The roles of the polyamines, putrescine, spermidine, and spermine in normal and malignant tissues

Despite the initial aversion to polyamine research which can be attributed to their peculiar nomenclature and to the erroneous idea that polyamines are products of bacterial decay, it appears that these ubiquitous amines play important roles in the physiological regulation of growth. Many of the definitive roles are yet to be elucidated, and these areas offer promise to biochemists. The evolution of the multifaceted ramifications of polyamines is not unlike that for cyclic AMP, which has profound effects at the cellular level. Further, we are at a stage at which basic knowledge of the roles of polyamines is becoming clinically relevant. We should begin to measure polyamines routinely to obtain clinical parameters which might allow for more efficacious treatment of cancer. No longer can the discussion of polyamines in biochemical textbooks be limited to a page and a half or no discussion at all, and no longer can well-informed scientists afford to neglect the importance and the far-reaching applications of polyamine research.     

Inhibition of putrescine uptake by polypyridinium quaternary salts in B16 melanoma cells treated with difluoromethylornithine.

Several bipyridinium, tetrapyridinium and hexapyridinium quaternary salts have been found to be potent inhibitors of putrescine uptake into B16 melanoma cells which had previously been treated with difluoromethylornithine. In general, the potency of inhibitors increased as the number of quaternary centres increased. A relationship between the distance apart of the positively charged nitrogen atoms and the potency of the salts as inhibitors of uptake has been established by comparison with a number of diaminoalkanes. It was found that an inter-nitrogen distance of 0.6-0.7 nm or 1.0-1.1 nm was optimal for high activity. This finding is significant in determining structural features of the polyamine transport system.     

Xylylated dimers of putrescine and polyamines: influence of the polyamine backbone on spermidine transport inhibition

Dimeric norspermidine and spermidine derivatives are strong competitive inhibitors of polyamine transport. A xylyl tether was used for the dimerization of various triamines and spermine via a secondary amino group, and of putrescine via an ether or an amino group. Dimerization of putrescine moieties potentiates their ability to compete against spermidine transport to a much greater extent than for triamine dimers.     

Melanocyte stimulating hormone activation of tyrosinase in B16 mouse melanoma cells. Evidence for a differential induction of two distinct isoenzymes.

Tyrosinase induction in murine malignant melanocytes by alpha MSH is well known, but its molecular basis has not been characterized. Treatment of B16 melanoma cells with theophylline or alpha MSH mediates a larger induction of tyrosine hydroxylase than of dopa oxidase activity in total cell extracts, and in the melanosomal and microsomal fractions. No evidence for the modulation of a tyrosinase effector was found. SDS-PAGE and specific activity stain demonstrated two forms of tyrosinase, with different degrees of induction by theophylline. These results agree with the recent proposal that two tyrosinases, encoded by different genes, are present in murine melanocytes.     

Tyrosinase-related protein-2 and -1 are trafficked on distinct routes in B16 melanoma cells

Tyrosinase related protein (TRP)-1 and -2 regulate the main steps in melanin synthesis and are immune targets in skin cancer or autoimmune pigmentary disorders. We found that ionophore monensin (Mon) and the quaternary amine chloroquine (CQ) discriminate between the traffic routes of TRP-2 and TRP-1. TRP-2 N-glycan processing is interrupted by Mon between ER and trans-Golgi, whereas this process continues for TRP-1. Mature TRP-2 is diverted by CQ treatment to a degradation pathway which depends on functional vacuolar ATPases. Conversely, the subcellular distribution and stability of TRP-1 were not affected by CQ. We propose that TRP-2 is sorted and trafficked in the early secretory pathway with a cargo which does not include TRP-1; post Golgi, TRP-2 intersects the endocytic pathway following a route via early endosomes, possibly by rapid recycling from the plasma membrane. These data show that highly structural homologous glycoproteins use distinct trafficking pathways in the same cell.     

Intracellular putrescine and spermidine deprivation induces increased uptake of the natural polyamines and methylglyoxal bis(guanylhydrazone).

Inhibition of polyamine synthesis by alpha-difluoromethylornithine in cultured Ehrlich ascites-carcinoma cells rapidly enhanced the uptake of exogenous putrescine, spermidine and spermine from the culture medium. In tumour cells exposed to the drug for 2 days, the intracellular concentration of spermidine was decreased to less than 10% of that found in untreated cells. However, the strikingly stimulated transport system brought the concentration of spermidine to the control values in less than 2h after supplementation of the cells with micromolar concentrations of the polyamine. In the absence of polyamine deprivation, tumour cells did not accumulate extracellular polyamines to any appreciable extent. Ascites-tumour cells deprived of putrescine and spermidine likewise concentrated methylglyoxal bis(guanylhydrazone) [1,1'-[methylethanedylidine)dinitrilo]diguanidine] at a greatly enhanced rate. A previous "priming of tumour cells with difluoromethylornithine followed by an exposure of the cells to methylglyoxal bis(guanylhydrazone) resulted in a marked and rapid anti-proliferative effect.     

