Intracellular Spermidine Research Articles

Polyamines and Polyamine Biosynthesis in Cells Exposed to Hyperthermia

The issue of how polyamines act to sensitize cultured cells to the lethal effects of hyperthermia was investigated using Chinese hamster cells which were induced to express thermotolerance. Intracellular levels of these naturally occurring polycations were manipulated in certain situations by treating whole cells with methylglyoxal bis-(guanylhydrazone), an inhibitor of the S-adenosyl-L-methionine decarboxylases. Exogenous spermine as low as 100 microM in the culture media dramatically sensitized cells expressing thermotolerance to the lethal effects of subsequent 42 degrees C exposures. When thermotolerance was differentially induced in cultures exposed to 42.4 degrees C by varying the rate of heating from 37 to 42.4 degrees C, the most resistant cells had the highest levels of intracellular spermidine and spermine. This finding was explainable in part by the observation that the putrescine-dependent S-adenosyl-L-methionine decarboxylase activity was minimally affected in cells expressing the greatest degree of thermotolerance. When this enzyme activity was inhibited by drug, lowered intracellular polyamine levels did not correspond with subsequent survival responses to heat. Interestingly, cultures treated with methylglyoxal bis-(guanylhydrazone) 24 hr previous to heat exposure showed a reduced capacity to express rate of heating-induced thermotolerance. Together, these results demonstrate that the polyamines, especially spermidine and spermine, enhance hyperthermia-induced cell killing by some mechanism involving the plasma membrane. Further, our data suggest that methylglyoxal bis-(guanylhydrazone) can act to affect thermal responses by a mechanism(s) other than modification of intracellular polyamine levels.

Regulation of polyamine content in cultured fibroblasts.

The content of putrescine and of the polyamines (spermidine and spermine) and the activities of their biosynthetic enzymes were measured in 3T3 mouse fibroblasts and SV40-transformed mouse fibroblasts over the entire period from subculturing in fresh medium until confluence. The transformed cells had a substantially higher content of putrescine and spermidine than the 3T3 cells and higher activities of all of the biosynthetic enzymes. However, the ratio of spermine synthase to spermidine synthase was higher in the 3T3 cells, which correlated with their higher spermine-to-spermidine ratio. All of the biosynthetic enzymes increased in activity during cell growth. Ornithine decarboxylase increased 20-fold with a maximum at 24-36 h after culturing whereas S-adenosylmethionine decarboxylase increased 3-fold at the same time. Spermidine synthase increased 10- to 16-fold during the growth period whereas spermine synthase increased 2- to 3-fold. The relative enzyme activities and the changes in total polyamine content suggested that 1) the activity of S-adenosylmethionine decarboxylase limited the production of the polyamines and 2) the relative amounts of spermidine and spermine synthase determined the predominant polyamine that the available decarboxylated S-adenosylmethionine is used to synthesize. When 3T3 cells become quiescent at confluence, there was a substantial fall in the intracellular spermidine level because of a greatly increased excretion of spermidine into the medium. Spermine content also fell because there was an increased conversion of spermine into spermidine, which was then excreted. The specific excretion of spermidine did not occur with the transformed SV-3T3 cells.

Methylglyoxal bis(guanylhydrazone) stimulates the cellular transport system of the polyamines

1. INTRODUCTION Methylglyoxal bis(guanylhydrazone) (MGBG), an inhibitor of polyamine biosynthesis [l], is ap- parently transported into the interior of the cell (by virtue of its structural resemblance to spermidine) via an inducible transport system used by the natu- ral polyamines spermidine and spermine [2-51. Cellular uptake of polyamines, and hence also of MGBG, is strikingly enhanced under conditions of intracellular putrescine and spermidine depletion, as a result of the use of inhibitors of ornithine de- carboxylase (EC 4.1.1.17) [5]. Even though the lat- ter phenomenon is obviously aimed at normalizing the reduced intracellular polyamine pools, it also offers a means of enhancing the cellular accumula- tion of the antiproliferatively acting MGBG when profound growth inhibition is desired [6]. Although intracellular polyamine depletion trig- gers a variety of compensatory mechanisms, such as induction and stabilization of ornithine and ad- enosylmethionine (EC 4.1.150) decarboxylases (for ref. see [7]), the greatly enhanced transport of extracellular polyamines can apparently alone abolish the antiproliferative effects of polyamine antimetabolites (inhibitors of polyamine bio- synthesis) in the presence of only trace amounts of exogenous polyamines. This fact may be prac- tically important since polyamine antimetabolites [MGBG and 2-difluoromethylornithine (DFMO)] have already been used alone (for ref. see [6]) or in combination [8] in the treatment of human malig- nancies. We will show here that when cultured Ehrlich ascites carcinoma cells were exposed to MGBG for not longer than a few hours, the uptake of poly- amines, especially that of diamines, was greatly en- hanced. Exposure to MGBG likewise stimulated the uptake of polyamines by tumor cells that have been brought to the state of severe polyamine de- privation with DFMO, a condition characterized by strikingly increased uptake of polyamines (51. The enhanced uptake of polyamines induced by the antimetabolites was apparently responsible for the appearance of substantial amounts of cadaver- ine (and putrescine) in L1210 leukemia cells har- vested from mice treated with MGBG alone or in combination with DFMO. 2. MATERIALS AND METHODS 2.1.

