Regulation of ornithine decarboxylase activity by spermidine and the spermidine analogue N1N8-bis(ethyl)spermidine.

Polyamine biosynthesis in intact cells can be exquisitely controlled with exogenous polyamines through the regulation of rate-limiting biosynthetic enzymes, particularly ornithine decarboxylase (ODC). In an attempt to exploit this phenomenon as an antiproliferative strategy, certain polyamine analogues have been identified [Porter, Cavanaugh, Stolowich, Ganis, Kelly & Bergeron (1985) Cancer Res. 45, 2050-2057] which lower ODC activity in intact cells, have no direct inhibitory effects on ODC, are incapable of substituting for spermidine (SPD) in supporting cell growth, and are growth-inhibitory at micromolar concentrations. In the present study, the most effective of these analogues, N1N8-bis(ethyl)SPD (BES), is compared with SPD in its ability to regulate ODC activity in intact L1210 cells and in the mechanism(s) by which this is accomplished. With respect to time and dose-dependence of ODC suppression, both polyamines closely paralleled one another in their response curves, although BES was slightly less effective than SPD. Conditions of minimal treatment leading to near-maximal ODC suppression (70-80%) were determined and found to be 3 microM for 2 h with either SPD or BES. After such treatment, ODC activity was fully recovered within 2-4 h when cells were re-seeded in drug-free media. By assessing BES or [3H]SPD concentrations in treated and recovered cells, it was possible to deduce that an intracellular accumulation of BES or SPD equivalent to less than 6.5% of the combined cellular polyamine pool was sufficient to invoke ODC regulatory mechanisms. Decreases in ODC activity after BES or SPD treatment were closely paralleled by concomitant decreases in ODC protein. Since cellular ODC mRNA was not similarly decreased by either BES or SPD, it was concluded that translational and/or post-translational mechanisms, such as increased degradation of ODC protein or decreased translation of ODC mRNA, were probably responsible for regulation of enzyme activity. Experimental evidence indicated that neither of these mechanisms seemed to be mediated by cyclic AMP or ODC-antizyme induction. On the basis of the consistent similarities between BES and SPD in all parameters studied, it is concluded that the analogue most probably acts by the same mechanisms as SPD in regulating polyamine biosynthesis.     

Mechanism of the inhibition of cell growth by N1,N12-bis(ethyl)spermine.

The mechanism of the antiproliferation effect of N1,N12-bis(ethyl)spermine (BESPM) was studied in detail using mouse FM3A cells, since this polyamine analogue mimics the functions of spermine in several aspects [Igarashi, K., Kashiwagi, K., Fukuchi, J., Isobe, Y., Otomo, S. & Shirahata, A. (1990) Biochem. Biophys. Res. Commun. 172, 715-720]. Our results indicate that not only the decrease in sperimine and spermine caused by BESPM but also its accumulation play important roles on the inhibition of cell growth by BESPM, since BESPM accumulated in cells at a concentration fivefold that of spermidine in control cells. In comparison with the polaymine-deficient cells caused by alpha-difluoromethylornithine, an inhibitor of ornithine decarboxylase, and ethylglyoxal bis(guanylhydrazone), an inhibitor of S-adenosylmethionine decarboxylase, the behavior of polyamine-deficient cells caused by BESPM was different as follows: the inhibition of cell growth by BESPM was not abrogated by spermine or spermidine; polyamine uptake, which is stimulated during polyamine deficiency, was greatly inhibited, while spermidine/spermine N1-acetyltransferase activity, which is inhibited during polyamine deficiency, was enhanced in BESPM-treated cells; thymidine kinase activity did not decrease in BESPM-treated cells; inhibition of cell growth and macromolecule synthesis by BESPM correlated with the swelling of mitochondria and the decrease in ATP content; BESPM caused cell death when incubated together for several days. The role of BESPM accumulation on inhibition of cell growth is discussed.     

Action of spermidine, N1-acetylspermidine, and N8-acetylspermidine at apurinic sites in DNA

The cleavage efficiency of spermidine and its acetyl derivatives (N1-acetylspermidine and N8-acetylspermidine) at apurinic sites in DNA were examined by PAGE-urea analysis. The three polyamines induced different rates of cleavage when compared at 1 mM concentrations. The order of effectiveness were: spermidine greater than N8-acetylspermidine greater than N1-acetylspermidine. Thus a decrease in efficiency was observed when the first order amino-groups of spermidine were blocked. The N-8amino-group of spermidine was less effective in inducing cleavage at AP-sites than the N1-amino-group. Among several proposed models of polyamine-DNA interactions, our results can best be explained by the model postulated by Liquori et al.     