Evidence for functionally distinct glucose transporters in basal and insulin-stimulated adipocytes

The activity and Km of glucose transport of rat adipocytes are quite variable in the basal state. This could be due to differing levels of highly saturable transport against a background of less saturable transport. Such heterogeneity could lead to differing conclusions as to the Km of basal cells compared to insulin-stimulated cells depending on the choice of substrate, the range of concentrations tested, and the rigor of data analysis. In the present work, we used a cell preparation which was stable and partially activated by constant agitation. We used a two-component model to fit the concentration dependence of D-glucose uptake. We defined two parallel pathways of glucose entry, a high-affinity/low-capacity pathway and a low-affinity/high-capacity pathway. Both pathways were stereospecific and were inhibited by cytochalasin B. The low-affinity pathway in basal cells had 97% of the total capacity (Vmax) with a high Km (greater than 50 mM). A second pathway had a very low Km (less than 1 mM) and only 3% of the total capacity, but contributed to 30-60% of glucose uptake at 8 mM glucose. In insulin-stimulated cells, a pathway with a Km of 4-5 mM dominated and contributed 85% of glucose transport. The low-affinity but not the very high affinity pathway persisted in stimulated cells, but its contribution was only 10-15% of transport at 8 mM glucose. These results suggest the presence of at least two functionally distinct transporters whose respective contributions can be characterized by nonlinear regression of data over a wide range of glucose concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)     

Affinity of putrescine,spermidine and spermine for pigmented tissues

Determinations of the amounts of putrescine, spermidine and spermine in tissues from mice and cows indicated that the eye melanocytes are very rich in these substances. The concentration of these di-and polyamines was also found to be much higher in pigmented than in albino hair of mice. The melanin polymer has the character of a polyanion — explaining the affinity of these cations for pigmented tissues. Further experiments indicated that these substances to a considerable extent may reach the melanin-containing tissues via the circulation.     

Binding sites of the polyamines putrescine, cadaverine, spermidine and spermine on A- and B-DNA located by simulated annealing

Molecular dynamics simulations with simulated annealing are performed on polyamine-DNA systems in order to determine the binding sites of putrescine, cadaverine, spermidine and spermine on A- and B-DNA. The simulations either contain no additional counterions or sufficient Na+ ions, together with the charge on the polyamine, to provide 73% neutralisation of the charges on the DNA phosphates. The stabilisation energies of the complexes indicate that all four polyamines should stabilise A-DNA in preference to B-DNA, which is in agreement with experiment in the case of spermine and spermidine, but not in the case of putrescine or cadaverine. The major groove is the preferred binding site on A-DNA of all the polyamines. Putrescine and cadaverine tend to bind to the sugar-phosphate backbone of B-DNA, whereas spermidine and spermine occupy more varied sites, including binding along the backbone and bridging both the major and minor grooves.     

Evidence that the small GTPase Rab8 is involved in melanosome traffic and dendrite extension in B16 melanoma cells

One of the major activities of melanocytes in skin is to produce melanin and transport it via dendrites to neighboring keratinocytes. Here, we present evidence that Rab8, a member of the small GTPase superfamily, is present in purified melanosomal fractions, and is upregulated by pigmentogenic agents like melanocyte-stimulating hormone/isobutylmethyl xanthine (MSH/IBMX) and ultraviolet radiation B (UVB). Confocal immunofluorescence microscopic studies revealed that Rab8 is colocalized with Mel5, a melanosomal protein, at the trans-Golgi area and in the cytoplasmic vesicles of B16 cells. During MSH/IBMX treatment, while a number of dendrites with numerous processes are formed, colocalization is extended towards the tips of protrusions. Since process formation is supported by cytoskeletal assembly as well as membrane transport, we tested the colocalization of Rab8 with actin filaments in B16 cells. Rab8, indeed, colocalized with phalloidin, mostly at the periphery, but when irradiated with UVB, cells were rounded instead of dendritic, and colocalization was found predominantly at the cytoplasmic area. Further, suppression of Rab8 expression by its antisense oligonucleotide revealed the reduction in staining intensity of Rab8 but not of Mel5, dendrite formation and melanosome transport towards the tips of the dendrites in B16 melanoma cells. Taken together, it is suggestive that Rab8, in B16 melanoma cells, might have a role in melanosome traffic and dendrite extension, both in constitutive and regulated fashion.This investigation was supported in part by Grants-In-Aid for Scientific Research from the Ministry of Education, Science, and Culture, Japan (grant 15591176)     