Death of tumor cells in response to the use of a system of stimulated polyamine uptake from the transport of methylglyoxal bis(guanylhydrazone).

Putrescine and spermidine depletion produced by α-difluoromethylornithine, an irreversible inhibitor of omithine decarboxylase (EC 4.1.1.17) greatly enhances the uptake of the anti-cancer drug methylglyoxal bis(guanylhydrazone) by cultured Ehrlich ascites carcinoma cells. The drug was also more effectively retained in the cells previously exposed to the inhibitor of ornithine decarboxylase. When untreated Ehrlich ascites carcinoma cells were shortly exposed to methylglyoxal bis(guanylhydrazone) and then transferred into drug-free medium, they rapidly reinitiated growth, while polyamine-deprived cells were irreversibly damaged upon similar exposure to the drug. Tumor cells with greatly reduced intracellular pools of putrescine and spermidine so effectively concentrated methylglyoxal bis(guanylhydrazone) that the drug largely disappeared from the medium resulting in a formation of concentration gradient of more than 5000-fold across the cell membrane. The tumor cells tolerated high intracellular concentrations (6-9 mM) of methylglyoxal bis(guanylhydrazone) for not more than 24 h whereafter they simply disintegrated and excreted the drug back into the medium. The final destruction of the polyaminedepleted cells in response to methylglyoxal bis(guanylhydrazone) was preceded by profound inhibition of protein synthesis, which occurred before any disturbances in thymidine incorporation were obvious. The inhibition of protein synthesis by methylglyoxal bis(guanylhydrazone) was more pronounced in cells previously exposed to difluoromethylornithine than in cells with normal intracellular polyamine pools. The extent of polyamine depletion produced by difluoromethylornithine and the rate of the uptake of methylglyoxal bis(guanylhydrazone) were positively correlated to the growth rate of the tumor cells, i.e. cells dividing slowly were more resistant towards the action of difluoromethylornithine, as regards the development of intracellular polyamine depletion, and they also accumulated methylglyoxal bis(guanylhydrazone) less effectively than did rapidly dividing cells. The rate of methylglyoxal bis(guanylhydrazone) transport was likewise related to the extent of intracellular putrescine and spermidine deprivation: tumor cells with greatly reduced putrescine and spermidine pools concentrated methylglyoxal bis(guanylhydrazone) more rapidly than cells containing only moderately decreased concentrations of the polyamines. Although the uptake of exogenous spermidine by polyamine-deprived tumor cells was initially at least as rapid as that of methylglyoxal bis(guanylhydrazone), the transport of the natural polyamine was automatically halted when the intracellular spermidine pool was brought to a normal level. It thus appears that the uptake of methylglyoxal bis(guanylhydrazone) by the stimulated transport system of polyamines leads to a suicidal accumulation of the drug and to rapid cell death.

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.

Effect of methylglyoxal bisguanylhydrazone on polyamine biosynthesis, growth, and differentiation of cultured keratinocytes.

