Regulation of polyamine acetylation and efflux in human cancer cells.
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Acetylation↗
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Polyamine metabolism.
Currently, two major pathways are distinguished along which the polyamines are metabolized: the interconversion pathway and the so-called terminal polyamine catabolism. In vertebrates, the interconversion pathway is a cyclic process which controls polyamine turnover. In conjunction with polyamine transport, it regulates intracellular polyamine homeostasis. In vertebrates, putrescine, the precursor of spermidine and spermine, is exclusively formed by decarboxylation of ornithine--as far as de novo synthesis is concerned. Spermidine and spermine synthase form spermidine from putrescine, and spermine from spermidine, by transfer of aminopropyl residues from decarboxylated S-adenosylmethionine. In the catabolic branch of the interconversion cycle, spermine is degraded to spermidine, and spermidine to putrescine. The first step in this sequence is acetylation in the N1 position. This is followed by oxidative splitting of the acetylated polyamines, whereby the aminopropyl residues which originated from decarboxylated S-adenosylmethionine are removed. The enzyme catalyzing this step is an FAD-dependent oxidase (polyamine oxidase). Ornithine decarboxylase, S-adenosylmethionine decarboxylase, and acetyl CoA:polyamine N1-acetyltransferase are highly regulated, inducible enzymes with a high turnover rate. Depending on the physiological situation, each of these enzymes may become rate limiting. Terminal polyamine catabolism is catalyzed by Cu2(+)-dependent amine oxidases, of which only diamine oxidase has been well defined. By oxidative deamination of a primary amino group, each intermediate of the interconversion cycle can be transformed into an aldehyde, which is further oxidized to an amino acid or a gamma-lactam. The products of the terminal catabolism as well as the acetylated polyamines are urinary excretory products. In addition to intracellularly synthesized polyamines, polyamines from various tissues and from exogenous sources (such as the gastrointestinal tract) may be utilized by those tissues which have a high demand. Polyamines play a paramount role in growth processes. In order to control growth (for example of tumors), it is necessary to block all major polyamine sources. If only one source is blocked, the remaining sources are usually capable of furnishing sufficient polyamines to support growth processes.
Acetylation↗
Regulation of cyclin B1 by estradiol and polyamines in MCF-7 breast cancer cells.
Recent studies have identified a family of proteins called cyclins that control cell cycle. Among these proteins, cyclin B synthesis and degradation are necessary and sufficient to cause a Xenopus egg cell-free system to oscillate between S and M. To understand the link between hormonal regulation of cell growth and the expression of B-type cyclins, we studied the effect of estradiol on cyclin B1 mRNA in a hormone-responsive breast cancer cell line, MCF-7. Cells were synchronized at G1 by isoleucine starvation, and estradiol was added along with the removal of cell cycle block. Flow cytometric analysis showed 81 +/- 7% cells in G1 after 30 h of isoleucine starvation. Significant population of cells progressed to S by 16 h after the addition of estradiol, whereas a comparable transition occurred in control cells by 36 h only. In cells progressing from G1-->S-->G2-->M under the influence of estradiol, there was a significant increase in cyclin B1 mRNA at 30 and 36 h, consistent with the accumulation of this cyclin in G2/M. In addition, we found that cyclin B1 mRNA degradation occurred early in G1, and this process was accelerated by estradiol. At 2 h after removal of the isoleucine block, there was a 40% reduction in the level of cyclin B1 mRNA in estradiol-treated cells compared to untreated controls. Cyclin B1 protein degradation followed a similar pattern, as determined by Western blots using a monoclonal anti-cyclin B1 antibody. Since previous studies suggested a polyamine pathway in the mechanism of action of estradiol, we questioned whether polyamines are important in controlling the level of cyclin B1 mRNA. Treatment of synchronized cells with the polyamine biosynthetic inhibitor, difluoromethylornithine attenuated cyclin B1 mRNA degradation in the presence of estradiol. This process was mostly reversed by exogenous putrescine and spermidine but not by putrescine homologues. Collectively, these data suggest that the mechanism of cell growth regulation by estradiol in MCF-7 cells includes alterations in cyclin B1 mRNA. Our data also indicate molecular pathways for the action of polyamines in estrogenic control of cell cycle.
