Modulation by polyamines of DNA-dependent DNA polymerase activity from human serum

1. Spermine, spermidine and putrescine activated DNA-dependent DNA polymerase from human sera by 47-125% at the concentrations of 0.2, 3 and 30 mM, respectively. 2. The polyamines shifted the optimal MgCl2 concentration for the polymerase activity from 10 mM to more physiological 5 mM. 3. Histamine having amino and imino groups at both ends of the molecule also increased the DNA polymerase activity, while cyclopentylamine and n-butylamine showed no effects on the enzyme activity. 4. The stimulatory effect of polyamines on the DNA polymerase activity was more evident with poly(dC)p(dG) used as a template/primer than with poly(dA)p(dT).     

Influence of polyamines on the activity of DNA polymerase I from Escherichia coli.

The influence of polyamines on the various activities of DNA polymerase I from Escherichia coli (EC 2.7.7.7) has been investigated. For all high molecular weight DNAs spermine and spermidine caused up to 80% inhibition when present in high concentrations, i.e. above 1 mM for spermine and 2 mM for spermidine. In the presence of low concentrations of polyamines a small activation was seen for some DNAs. The diamines cadaverine and putrescine had little influence on the rate of synthesis with natural occurring DNAs. In the case of d(A--T)n the activation/inhibition was found to be markedly dependent on the molecular weight of the samples used. With a low molecular weight DNA, 5.6 S, addition of spermidine resulted in up to 3-fold stimulation of activity. The activation was dependent on the concentration of MgCl2 and ionic strength; increasing concentration of these gave a decrease in the degree of activation. Polyamines also had a dramatic effect on the rate of synthesis using the homopolymers (dA)n . (dT)10 and (rA)n . (dT)10 . (20:1) as primers. Putrescine, in particular, increased the activity up to 10-fold with (rA)n . (dT)10 and somewhat less for (dA)n . (dT)10. The apparent Km for the primer (rA)n . (dT)10 decreased approx. 35-fold in the presence of 6.6 mM putrescine. There was no influence on the apparent Km for dTTP. The influence of polyamines on both the 5' leads to 3' and 3' leads to 5' nuclease activity was also investigated. Inhibition of nuclease activity was observed in the presence of polyamines, particularly with spermine. Thus with d(A--T)n and T7 DNA as substrates addition of 0.7 mM spermine resulted in almost complete inhibition of the activity. The dramatic inhibition observed with high concentrations of spermine (spermidine) both in the case of polymerizing and nuclease activity is thought to be due to polyamine-induced aggregation of DNA molecules.     

Effects of polyamines on DNA synthesis using various subcellular DNA polymerases extracted from normal rat liver, tumour-bearing rat liver, and tumour cells

The effects of polyamines on DNA synthesis in vitro using various subcellular DNA polymerase fractions from normal and tumour-bearing rat livers, and tumour cells were investigated. When nuclear and mitochondrial DNA polymerase fractions were used, DNA synthesis on activated DNA was increased 3.5-8-fold by the addition of 20 mM putrescine or cadaverine. However, DNA synthesis was not stimulated by the addition of spermidine or spermine at any concentration tested. In contrast, DNA synthesis using the cytoplasmic DNA polymerase fraction was not stimulated at various concentrations of any of the four polyamines tested. The stimulatory effects of putrescine and cadaverine were absent when nuclear fractions from tumour-bearing rat liver or from tumour cells were used. In addition, in vitro DNA synthesis was not stimulated by 20 mM putrescine or cadaverine when nuclear extracts from the livers of rats administered putrescine subcutaneously were used. The specific activities of DNA polymerases extracted from tumour cells and tumour-bearing rat liver were already fully stimulated. These results suggest that DNA polymerases in tumour cells and tumour-bearing liver cells are stimulated by trapped putrescine produced in tumour cells and are thus no longer activated by exogenous putrescine.     