Properties of the spermidine/spermine N1-acetyltransferase mutant L156F that decreases cellular sensitivity to the polyamine analogue N1, N11-bis(ethyl)norspermine

Properties of a mutant form of spermidine/spermine N(1)-acetyltransferase, L156F (L156F-SSAT), that is present in Chinese hamster ovary cells selected for resistance to the polyamine analogue N(1,) N(11)-bis(ethyl)norspermine (BE 3-3-3) were investigated. Increased K(m) values, decreased V(max) values, and decreased k(cat) values with both polyamine substrates, spermidine and spermine, indicated that L156F-SSAT is an inferior and less efficient acetyltransferase than wild-type SSAT. Transfection of L156F-SSAT into C55.7Res cells indicated that cellular SSAT activity per nanogram of SSAT protein correlated well with the in vitro data and was also approximately 20-fold less for the mutant protein than for wild-type SSAT. Increased expression of L156F-SSAT was unable to restore cellular sensitivity to BE 3-3-3 despite providing measurable basal SSAT activity. Only a 4-fold induction of L156F-SSAT activity resulted from the exposure of cells to the polyamine analogue, whereas wild-type SSAT was induced approximately 300-fold. Degradation studies indicated that BE 3-3-3 cannot prevent ubiquitination of L156F-SSAT and is therefore unable to protect the mutant protein from degradation. These studies indicate that the decreased cellular sensitivity to BE 3-3-3 is caused by the lack of SSAT activity induction in the presence of the analogue due to its inability to prevent the rapid degradation of the L156F-SSAT protein.     

Isolation of peptides containing citrulline and the cross-link, epsilon-(gamma-glutamyl)lysine, from hair medulla protein.

The epsilon-(gamma-glutamyl)lysine cross-link has been unequivocally localized in the citrulline-containing protein of hair medulla tissue. This was achieved by the isolation of cross-linked peptides that contain citrulline. The peptides were purified by molecular sieving, cation-exchange and paper chromatography and paper electrophoresis and their amino acid compositions are reported. They were detected by analyzing a large number obtained from a tryptic digest of thermolytic peptides of the medulla protein. There is no evidence at present as to whether the cross-link is in fact inter- or intrachain, or both.     

Induction of spermidine/spermine N1-acetyltransferase by methylglyoxal bis(guanylhydrazone)

The anti-tumor agent methylglyoxal bis(guanylhydrazone) was found to be a competitive inhibitor of spermidine/spermine N1-acetyltransferase with a Ki of about 8 microM. Treatment of rats with this drug lead to a very large increase in the total amount of spermidine/spermine N1-acetyltransferase in liver, kidney and spleen. The total increase as measured using a specific antiserum amounted to 700-fold in liver and 100-fold in kidney within 18 h of treatment with 80 mg/kg doses. At least part of this induction was due to a pronounced increase in the half-life of the acetyltransferase which increased from 15 min to more than 12 h. The very large increase in the amount of the enzyme is likely to overwhelm the direct inhibition, and a net increase in the acetylation of polyamines by this enzyme would be expected to occur after treatment with methylglyoxal bis(guanylhydrazone). The acetylated polyamines are known to be rapidly degraded by polyamine oxidase producing putrescine. Direct evidence that a substantial part of the increase in the content of putrescine in the liver of rats treated with methylglyoxal bis(guanylhydrazone) occurs via the induction of this acetylase/oxidase pathway was obtained. These results indicate that methylglyoxal bis(guanylhydrazone) affects cellular polyamine levels not only by means of its inhibitory effect on S-adenosylmethionine decarboxylase and diamine oxidase but also by the induction of spermidine/spermine N1-acetyltransferase. They also raise the possibility that the enormous increase in this enzyme which occurs with higher doses may contribute to the very severe toxicity of methylglyoxal bis(guanylhydrazone).     

Formation of N epsilon-(gamma-glutamyl)-lysine isodipeptide in Chinese-hamster ovary cells.

N epsilon-(gamma-Glutamyl)-lysine isodipeptide was detected in a protein-free fraction of Chinese-hamster ovary cells and their culture fluid by using radioactive lysine as a tracer. The identity of the isodipeptide was established by its separation on ion-exchange chromatography, analysis by h.p.l.c. after derivatization, recovery of lysine after acidic hydrolysis or after cleavage by a specific enzyme, namely gamma-glutamylamine cyclotransferase. The amount of isodipeptide was raised (460 pmol/10(7) cells and 61 pmol/ml of culture fluid were observed as highest values) as the cell density increased. Effects of inhibitors of intracellular protein degradation have shown that the isodipeptide derives from cross-linking N epsilon-(gamma-glutamyl)-lysine bonds formed by tissue transglutaminase. Estimated half-life values of cross-linked proteins were about 3 h. gamma-Glutamylamine cyclotransferase, which may split the isodipeptide formed during the continuous turnover of cross-linked proteins, was also found in Chinese-hamster ovary cells. Isodipeptide may have been accumulated when either its generated amount is beyond the capacity of gamma-glutamylamine cyclotransferase or it is generated in cell compartments where this enzyme is not present.     