Biogenic Polyamines↗
Chemical highlights of polyamine transport.
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Animals↗
Spider toxins affecting glutamate receptors: polyamines in therapeutic neurochemistry.
Polyamine amide toxins obtained from venous of spiders and wasps interact selectively with ionotropic glutamate receptors (GLU-R) of vertebrate central nervous systems. The sites and modes of action of these polyamine amide toxins are reviewed with particular reference to their structure-activity relationships. Qualitatively, their effects on GLU-R are identical to those exerted by polyamines such as spermine, but the polyamine amides are more potent. These compounds (a) potentiate and (b) antagonize GLU-R, the latter arising through open channel block. For the N-methyl-D-aspartate receptor this non-competitive antagonism probably arises through binding of toxin to the Mg2+ site(s) located in the channel gated by this receptor. Similarities and differences between GLU-R in vertebrates and in invertebrates with respect to their interactions with polyamines and polyamine amide toxins are discussed. In both groups the low specificity of these compounds is illustrated by their antagonism at nicotinic acetylcholine receptors in addition to GLU-R. Electrophysiological studies, including those employing Xenopus oocytes, are reviewed and future prospects for the use of polyamine amides in therapy are discussed.
Animals↗
Control of ornithine decarboxylase activity by polyamines and absence of antizyme in Tetrahymena.
1. In cells of Tetrahymena pyriformis and thermophila, ODC activity was significantly suppressed but ODC decay was not stimulated by putrescine. 2. Free antizyme and ODC-antizyme complex were both not detected in extracts of cells of T. pyriformis treated with putrescine. 3. It was concluded that in Tetrahymena, unlike vertebrate cells, ODC is not subject to polyamine-induced destabilization mediated by antizyme.
Animals↗
Combined action of inhibitors of polyamine biosynthetic pathway with a known antimalarial drug chloroquine on Plasmodium falciparum.
Difluoromethylornithine (alpha-DFMO), a specific enzyme activated inhibitor of ornithine decarboxylase activity, a bis (benzyl) polyamine analogue (MDL 27695) and chloroquine resulted in a dose-dependent inhibition of the parasite. The IC50 value of alpha-DFMO, MDL 27695 and chloroquine was 1.85 mM, 2.0 microM and 23 ng ml-1 respectively. The combined action of MDL 27695 (3 microM) and chloroquine (5 ng ml-1) on P. falciparum growth showed near additive effect, whereas the combination of alpha-DFMO (1.0 mM) and chloroquine (5 ng ml-1) was more than the effect of each drug individually but not additive. Similarly the combined effect of MDL 27695 (3 microM) and alpha-DFMO (1.0 mM) on P. falciparum growth was not additive. The effect of these inhibitors alone and in combination on polyamine biosynthesis is also reported.
Animals↗
Methylthioadenosine phosphorylase, a gene frequently codeleted with p16(cdkN2a/ARF), acts as a tumor suppressor in a breast cancer cell line.
The human methylthioadenosine phosphorylase (MTAP) gene is located on 9p21 and is frequently homozygously deleted, along with p16(cdkN2a/ARF), in a wide variety of human tumors and human tumor-derived cell lines. The function of MTAP is to salvage methylthioadenosine, which is produced as a byproduct of polyamine metabolism. We have reintroduced MTAP into MCF-7 breast adenocarcinoma cells and have examined its effect on the tumorigenic properties of these cells. MTAP expression does not affect the growth rate of cells in standard tissue culture conditions but severely inhibits their ability to form colonies in soft agar or collagen. In addition, MTAP-expressing cells are suppressed for tumor formation when implanted into SCID mice. This suppression of anchorage-independent growth appears to be because of the enzymatic activity of MTAP, as a protein with a missense mutation in the active site does not exhibit this phenotype. MTAP expression causes a significant decrease in intracellular polyamine levels and alters the ratio of putrescine to total polyamines. Consistent with this observation, the polyamine biosynthesis inhibitor alpha-difluoromethylornithine inhibits the ability of MTAP-deficient cells to form colonies in soft agar, whereas addition of the polyamine putrescine stimulates colony formation in MTAP-expressing cells. These results indicate that MTAP has tumor suppressor activity and suggest that its effects may be mediated by altering intracellular polyamine pools.