Protein synthesis in polyamine-deficient bacteria during amino-acid starvation

Amino-acid starvation in polyamine-auxotrophic bacteria grown in the presence of putrescine provokes a marked inhibition of protein synthesis. This inhibition is almost completely relieved in polyamine-depleted cells. The differential behaviour of bacterial protein synthesis depending on the endogenous levels of polyamines is not due to a change in the uptake of amino acids used to measure protein synthesis, nor to the decreased growth rate of polyamine-depleted cells. During leucine starvation, cells grown with putrescine synthesized a somewhat lower amount of high-molecular-weight proteins than polyamine-depleted bacteria. In addition, cells with normal endogenous levels of polyamines accumulated significant amounts of 62 and 41 kDa polypeptides as well as several low-molecular-weight peptides.     

Effects of polyamines and methylglyoxal bis(guanylhydrazone) on hepatic nuclear structure and deoxyribonucleic acid template activity.

1. The interaction of polyamines and methylglyoxal bis(guanythydrazone) (1, 1'-[(methylethanediylidene)-dinitrilo]diguanidine) with isolated rat liver nuclei was investigated by electron microscopy. 2. At 4mM, putrescine was without effect; however, spermidine, spermine or methylglyoxal bis(guanythydrazone) resulted in dispersed chromatin and alterations in nucleolar structure. In addition, spermidine or methylglyoxal bis(guanylhydrazone) caused marked aggregation of interchromatin granules. 3. The DNA template property of calf thymus DNA was examined by using DNA polymerases from Escherichia coli, Micrococcus lysodeikticus and calf thymus in the presence of 0-5 mM-amine. 4. In the presence of DNA polymerase, spermine or methylglyoxal bis(guanylhydrazone) inhibited activity, whereas putrescine or spermidine had much less effect or in some cases stimulated [3H]dTMP incorporation. 5. Template activity which was inhibited by spermine or methylglyoxal bis(guanylhydrazone) could be partially restored by additional DNA or enzyme. 6. When mixed with calf thymus DNA, calf thymus histone inhibited template activity as measured with E. coli DNA polymerase. The template activity of such a 'histone-nucleate' could not be restored by putrescine, spermidine, spermine or methylglyoxal bis(guanylhydrazone). 7. DNA template activity of isolated rat liver nuclei was tested by using E. coli DNA polymerase. None of the amines was able to increase the template activity of the nuclear DNA in vitro.     

The level of substrate ornithine can alter polyamine-dependent DNA synthesis following phorbolester stimulation of cultured hepatoma cells

Although the precise intracellular function(s) of the polyamines remain incompletely defined, a myraid of evidence now shows that the polyamines must accumulate or be maintained at a specific intracellular concentration in order for all mammalian cells to grow or divide. The initial step in polyamine biosynthesis normally involves the decarboxylation of ornithine by the enzyme ornithine decarboxylase (ODCase E.C. 4.1.1.17) to yield putrescine. Increases in the steady-state level of intracellular ornithine have been reported to markedly alter the accumulation of the polyamines following stimulation of Reuber H35 Hepatoma cells with 12-O-tetradecanoylphorbol-beta-acetate (TPA) in the presence of serum (Wu and Byus: (Biochem. Biophys. Acta 804:89-99, 1984); Wu et al.: (Cancer Res. 41:3384-3391, 1981). We wished to determine whether or not incubation of H35 hepatoma cells with exogenous ornithine would result in a stimulation of DNA synthesis following treatment with the mitogens TPA and insulin. For these studies, H35 cells were maintained under serum-free conditions for 2-3 days in order to obtain synchronous cultures suitable for analysis of the level of DNA synthesis. Cultures treated in this manner were highly viable, maintained similar growth rates, and possessed the equivalent levels of intracellular ornithine and polyamines as the serum-containing cultures. Arginine levels, however, were approximately twofold higher following culture under serum-restricted conditions for 3 days. The addition of exogenous ornithine (0.5 mM) was accompanied by a 4-5-fold increase in intracellular steady-state ornithine levels and by a 6-8-fold increase in the presence of TPA and ornithine. In a manner identical to the serum-containing cultures (Wu and Byus (1984] the addition of TPA and exogenous ornithine to the serum-free cells caused a dose-dependent increase in intracellular putrescine (up to 5-fold) and a concomitant decrease in ODC activity in comparison to stimulation with TPA alone. The addition of TPA led to a 3-5-fold increase in the incorporation of tritiated thymidine into DNA. In the presence of exogenous ornithine, TPA-induced DNA synthesis was further stimulated more than twofold in a dose-dependent manner. Insulin (10(-10)-10(-8) M) proved to be more efficacious as a mitogen in the H35 cells and led to greater stimulation of DNA synthesis than TPA. Insulin alone also resulted in a higher steady-state level of ornithine and putrescine in comparison with TPA alone.(ABSTRACT TRUNCATED AT 250 WORDS)     