Increased breast cancer cell toxicity by palladination of the polyamine analogue N 1,N 11-bis(ethyl)norspermine

Breast cancer is one of the most common malignant tumor forms among women and many women succumb to their disease. Thus, new anticancer agents that can efficiently improve patient survival are of the utmost importance. In this study, the effects of the polyamine analogues N ¹,N ¹¹-bis(ethyl)norspermine (BENSpm) and N ¹-cyclo-propylmethyl-N ¹¹-ethylnorspermine (CPENSpm) and the synthesized dinuclear complexes Pd₂BENSpm (Pd-BENSpm), Pt₂CPENSpm (Pt-CPENSpm) and Pd₂Spm (Pd-Spm) were investigated in normal-like breast epithelial MCF-10A cells and the breast cancer cell lines JIMT-1 and L56BR-C1. The overall data show that palladination of BENSpm resulted in enhanced cytotoxicity, in contrast to platination of CPENSpm that reduced cytotoxicity, which might be explained by differences in the cellular uptake of Pd-BENSpm and Pt-CPENSpm. BENSpm and Pd-BENSpm treatment reduced the CD44⁺CD24^? putative cancer stem cell population, evaluated by flow cytometry. Furthermore, Pd-BENSpm was the most efficient compound regarding induction of DNA damage and decrease in colony formation in soft agar. Pt-CPENSpm and Pd-Spm, on the other hand, were shown to be the least toxic compounds of all tested. Pd-Spm efficiently reduced the cellular glutathione levels, which probably was a consequence of its metabolic inactivation by conjugation to this endogenous thiol. The normal-like cells were found to be less sensitive to the agents than the breast cancer cells. Our findings show that Pd-BENSpm exhibits promising anticancer effects which render it suitable for further optimization to develop a new metal-based chemotherapeutic drug for breast cancer treatment.     

High specific induction of spermidine/spermine N1-acetyltransferase in a human large cell lung carcinoma.

The effects of insulin and anti-(insulin receptor) monoclonal antibodies on tyrosine phosphorylation were investigated in fibroblasts transfected with human insulin receptor cDNA (NIH 3T3HIR3.5 cells) using anti-phosphotyrosine immunoblotting. Insulin increased levels of tyrosine phosphorylation in two major proteins of molecular mass 97 kDa (pp97, assumed to be the insulin receptor beta-subunit) and 185 kDa (pp185). Insulin-mimetic anti-receptor antibodies also stimulated tyrosine phosphorylation of both pp97 and pp185. The observation of antibody-stimulated pp97 phosphorylation, as detected by immunoblotting, is in contrast with previous data which failed to show receptor autophosphorylation in NIH 3T3HIR3.5 cells labelled with [32P]P1. The effect of insulin on pp97 was maximal within 1 min, but the response to antibody was apparent only after a lag of 1-2 min and rose steadily over 20 min. The absolute level of antibody-stimulated phosphorylation of both pp97 and pp185 after 20 min was only about 20% of the maximum level induced by equivalent concentrations of insulin, even at concentrations of antibody sufficient for full occupancy of receptors. Another insulin-mimetic agent, wheat-germ agglutinin, stimulated receptor autophosphorylation with kinetics similar to those produced by the antibody. It is suggested that the relatively slow responses to both agents may be a function of the dependence on receptor cross-linking. These data are consistent with a role for the insulin receptor tyrosine kinase activity in the mechanism of action of insulin-mimetic anti-receptor antibodies.     

Synthesis, characterization and studies on DNA-binding of a new Cu(II) complex with N1,N8-bis(l-methyl-4-nitropyrrole-2-carbonyl)triethylenetetramine

A new Cu(II) complex of CuLCl(2) (here, L=N(1),N(8)-bis(1-methyl-4-nitropyrrole-2- carbonyl)triethylenetetramine) had been synthesized and characterized. The structure of the complex was investigated with density functional theory (DFT) calculations. DNA-binding of the Cu(II) complex and its effects on tumor cell viability were firstly studied. The interactions between the complex and calf thymus DNA had been investigated using UV spectra, fluorescent spectra, viscosity and CV (cyclic voltammetry). The cleavage reaction on plasmid DNA has been monitored by agarose gel electrophoresis. The experimental results show that the mode of binding of the complex to DNA is classical intercalation and the complex can cleave pBR322 DNA. The effects of the CuL on cell viability were tested using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide) dye assay and the results indicate that the CuL had certain effect on cancer cells.     