The putrescine, spermidine, and spermine content of subcultured human newborn foreskin keratinocytes was determined during growth and early plateau phase and found to be highest during growth. Exposure of the cells to methylglyoxal bis(guanylhydrazone) during growth phase caused a dose-dependent fall in intracellular spermidine and spermine levels and an increase in putrescine levels at higher concentrations. These effects reflect inhibition of S-adenosyl methionine decarboxylase by the drug. At 8 X 10(-6) M the drug reduced incorporation of leucine into protein, lowered or stopped the accumulation of DNA per dish, inhibited mitotic activity, and increased the histidine/leucine incorporation into protein. The last effect is regarded as induction of keratinization. All these effects were reversible if the use of the drug was discontinued after 3 days. Inhibition of the enzymes of polyamine biosynthesis may have value in psoriasis therapy.

Inhibition of ornithine decarboxylase of HeLa cells by diamines and polyamines. Effect on cell proliferation.

1. Ornithine decarboxylase activity is stimulated in high-density HeLa-cell cultures by dilution of or replacement of spent culture medium with fresh medium containing 10% (v/v) horse serum. 2. After stimulation, ornithine decarboxylase activity reaches a peak at 4-6h, then rapidly declines to the low enzyme activity characteristic of quiescent cultures, where it remains during the remainder of the cell cycle. 3. The stimulation of ornithine decarboxylase is eliminated by the addition of 0.5mum-spermine or -spermidine or 10mum-putrescine to the HeLa-cell cultures at the time of re-feeding with fresh medium. Much higher concentrations (1mm) of the non-physiological diamines, 1,3-diamino-propane or 1,3-diamino-2-hydroxypropane, are required to eliminate the stimulation of ornithine decarboxylase in re-fed HeLa-cell cultures. 4. A heat-labile, non-diffusible inhibitor, comparable with the inhibitory protein ornithine decarboxylase antizyme, is induced in HeLa cells by the addition of exogenous diamines or polyamines. 5. Intracellular putrescine is eliminated, intracellular spermidine and spermine are severely decreased and proliferation of HeLa cells is inhibited when cultures are maintained for 48h in the presence of the non-physiological inducer of ornithine decarboxylase antizyme, 1,3-diamino-2-hydroxypropane. Exogenous putrescine, a physiological inducer of the antizyme, does not decrease intracellular polyamines or interfere with proliferation of HeLa cells.

The estimation of turnover of spermidine in Anacystis nidulans

The turnover of spermidine in Anacystis nidulans was studied using [2-14C]methionine to prelabel intracellular spermidine. It was found that there is essentially neither excretion nor degradation of spermidine in exponentially growing Anacystis nidulans. Spermidine was degraded rapidly in stationary phase cells. The half-life of specific activity of spermidine in exponential phase was 8.3 h, a period similar to that of the doubling time (7.5 h) of the bacterium. The rate of synthesis of spermidine was calculated to be 0.04 nmol/108 cells/h.

Depletion of intracellular putrescine and spermidine by α-difluoromethylornithine does not inhibit proliferation of 9L rat brain tumor cells

Incubation of 9L rat brain tumor cells with 25 mM DL-α-difluoromethylornithine inhibits cell proliferation, while treatment with 10 mM and 1 mM do not. All three concentrations cause equal degrees of depletion of intracellular putrescine and spermidine content, but have no effect on spermine content. These observations show that 9L cells can continue to proliferate in spite of significant polyamine depletion and leads one to question the role of polyamines in 9L cell replication. These observations also suggest that inhibition of 9L cell proliferation by 25 mM DL-α-difluormethylornithine is probably not due to its effect on ornithine decarboxylase or on intracellular polyamine content.

Initial characterization of a HTC cell variant partially resistant to the anti-proliferative effect of ornithine decarboxylase inhibitors

Under the selective pressure of dl-α-methylornithine (α-MeOrn), a competitive inhibitor of ornithine decarboxylase (ODC) (EC 4.1.1.17) a clone of rat hepatoma tissue culture (HTC) cells has been isolated and designated HMO A. The growth of this clone is affected by the drug only after a lag period of three generations. The same partial resistance was observed to dl-α-difluoromethyl ornithine (α-DFMeOrn), an irreversible inhibitor of ODC. HMO A cells showed elevated ODC activity with a concomitant increase of the putrescine content but no change in S-adenosyl- l-methionine decarboxylase (SAM-DC) (EC 4.1.1.50) activity. Evidence is given that this overproduction of putrescine may be responsible for the partial resistance of HMO A cells to the anti-proliferative effect of the ODC inhibitors. α-MeOrn increases ODC and SAM-DC activities and α-DFMeOrn raises SAM-DC activity in a time and cell line-dependent manner. These findings may support the concept that intracellular putrescine and spermidine play a direct or indirect regulatory role in the expression of ODC and SAM-DC. Thus, the variant cell should be useful for studies on the genetic regulation of polyamine metabolism in eukaryotic cell systems.

