to maintain repression of polyamine transporter proteins that had been inhibited previously by normal or elevated intracellular polyamines. These results suggest that a major factor in the regulation of polyamine uptake is the rapid, reversible inactivation of existing polyamine carrier molecules by an unstable protein whose synthesis is stimulated by intracellular polyamines.     

Polyamine auxotrophs of Saccharomyces cerevisiae.

Strains of yeast have been constructed that are unable to synthesize ornithine and are thereby deficient in polyamine biosynthesis. These strains were used to develop a protocol for isolation of mutants blocked directly in polyamine synthesis. There were seven mutants isolated that lack ornithine decarboxylase activity; these strains exhibited greatly decreased pool levels of putrescine, spermidine, and spermine when grown in the absence of polyamines. Three of the mutants lack S-adenosylmethionine decarboxylase activity; polyamine limitation of a representative mutant resulted in an accumulation of putrescine and a decrease in spermidine and spermine. When the mutants were cultured in the absence of polyamines, a continuously declining growth rate was observed.     

The role of polyamine reutilization in depletion of cellular stores of polyamines in non-proliferating tissues

It was known from previous work that specific inhibition of neither ornithine decarboxylase activity nor polyamine oxidase activity produces spermidine depletion by more than 20% in non-growing organs, which are in a steady state with regard to polyamine metabolism. Combined treatment with inactivators of both ornithine decarboxylase and polyamine oxidase for a prolonged time caused, however, a gradual decrease of spermidine levels in liver, kidney and brain of mice by 50% and more. The method is in accordance with the previously suggested role of polyamine interconversion. Inhibition of polyamine oxidase prevents the reutilization for de novo polyamine biosynthesis of putrescine and spermidine, which are formed by oxidative splitting of N1-acetylspermine and N1-acetylspermidine, respectively, and the ornithine decarboxylase inhibitor prevents the compensatory increase of putrescine from ornithine. The findings are further evidence for the physiological significance of polyamine reutilization.     

Changes in polyamine metabolism during glucocorticoid-induced programmed cell death in mouse thymus

When mice are injected with dexamethasone, cortical thymocytes are deleted through programmed cell death (PCD). We have used this in vivo model system to investigate the kinetics of PCD and cell proliferation in relation to polyamine metabolism for 16 h after injection of dexamethasone. As a marker for PCD, we used the appearance of a sub-G(1)peak in the DNA histogram. When a sub-G(1)peak appeared at 4 h after dexamethasone treatment, the activity of the polyamine catabolic enzyme spermidine/spermine N(1)-acetyltransferase (SSAT) was significantly increased and the activity of the polyamine biosynthetic enzyme S-adenosylmethionine decarboxylase (AdoMetDC) was significantly decreased compared to the activities found in the thymi of control mice. Despite the significant changes in the activities of SSAT and AdoMetDC, the only change in the polyamine pool during the experimental period was that of putrescine. Presumably the complexity of this in vivo system masks changes in the spermidine and spermine pools that were expected in relation to the increased SSAT activity and decreased AdoMetDC activity.     

Effects of glucocorticoids on polyamine metabolism in liver and spleen of guinea pig during sensitization

Summary. Glucocorticoids are potent anti-inflammatory and immunosuppressive agents. As endogenous inhibitors of cytokine synthesis, glucocorticoids suppress immune activation and uncontrolled overproduction of cytokines, preventing tissue injury. Also, polyamine spermine is endogenous inhibitor of cytokine production (inhibiting IL-1, IL-6 and TNF synthesis). The idea of our work was to examine dexamethasone effects on the metabolism of polyamines, spermine, spermidine and putrescine and polyamine oxidase activity in liver and spleen during sensitization of guinea pigs. Sensitization was done by application of bovine serum albumin with addition of complete Freund’s adjuvant. Our results indicate that polyamine amounts and polyamine oxidase activity increase during immunogenesis in liver and spleen. Dexamethasone application to sensitized and unsensitized guinea pigs causes depletion of polyamines in liver and spleen. Dexamethasone decreases polyamine oxidase activity in liver and spleen of sensitized guinea pigs, increasing at the same time PAO activity in tissues of unsensitized animals.     

Synthesis and cytotoxic activities of usnic acid derivatives

Nine usnic acid-amine conjugates were evaluated on murine and human cancer cell lines. The polyamine derivatives showed significant cytotoxicity in L1210 cells. Their activities appeared to be independent of the polyamine transport system (PTS). Indeed, their activities were similar in chinese hamster ovary (CHO) and in the PTS deficient CHO-MG cells. In addition, alpha-difluoromethylornithine, an ornithine decarboxylase inhibitor known to indirectly enhance the activity of the PTS and consequently increase the cytotoxicity of cytotoxic drugs entering cells via the PTS, had no effect on the activity of the polyamine derivatives. The more active derivative (1,8-diaminooctane derivative) displayed similar activities on all cancer cell lines studied and induced apoptosis.     

Uptake of polyamines by the unicellular green alga Chlamydomonas reinhardtii and their effect on ornithine decarboxylase activity

Uptake of exogenous polyamines by the unicellular green alga Chlamydomonas reinhardtii and their effects on polyamine metabolism were investigated. Our data show that, in contrast to mammalian cells, Chlamydomonas reinhardtii does not contain short-living, high-affinity polyamine transporters whose cellular level is dependent on the polyamine concentration. However, exogenous polyamines affect polyamine metabolism in Chlamydomonas cells. Exogenous putrescine caused a slow increase of both putrescine and spermidine and, vice versa, exogenous spermidine also led to an increase of the intracellular levels of both spermidine and putrescine. No intracellular spermine was detected under any conditions. Exogenous spermine was taken up by the cells and caused a decrease in their putrescine and spermidine levels. As in other organisms, exogenous polyamines led to a decrease in the activity of ornithine decarboxylase, a key enzyme of polyamine synthesis. In contrast to mammalian cells, this polyamine-induced decrease in ornithine decarboxylase activity is not mediated by a polyamine-dependent degradation or inactivation, but exclusively due to a decreased synthesis of ornithine decarboxylase. Translation of ornithine decarboxylase mRNA, but not overall protein biosynthesis is slowed by increased polyamine levels.     

Topoisomerase II is nonfunctional in polyamine-depleted cells.

The polyamines-putrescine, spermidine, and spermine-are essential for normal cell proliferation. Polyamine depletion affects DNA structure and synthesis. Topoisomerase II (topo II) is also necessary for normal cell proliferation, and it has been shown in vitro that polyamines may affect topo II activity. In order to investigate the effect of polyamine depletion on topo II activity, we treated Chinese hamster ovary cells with either alpha-difluoromethylornithine (DFMO) or 4-amidinoindan-1-one-2'-amidinohydrazone (CGP 48664), which are polyamine biosynthesis inhibitors. Treatment with the topo II inhibitor etoposide results in DNA strand breaks only if there is active topo II in the cells. By quantitating DNA strand breaks after etoposide treatment using single cell gel electrophoresis, we were able to estimate intracellular topo II activity. We also quantitated topo II activity in crude nuclear extracts from control and polyamine biosynthesis inhibitor-treated cells. Using single cell gel electrophoresis, we noted a clear decrease in the function of topo II in polyamine biosynthesis inhibitor-treated cells, as compared with untreated control cells. However, the topo II activity in crude nuclear extracts did not differ significantly in control versus polyamine biosynthesis inhibitor-treated cells. Taken together, these results indicate that although the function of topo II in polyamine-depleted cells was impaired, topo II remained functional in an in vitro assay. Using the single cell gel electrophoresis assay, we also found that spermine depletion itself caused DNA strand breaks.