Polyamines and HeLa-cell DNA replication.

HeLa cells were synchronized for S-phase DNA synthesis by the double thymidine-block procedure. A comparison was made of the polyamine content and S-phase DNA synthesis in cells from control cultures and cultures to which an inhibitor of polyamine biosynthesis, alpha-difluoromethylornithine, was added to the synchronization medium. Control cells showed a peak of synchronous DNA synthesis at 3 h and a maximum concentration of polyamines at 6-9 h after release of the second thymidine block. Cells from cultures containing the inhibitor were severely inhibited in the synthesis of DNA and contained no putrescine and only traces of spermidine while the spermine content was lowered by as much as 80%. Supplementation of cultures containing alpha-difluoromethylornithine with a polyamine, at the time of release of the second thymidine block, replenished the intracellular pool of the administered polyamine and partially restored S-phase DNA synthesis, with a lag of 3-6 h. Almost complete restoration of DNA synthesis in cells depleted of polyamines was achieved by the addition of a polyamine to cultures at least 10 h before release of the second thymidine block. The lag in initiation of synchronous S-phase DNA synthesis was eliminated in these cells. It is concluded that reversal by polyamines of the deficiency in S-phase DNA synthesis, in polyamine-depleted HeLa cells, is a time-dependent process indicative of the necessity for the replenishment of replication factors or their organization into an active replication complex.     

Inhibition of protein synthesis by aminoglycoside antibiotics in polyamine-requiring bacteria

The effect of streptomycin and other aminoglycosides on protein synthesis has been studied using various streptomycin-sensitive strains unable to synthesize polyamines. We have confirmed and extended our previous results showing that the strong inhibition of translation caused by the antibiotic in polyamine-supplemented bacteria was markedly reduced in polyamine-starved cells. The analysis of polypeptides synthesized in the absence and presence of streptomycin in bacteria grown with and without putrescine has shown that the antibiotic provoked the accumulation of low molecular weight peptides partially bound to ribosomes in polyamine-unstarved cells. On the contrary, the drug did not induce major alterations in the patterns of proteins obtained from polyamine-depleted bacteria. The addition of the antibiotic did not evoke any change of proteolytic activity.     

Synthesis of Semliki-forest virus in polyamine-depleted baby-hamster kidney cells.

The role of polyamines in macromolecular synthesis has been studied using the synthesis of Semliki-Forest virus (SF virus) in normal and alpha-difluoromethylornithine-treated baby-hamster kidney (BHK21) cells as a model system. The activities of ornithine decarboxylase and S-adenosylmethionine decarboxylase, the rate-limiting enzymes in polyamine biosynthesis, decreased rapidly in mock- and SF-virus-infected cells, indicating that virus production in BHK21 cells was not dependent on polyamines formed after infection. A prolonged treatment of BHK21 cells with alpha-difluoro-methylornithine, a specific inhibitor of polyamine synthesis, resulted in a marked inhibition of the initial rate of virus production, which appeared 72 h after the beginning of the treatment. This inhibition was reversed by putrescine, spermidine and spermine, and at last partially by several other diamines and polyamine homologues. Polyamine-depletion also markedly reduced viral RNA polymerase activity in SF-virus infected cells. Addition of spermidine to the culture medium rapidly increased viral RNA polymerase activity in the inhibitor-treated cells but had no effect on the enzyme activity when added directly to the assay mixture. The results indicated that polyamines are needed for maximum initial rate of SF-virus replication and suggest that the inhibition of virus production in polyamine-depleted cells is at least partly due to malfunction of the protein-synthetic machinery of the host cell.     