Biogenic Polyamines↗
[Effect of inhibition of polyamine biosynthesis on the growth and telomerase activity of human laryngeal carcinoma cells Hep-2].
OBJECTIVE: To observe the effect of difluoromethylornithine (DFMO), an inhibitor of polyamine biosynthesis, on the growth and telomerase activity of human laryngeal carcinoma cells Hep-2, and try to find a new clue to search for the mechanism of reversion of tumor malignant phenotype via polyamine biosynthesis inhibition. METHODS: The growth characteristics of Hep-2 cells treated with DFMO were studied by morphological observation, cell growth curve and FCM analysis. Telomerase activity was detected by Telomerase Repeat Amplification Protocol (TRAP). RESULTS: The inhibition of cell proliferation, a marked increase in G1 and a decrease in S phase populations in the cell cycle of 2.5 mmol/L or 5 mmol/L DFMO treated Hep-2 cells were demonstrated; The cell apoptosis was shown by cell morphology and apoptosis peak in FCM; The telomerase activity of the treated cells was suppressed, while these changes were prevented by provision of exogenous putrescine(Pu) along with DFMO. CONCLUSION: Inhibition of polyamine biosynthesis could result in the proliferation inhibition and apoptosis induction of Hep-2 cells, which was associated with suppressed telomerase activity. It is suggested that inactivation of telomerase would be one of the important molecular events in reversion of tumor malignant phenotype via inhibition of polyamine biosynthesis.
Antineoplastic Agents↗
alpha-Difluoromethylornithine alters calcium signaling in platelet-derived growth factor-stimulated A172 brain tumor cells in culture.
alpha-Difluoromethylornithine (DFMO), an irreversible inhibitor of the polyamine biosynthetic enzyme ornithine decarboxylase, inhibits the growth of brain tumor cell lines and is undergoing clinical trials as a treatment for brain tumors. Platelet-derived growth factor (PDGF) is thought to regulate the growth and development of precursors of both normal and neoplastic astrocytic cells; calcium signaling is thought to play a role in the transduction of PDGF signals. Using laser fluorescence image cytometry, flow cytometry, and spectrofluorometry, we studied the effect of DFMO on the calcium signals induced by PDGF in A172 human glioblastoma cells. Four days of treatment with 5 mM DFMO substantially shortened PDGF-induced calcium signals. The effect was reversed more than 10 h but less than 24 h after putrescine treatment, even though polyamines were repleted 4 h after putrescine and spermidine were added. DFMO did not substantially affect intracellular calcium release or the timing of the opening and closing of plasma membrane calcium channels. These findings support the notion that calcium signaling may be a target for inhibitors of polyamine metabolism.
Biogenic Polyamines↗
Antiproliferative effect of spermine depletion by N-cyclohexyl-1,3-diaminopropane in human breast cancer cells.