Relations between spermidine content and RNA stability in rifampicin treated [formula omitted

During incubation in the presence of chloramphenicol (CM), E. coli synthesizes RNA (CM-RNA). When CM is removed and further RNA synthesis is blocked with rifampicin, cells lose spermidine and CM-RNA degrades with a 15 minute half-life. However, if RNA synthesis is blocked by starving for a required amino acid, spermidine is retained by the cells and CM-RNA degrades with a 24 minute half-life. Raising the intracellular spermidine concentration in rifampicin treated cultures by the addition of spermidine to the growth medium stabilizes CM-RNA to the level observed in histidine starved cultures.

The complete conversion of spermidine to a peptide derivative in Escherichia coli

When full-grown cultures of Escherichia coli were incubated anaerobically, all of the intracellular spermidine was converted to a derivative that has been identified tentatively as glutathionylspermidine. This is in contrast to E. coli growing logarithmically, in which little, if any, of this derivative was present. This spermidine derivative was very rapidly converted to spermidine when cells containing the derivative were transferred to fresh medium, and subjected to vigorous aeration. An enzyme has been demonstrated in E. coli sonicates which carries out this cleavage.

Metabolism of 1,4-Diaminobutane and Spermidine in Escherichia coli: The Effects of Low Temperature during Storage and Harvesting of Cultures

Abstract 1,4-Diaminobutane and spermidine are the predominant polyamines in Escherichia coli that are growing in log phase on minimal medium. Contrary to earlier reports, significant acetylation does not occur when E. coli is grown under these conditions. The intracellular amines are completely retained by the cell during logarithmic growth. There is no evidence of oxidative degradation or of significant loss to the medium. Significant acetylation of the amines has been found in two situations: (a) storage of a culture in the cold is associated with a continuous increase in the monoacetylspermidine at the expense of intracellular free spermidine; and (b) growth in a medium containing high concentrations of spermidine results in the conversion of a significant percentage of the cellular spermidine to monoacetylspermidine. Storage of a culture in the cold also leads to a progressive loss of 1,4-diaminobutane to the medium, resulting in a marked decrease in its cellular concentration.

The biochemical role of naturally occurring polyamines in nucleic acid synthesis.

Summary1. The polyamines, putrescine, spermidine and spermine occur in free or acetylated form in a wide variety of living organisms. Putrescine is biosynthesized from ornithine or arginine; spermidine and spermine from methionine and either ornithine or arginine.2. It is difficult to determine the intracellular distribution of polyamines since they are all very soluble in water and they are readily redistributed when cells are disrupted. Evidence suggests that a substantial proportion of the intracellular polyamines is attached to the ribosomes and that spermidine is not concentrated in the nucleus.3. Polyamines bind strongly to both DNA and RNA. The strength of binding is:spermine > spermidine > putrescine. Polyamines stabilize the double helix of DNA, probably by forming a bridge across the narrow groove, by involving electrostatic bonding with the phosphate group. However, they do not appear to alter the overall conformation of DNA. Spermine enables single‐stranded RNA to fold into a more compact configuration which is less susceptible to attack by ribonuclease.4. Spermine and spermidine are able to stimulate the DNA primed RNA polymerase. They facilitate the removal of RNA from the DNA‐RNA‐enzyme complex.5. Polyamines promote the association of ribosomal subunits and also the binding of amino acyl transfer RNA to ribosomes. They cause increased coding ambiguities in the process of translation in certain bacterial systems.6. There is a close correlation between the intracellular concentration of spermidine and the rate of RNA synthesis both in rat liver and in Escherichia coli. Conditions which affect the rate of RNA synthesis also affect the concentration of free intracellular spermidine.7. Bacteria usually contain putrescine and spermidine, whereas animal tissues contain spermine and spermidine. Spermidine probably fulfils the same role in both bacteria and animal tissues, but the presence of spermine, which is common to eucaryotes, is possibly associated with their more complex mechanisms for regulating RNA and protein synthesis.