Effect of polyamines on ADP-ribosylation by chick-embryo-liver nuclei

Effects of polyamines on poly(ADP-ribose) formation and DNA synthesis in the chick-embryo-liver nuclei were investigated. When 14-day chick-embryo-liver nuclei were incubated with [3H]NAD in the presence of 1 mM spermine, 2.5 mM spermidine, or 3.5 mM putrescine, a 9-fold increase in poly)ADP-ribose) formation was observed. Nuclei treated with nuclease showed high poly(ADP-ribose) synthetase activity as spermine-treated nuclei. However, no further increase in the polymer formation by polyamines was detected in the nuclease-treated nuclei. We found that an increase in the polymer formation by spermine was the result of an increase in both chain length and chain number of the polymer at 2.3- and 6-fold, respectively. The major ADP-ribosylated proteins were determined as two non-histone proteins of Mr 130 000 and 70 000. The experiment of DNA synthesis with nuclei ADP-ribosylated in the presence of spermine showed a 7-fold increase in [3H]dTMP incorporation into the acid-inaoluble fraction. A similar stimulation was also found with nuclei treated with other polysmines, spermidine and putrescine, in the presence of NAD. These results indicate that DNA synthesis in growing tissues containing polyamines at high levels, such as is the case with tumors and the fetus, is stimulated by polyamine-mediated ADP-ribosylation of the nuclear proteins.     

Putrescine and spermidine sensitivity of lysine decarboxylase in Escherichia coli: evidence for a constitutive enzyme and its mode of regulation

Cells of Escherichia coli grown under physiological (noninducing) conditions have a low level of lysine decarboxylase activity. This activity differs from the enzyme found in induced cells in its sensitivity to putrescine (33% of control in the presence of 20 mM putrescine). It is also sensitive to spermidine (20% of control in the presence of 6 mM spermidine). A mixture of putrescine and spermidine completely eliminated lysine decarboxylase activity. This provides evidence for the existence of a biosynthetic enzyme and suggests a mechanism to explain the appearance of cadaverine in polyamine-depleted cells.     

Polyamine levels and the activity of their biosynthetic enzymes in human erythrocytes infected with the malarial parasite, Plasmodium falciparum.

Human erythrocytes contain only trace amounts of polyamines and lack active polyamine biosynthetic enzymes. A remarkable increase in polyamine content, and in the activity of ornithine and S-adenosyl-L-methionine decarboxylases, is noted in synchronous cultures of the malarial parasite, Plasmodium falciparum. Polyamine biosynthesis reached peak values during the early trophozoite stage, whereas nucleic acid and protein synthesis occurred later in mature trophozoites. DL-alpha-Difluoromethylornithine, an irreversible inhibitor of ornithine decarboxylase, did not interfere with merozoite invasion and with ring-form development, but prevented the transformation of trophozoites to schizonts. Concomitantly, the synthesis of proteins and nucleic acids was significantly inhibited. These inhibitory effects could be readily reversed by the diamine putrescine. Macromolecular synthesis and schizogony were normal when 5-10 mM-DL-alpha-difluoromethylornithine and 0.1 mM-putrescine were added to the cultures simultaneously.     

Alterations in amounts and covalent modifications of low-molecular-weight chromosomal proteins in Chinese hamster ovary cells during polyamine depletion