Spermine is often the most abundant polyamine in human tumors such as breast carcinomas. However, its specific role in tumor biology is still uncertain, since inhibitors of ornithine decarboxylase such as alpha-difluoromethylornithine depress cell growth while leaving spermine content mostly unaffected. We have assessed the specific role of spermine in breast cancer cell growth using N-cyclohexyl-1,3-diaminopropane (C-DAP), a potent spermine synthase inhibitor. In ZR-75-1 cells, C-DAP decreased net cell growth after 14 days by 65% at 50 microm, with an IC50 of about 5 microM, and was about 10 times more potent than N-(n-butyl)-1,3-diaminopropane, another spermine synthase inhibitor. C-DAP acted as a specific inhibitor of spermine biosynthesis, since (a) it depleted spermine content while causing an equal or greater accumulation of spermidine on a molar basis, (b) it rapidly induced S-adenosylmethionine decarboxylase activity and the accumulation of its products due to relief of spermine-dependent inhibition of enzyme expression, and (c) exogenous spermine (1 microM) completely reversed C-DAP-induced growth inhibition. C-DAP and related compounds were accumulated, at least in part, through a mechanism distinct from the polyamine transport system, while also blocking putrescine and spermidine uptake with various potencies. Reversibility of C-DAP-induced growth inhibition by exogenous spermine was progressively lost on prolonged treatment, in association with marked morphological changes. In 4 different human breast cancer cell lines (ZR-75-1, T47-D, MCF-7, and MDA-MB-231), relative growth sensitivity to C-DAP was inversely related to the extent of spermidine accumulation caused by spermine synthase inhibition, suggesting that spermidine overaccumulation can functionally replace spermine. Interestingly, C-DAP strongly potentiated growth inhibition caused by alpha-difluoromethylornithine in all cell lines tested by preventing conversion of residual spermidine to spermine, indicating that spermine synthesis limits alpha-difluoromethylornithine action and that under some critical threshold, spermidine cannot fulfill cellular needs for spermine. Thus, spermine plays specific and important functions in breast tumor growth, and spermine synthase inhibitors could markedly improve the therapeutic effectiveness of existing polyamine depletion strategies, especially in spermine-rich tumors.
Biogenic Polyamines↗
Inhibition of voltage-activated K+ currents in cultured sensory neurons by the polyamine spider toxin argiotoxin-636 may involve a polyamine transporter and an intracellular site of action.
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Action Potentials↗
Polyamine uptake by vascular endothelial cells does not alter intracellular pH.
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Biogenic Polyamines↗
JAR human placental choriocarcinoma cells actively synthesize, take up and release polyamines.
Uptake of polyamines has been investigated extensively in many cells, but not in placenta, where the polyamine-polyamine oxidase system is supposed to have an immunoregulatory function in pregnancy. Due to the importance of the transfer in this tissue, we have started this study. JAR human placental choriocarcinoma cells in monolayer at confluency were used as a model for measuring the key enzymes of polyamine synthesis and interconversion, rate of uptake and efflux, and the polyamine content. Polyamines were taken up by JAR cells and released by an independent mechanism. Ornithine decarboxylase and spermidine acetyltransferase activities and the rate of transport in and out of the cell were much higher than in other cells, such as L1210 cells. However the systems used for uptake and release appear in many respects to be similar to those observed in L1210 cells, but different from others. The uptake appears to be regulated by an inhibitory protein. Moreover, protein kinase C appears to be involved in the process. The efflux also is regulated as in L1210 cells, through control of H+ and Ca2+ concentration. In conclusion, this study shows that, in JAR cells, ornithine decarboxylase and spermidine acetyltransferase activities were much higher than in other cells, and so was the rate of transport in and out of the cells. As a result, a much higher polyamine content was observed.
Animals↗
Ectomycorrhizal fungal species and strains differ in their ability to produce free and conjugated polyamines.
Production of free and conjugated polyamines by one strain of Laccaria proxima (Boud.) Maire, three strains (H, O, K) of Paxillus involutus (Batsch) Fr., and one strain of Pisolithus tinctorius was studied in vitro. Spermidine (Spd) was the main polyamine in the 4-week-old mycelium of all the fungi. It was mainly present in the free form, but it also occurred in conjugated forms. Paxillus involutus strain H released large amounts of free putrescine (Put), and the Pisolithus tinctorius released a compound probably related to cadaverine (Cad). On the other hand, these two fungi contained less conjugated polyamines than the other fungi. In addition to the amounts, the forms (perchloric acid soluble and insoluble) of conjugated polyamines in the mycelium varied between species and strains. L. proxima contained nearly as much insoluble conjugated Spd as free Spd, whereas Paxillus involutus strains O and K contained relatively large amounts of soluble conjugated Spd. The results suggest that ectomycorrhizal fungal species and strains differ in their ability and need to produce conjugated polyamines. The small amounts of soluble conjugated polyamines found in the culture filtrates indicate that some specific conjugated polyamines may be involved in polyamine translocation across the plasma membrane.