Studies on the accumulation of putrescine and spermidine in Escherichia coli

The rate of accumulation of the polyamines spermidine and putrescine by E. coli depended on growth rate. Spermidine accumulation was faster in chemostat cultures with high dilution rates than in those with low dilution rates and was slower in bacteria that had been grown for several generations with either putrescine or spermidine, suggesting that the spermidine-uptake system was repressed by exogenous polyamines. The uptake of spermidine required metabolic energy. Thus accumulation occurred in an energy-starved unc strain only upon addition of glucose (or D-lactate to a smaller extent). With glucose present accumulation occurred in an unc, frd strain under anaerobic conditions, suggesting that ATP drives uptake. However, accumulation was generally sensitive to carbonylcyanide m-chlorophenylhydrazone (CCCP), indicating that the proton motive force was involved in uptake. Unlike spermidine, putrescine accumulation was faster in slow-growing than in fast-growing cultures. This may have been due to greater efflux of putrescine at faster growth rates. Accumulation of putrescine was faster following prolonged growth with either putrescine or spermidine, suggesting induction of the putrescine-uptake system by exogenous polyamines. Like spermidine accumulation, putrescine accumulation required metabolic energy. Accumulation was insensitive to CCCP and occurred only when glucose was added to energy-starved unc bacteria, suggesting that high-energy bonds may drive the uptake of putrescine.     

Polyamines and cellular metabolism in plants: transgenic approaches reveal different responses to diamine putrescine versus higher polyamines spermidine and spermine

Distribution of biogenic amines—the diamine putrescine (Put), triamine spermidine (Spd), and tetraamine spermine (Spm)—differs between species with Put and Spd being particularly abundant and Spm the least abundant in plant cells. These amines are important for cell viability and their intracellular levels are tightly regulated, which have made it difficult to characterize individual effects of Put, Spd and Spm on plant growth and developmental processes. The recent transgenic intervention and mutational genetics have made it possible to stably alter levels of naturally occurring polyamines and study their biological effects. We bring together an analysis of certain metabolic changes, particularly in amino acids, to infer the responsive regulation brought about by increased diamine or polyamine levels in actively growing poplar cell cultures (transformed with mouse ornithine decarboxylase gene to accumulate high Put levels) and ripening tomato pericarp (transformed with yeast S-adenosylmethionine decarboxylase gene to accumulate high Spd and Spm levels at the cost of Put). Our analysis indicates that increased Put has little effect on increasing the levels of Spd and Spm, while Spd and Spm levels are inter-dependent. Further, Put levels were positively associated with Ala (α and β), Ile and GABA and negatively correlated with Gln and Glu in both actively growing poplar cell cultures and non-dividing tomato pericarp tissue. Most amino acids showed positive correlations with Spd and Spm levels in actively growing cells. Collectively these results suggest that Put is a negative regulator while Spd–Spm are positive regulators of cellular amino acid metabolism.     

Involvement of calpain in melanogenesis of mouse B16 melanoma cells

In the current study, the involvement of calpain, a cysteine proteinase in the regulation of melanogenesis was examined using mouse B16 melanoma cells. In response to α-melanocyte-stimulating hormone (α-MSH), B16 melanoma cells underwent differentiation characterized by increased melanin biosynthesis. The total calapain activity was decreased within 2 h following α-MSH-treatment, and restored to the initial level in 6–12 h. To further investigate the involvement of calpain in the regulation of melanogenesis, the effect of calpain inhibitors on α-MSH-induced melanogenesis was examined. Inhibition of calpain by either N-acetyl-Leu-Leu-norleucinal (ALLN) or calpastatin (CS) peptide blocked α-MSH-induced melanogenesis. The magnitude of inhibition of melanin biosynthesis was well correlated with a decrease in the activity of tyrosinase, a key regulatory enzyme in melanogenesis. Treatment of B16 cells with ALLN caused marked decrease in both tyrosinase protein and mRNA levels. These results indicate that calpain would be involved in the melanogenic signaling by modulating the expression of tyrosinase in mouse B16melanoma cells.     

Inducible macropinocytosis of hyaluronan in B16-F10 melanoma cells

Hyaluronan (HA) is a glycosaminoglycan composed of N-acetylglucosamine and glucuronic acid subunits. Endocytosis is thought to play an essential role in the catabolism of HA due to the intracellular compartmentalization of the HA degrading hyaluronidase enzymes. Previous investigations have shown that keratinocytes, chondrocytes and breast tumor cell lines endocytose HA via the cell surface glycoprotein, CD44. However, other cell types endocytose HA using a CD44-independent mechanism that remains to be defined. The purpose of this study was to investigate HA endocytosis in B16-F10 melanoma cells. We found that B16-F10 melanoma cells expressed CD44 on their surfaces. Unexpectedly, CD44 did not play a role in the endocytosis of HA. Electron microscopy studies revealed that B16-F10 melanoma cells exhibited membrane ruffling, a characteristic feature of macropinocytosis, only after incubating the cells with the HA co-polymer. Moreover, B16-F10 melanoma cells endocytosed HA via macropinocytosis as assessed by drug inhibition studies and the co-localization of fluorescently labeled HA with fluorescent tracers under confocal microscopy. Based on these results, we conclude that induced macropinocytosis may provide a previously unrecognized avenue for HA endocytosis in some cell types.