The effect of polyamine depletion on phosphorylation and ADP-ribosylation of low-M_r chromosomal proteins was studied in intact, mutant Chinese hamster ovary cells (CHO-P22) devoid of ornithine decarboxylase activity. When starved of polyamines for 6 days, severe polyamine deficiency develops and the cells gradually stop growing. The rate of DNA synthesis was retarded to 16% of the control value and to 29% in density-inhibited cells. The synthesis of high-mobility-group (HMG) proteins was decreased by 65% in polyamine-depleted cells and by 40% in density-inhibited cells. The synthesis of core histones was decreased by 40% both in polyamine-depleted and density-inhibited cells. In polyamine-depleted cells the molar ratio of the higher-M_r HMG proteins (HMG 1 + 2) to the lower-M_r HMG proteins (HMG 14 + P) was about one-half of that found in cells grown in the presence of putrescine or in density-inhibited cells. In contrast to HMG proteins, no major differences were found in the content of core histones in these cell populations. In the perchloric acid-soluble fraction of nuclear proteins, ³²P was incorporated mainly into histone H1, HMG P and a protein migrating more slowly than HMG 1 (protein P1). Specific changes in the ³²P-labeling and migration of a number of protein bands, including histone H1, was observed in polyamine-depleted cells as compared to cells grown in the presence of putrescine or to density-inhibited cells. ADP-ribosylation experiments using [³H]adenosine showed a different pattern of label distribution; the higher-M_r HMG proteins from polyamine-depleted cells contained about one-half the amount of label found in the proteins from control cells. The lower-M_r HMG proteins and histone H1 were the preferentially labeled proteins in polyamine-depleted cells. Labeling of core histones with [³²P]orthophosphate or [³H]adenosine did not differ markedly in the two cell populations. The results obtained using intact polyamine auxotrophic cells indicated that polyamine depletion is connected with more severe alterations in amounts and covalent modifications (phosphorylation and ADP-ribosylation) of HMG chromosomal proteins and histone H1 than core histones.     

Acquisition of polyamines by the obligate intracytoplasmic bacterium Rickettsia prowazekii.

Both the polyamine content and the route of acquisition of polyamines by Rickettsia prowazekii, an obligate intracellular parasitic bacterium, were determined. The rickettsiae grew normally in an ornithine decarboxylase mutant of the Chinese hamster ovary (C55.7) cell line whether or not putrescine, which this host cell required in order to grow, was present. The rickettsiae contained approximately 6 mM putrescine, 5 mM spermidine, and 3 mM spermine when cultured in the presence or absence of putrescine. Neither the transport of putrescine and spermidine by the rickettsiae nor a measurable rickettsial ornithine decarboxylase activity could be demonstrated. However, we demonstrated the de novo synthesis of polyamines from arginine by the rickettsiae. Arginine decarboxylase activity (29 pmol of 14CO2 released per h per 10(8) rickettsiae) was measured in the rickettsiae growing within their host cell. A markedly lower level of this enzymatic activity was observed in cell extracts of R. prowazekii and could be completely inhibited with 1 mM difluoromethylarginine, an irreversible inhibitor of the enzyme. R. prowazekii failed to grow in C55.7 cells that had been cultured in the presence of 1 mM difluoromethylarginine. After rickettsiae were grown in C55.7 in the presence of labeled arginine, the specific activities of arginine in the host cell cytoplasm and polyamines in the rickettsiae were measured; these measurements indicated that 100% of the total polyamine content of R. prowazekii was derived from arginine.     

Cell-free synthesis of herpes simplex virus DNA: the influence of polyamines.

The effect of polyamines on cell-free DNA synthesis of herpes simplex virus DNA in two different systems is investigated. Purified nuclei from infected cells are devoid of measurable amounts of putrescine, spermidine, and spermine, while an unfractionated lysate contains the polyamines at close to their respective cellular concentrations. Spermine, 0.3 mM, and 0.5 mM spermidine, when added to the nuclear system, decrease the extent of viral DNA synthesis to the level found in the lysate system, the size of the cell-free viral DNA product is increased, and a specific inhibition of repair-type DNA synthesis is observed. These effects of the polyamines occur only in the presence of ATP and not the other three ribonucleoside triphosphates.     

Polyamines secreted by cancer cells possibly account for the impairment of the human erythrocyte sodium pump activity.