Agaricales↗
An evaluation of the role of polyamines in different models of kidney hypertrophy in mice.
The role of ornithine decarboxylase (ODC) and polyamines in kidney hypertrophy is controversial. Since part of this controversy could be related to differences in the model system used by the different authors, we studied the changes in renal ODC and polyamines in six different models of kidney hypertrophy in mice, including compensatory renal hypertrophy produced by unilateral nephrectomy, experimental diabetes, potassium depletion and treatment with hormones such as testosterone, thyroxine and fluorocortisone. Only in the case of renal hypertrophy produced by testosterone administration was there a significant increase in ODC activity and putrescine content in the kidneys. However, the concomitant treatment with difluoromethylornithine (DFMO), an irreversible inhibitor of ODC, as a 2% solution in the drinking water completely abolished the increase of renal ODC, but the kidney weights increased and other androgenic effects, such as the induction of renal beta-glucuronidase, were not affected. Moreover, DFMO-treatment did not prevent the kidney enlargement produced in other types of hypertrophy, even in the cases associated with hyperplasia. The present results support the premise that, at least in mice, the increase in ODC activity and polyamine biosynthesis is not required for kidney growth, and also that in most cases renal enlargement is not accompanied by any increase in the polyamine content.
Alloxan↗
Polyamine analogues as anticancer drugs.
Just over 30 years ago, the late Diane Russell published the first in a series of papers linking polyamines and cancer. These early studies led to a flurry of research activity in the polyamine field that continues to this day attempting to identify a role for the polyamines in cancer development, treatment and/or prevention. The recognition that polyamines are critical for the growth of cancer cells, and consequently the identification of their metabolic pathways as a target for therapeutic intervention, led to the development of a number of useful inhibitors of polyamine biosynthesis. Arguably the most significant addition to the polyamine field in the last 30 years was the synthesis of alpha-difluoromethylornithine (DFMO), which is being tested currently as a cancer chemopreventative agent in man and is used also as a highly effective trypanocidal agent. Although an extremely useful tool experimentally, DFMO has been disappointing in clinical trials with little therapeutic efficacy. Despite this setback, the polyamine pathway is still considered a viable target for chemotherapeutic intervention. This has led to the development of the polyamine analogues as multifunctional inhibitors that will produce inhibition of tumour cell growth, polyamine depletion and optimum therapeutic efficacy.
Antineoplastic Agents↗
Decrease in polyamines with aging and their ingestion from food and drink.
Changes in polyamine levels during aging were measured in 3-, 10- and 26-week-old female mice. The level of polyamines in pancreas, brain, and uterus was maintained during these periods. The level of spermidine slightly decreased in intestine, and decreased significantly in thymus, spleen, ovary, liver, stomach, lung, kidney, heart and muscle during these periods. In skin, the level of spermidine was maximal in 10-week-old mice and markedly reduced in 26-week-old mice. The results suggest that maintenance of polyamine levels may play important roles in the function of the pancreas, brain and uterus in 3- to 26-week-old mice. We next looked for polyamine-rich food materials as a dietary source of polyamines. Foods found to be rich in polyamines included wheat germ, rice bran, black rice, Philippine mango, green pepper, Japanese pumpkin, nuts, fermented pickles, pond smelt, turban shell viscera, whelk viscera, salted salmon roe, salted cod roe, beef intestine (boiled) and liver of eel, beef, pork and chicken; and, as previously reported, soybean, fermented soybean (natto), mushrooms, orange and green tea leaf. These results offer useful information when it becomes necessary to ingest polyamines from food.
Aging↗