Using an original microcalorimetric method, we previously showed that in erythrocytes from cancer patients, the sodium pump activity was decreased and returned to normal in patient in remission. In addition we suggested that a plasma-borne factor probably secreted by cancer cells accounted for this impairment of the sodium transporter. In the present study we sought to identify this factor as well as its mechanism of action. First we determined the effect of culture media from undifferentiated and differentiated colon cancer cell lines (Caco-2 and HT29-D4) on the sodium pump activity of normal human erythrocytes. The inhibitory powers of culture media from undifferentiated cells were higher than those of differentiated cells (38.6 +/- 3.5% vs 6.9 +/- 4.6%, p<0.05 for Caco-2 and 45.8 +/- 6.2% vs 9.0 +/- 5.0%, <0.05 for HT29-D4). The use of alpha difluoro-methylomithine (2 mM) to inhibit ornithine decarboxylase, the rate-limiting enzyme for polyamine biosynthesis, dramatically reduced the sodium pump inhibition induced by the two undifferentiated cell lines (75% for Caco-2 and 89% for HT29-D4). Polyamines secreted by undifferentiated cells and then taken up by human erythrocytes thus appeared as inhibitors of sodium pump of these red blood cells. Putrescine, spermidine, and spermine (the main polyamines) exerted a similar inhibitory effect (33 +/- 2%). Tested in vitro on Na,KATPase, these polyamines (3 mM) were inhibitors (putrescine = 23 +/- 2%; spermidine= 48 +/- 3%; spermine= 55 +/- 2%) when assay condition for the ATPase reaction was suboptimal (Na+ = 10 mM; K+ = 1 mM). The inhibitory effect appeared to be related to their charge and their aliphatic chain length. The effect of spermidine and spermine on the ionic substrates and ATP-Mg showed that molecules decreased the affinity (Km) of the Na,K-ATPase for Na+ (11.24 +/- 0.49 mM for control vs 23.51 +/- 1.53 mM for spermine and 18.86 +/- 0.98 mM for spermidine), indicating that polyamines exerted their inhibitory effect in a competitive manner.     

Polyamines in the Synthesis of Bacteriophage Deoxyribonucleic Acid II. Requirement for Polyamines in T4 Infection of a Polyamine Auxotroph

Polyamine depletion produced by exogenous arginine in Escherichia coliK-12 cultures defective in agmatine ureohydrolase activity resulted in a marked inhibition of the rates of growth and nucleic acid synthesis. Addition of putrescine or spermidine to such depleted cultures restored the control rate of growth and nucleic acid accumulation. The omission of lysine resulted in a further decrease in the rates of growth and nucleic acid synthesis in polyamine-depleted cells. The addition of exogenous cadaverine increased the rates of growth and ribonucleic acid synthesis to those observed in lysine-supplemented cultures, suggesting that lysine or a derivative of lysine serves a function similar to cadaverine. Addition of lysine to polyamine-depleted cultures at neutral pH results in the synthesis of cadaverine and a new spermidine analogue, both containing lysine carbon. This new metabolite has been isolated and identified as N-3-aminopropyl-1, 5-diaminopentane. T4D infection of the polyamine-depleted mutant resulted in a very low rate of DNA synthesis and phage maturation. The addition of putrescine or spermidine 15 min before infection restored phage DNA synthesis and phage maturation to control rates, i.e., rates observed in infected cells grown in the absence of arginine.     

Na K ATPase activity in mammalian erythrocytes

The effects of membranotropic substances--nonionic detergent Tween-20 and EDTA--on the activity and some properties of Na,K-ATPase from mammalian erythrocytes were studied. It was shown that pretreatment of whole erythrocytes with Tween-20 (5 mg/ml) allows a detection of the enzyme activity, which cannot be detected in intact cells. It was also found that erythrocyte ghosts with a high and stable activity of Na,K-ATPase can be obtained by injections of EDTA (1-2 mM) into the hemolysis medium. Although the enzyme activity in whole erythrocytes and their ghosts was detected by the use of various membranotropic agents, the type of the dependence of the Na,K-ATPase activity on MgCl2 and EDTA concentration in the incubation medium was essentially the same for both cell preparations, the optimal concentrations of MgCl2 and EDTA being 3 and 1 mM, respectively. A rise in MgCl2 concentration above 3 mM caused a decrease of the enzymatic activity. Simple techniques have been developed for the detection of the Na,K-ATPase activity in mammalian erythrocytes which allow the determination of a higher enzymatic activity than those described in literature.