Dolichos biflorus agglutinin (DBA) reveals a similar basal cell differentiation in normal and psoriatic epidermis

Summary Binding of N-acetyl galactosamine (GalNAc)-specific Dolichos biflorus agglutinin (DBA) conjugates to frozen sections of normal epidermis and of psoriatic uninvolved and lesional skin was studied in fluorescence microscopy. The DBA conjugates bound only to single basal cell layer in normal and uninvolved psoriatic epidermis from patients with different blood group status. In the lesional area of psoriatic skin a similar reaction with a single basal cell layer was revealed. Other lectin-conjugates applied, presenting also GalNAc specificity, reacted with most cell layers of normal and both uninvolved and lesional psoriatic epidermis and gave an attenuated reaction with the middle epidermal layers. The results show that the basal cell characteristics are confined only to the cells along the basal membrane also in psoriatic epidermis, although cells in three lowest layers may be able to proliferate.     

Dolichos biflorus agglutinin (DBA) reveals a similar basal cell differentiation in normal and psoriatic epidermis

Binding of N-acetyl galactosamine (GalNAc)-specific Dolichos biflorus agglutinin (DBA) conjugates to frozen sections of normal epidermis and of psoriatic uninvolved and lesional skin was studied in fluorescence microscopy. The DBA conjugates bound only to single basal cell layer in normal and uninvolved psoriatic epidermis from patients with different blood group status. In the lesional area of psoriatic skin a similar reaction with a single basal cell layer was revealed. Other lectin-conjugates applied, presenting also GalNAc specificity, reacted with most cell layers of normal and both uninvolved and lesional psoriatic epidermis and gave an attenuated reaction with the middle epidermal layers. The results show that the basal cell characteristics are confined only to the cells along the basal membrane also in psoriatic epidermis, although cells in three lowest layers may be able to proliferate.     

Combined regulation of ornithine and S-adenosylmethionine decarboxylases by spermine and the spermine analogue N1 N12-bis(ethyl)spermine.

Glycogenolysis was studied in glycogen-rich perfused livers in which glycogen phosphorylase was fully converted into the a form by exposure of the livers to dibutyryl cyclic AMP. We monitored intracellular Pi by 31P n.m.r. Perfusion with Pi-free medium during 30 min caused a progressive decrease of the Pi signal to 50% of its initial value. In contrast, exposure of the livers to KCN and/or 2,4-dinitrophenol resulted in a rapid doubling of the Pi signal. Alterations in the intracellular Pi coincided with proportional changes in the rate of hepatic glycogenolysis (measured as the output of glucose plus lactate). The results indicate that the rate of glycogenolysis catalysed by phosphorylase a depends linearly on the hepatic Pi concentration. Hence the Km of phosphorylase a for its substrate Pi must be considerably higher than the concentrations that occur in the cytosol, even during hypoxia.     

The formation of radiolabeled N1- and N8-acetylspermidine in various rat organs following [14C]spermidine administration

To detect the in vivo formation of acetylspermidine, three female Sprague-Dawley rats were injected intravenously with 20 muCi of [14C]spermidine. Twenty-four hours after the injection of the radiolabel, the kidneys, liver, lungs, pancreas, small intestine, spleen, stomach and thymus were removed under Halothane anesthesia. High performance liquid chromatography (HPLC) of the pooled radiolabeled extracts from each tissue demonstrated the presence of a 14C-labeled material which co-chromatographed with an acetylspermidine standard. Thin layer chromatography (TLC) of the HPLC eluent demonstrated the presence of both radiolabeled N1- and N8-acetylspermidine in the tissues. Concentrations of labeled acetylspermidine ranged from 0.27 to 1.9% of the total tissue radiolabel. The N1-to N8-acetylspermidine ratio in tissues ranged from approx. 5 to 1 in the thymus to 1.5 to 1 in the liver.     

Heterologous cross-linking of Lyt-2 (CD8) to the alpha beta-T cell receptor is more effective in T cell activation than homologous alpha beta-T cell receptor cross-linking

Ag recognition of Lyt-2 (CD8)-positive T lymphocytes requires the presentation by APC of a suitably processed Ag in association with MHC class I molecules. In previous studies we have obtained evidence that, for optimal activation, both the alpha beta-TCR and Lyt-2 have to participate in this recognition process. In the current study we investigate the functional consequences of limited cross-linking of these cell surface molecules by using soluble, dimeric hetero- and homoconjugates of mAb to Lyt-2 and to the TCR beta-chain (F23.1). Heterologous cross-linking of Lyt-2 to the TCR induced a vigorous, selective Lyt-2+ T cell proliferative response. Functionally active cytotoxic cells were generated, and a high frequency of responding cells was observed in limiting dilution analyses. In contrast, homologous TCR cross-linking initiated a less pronounced proliferation with a relatively low frequency of response, whereas Lyt-2 cross-linking resulted in no cellular proliferation. Significant T cell activation occurred with exposure to anti-Lyt-2: F23.1 mAb dimers at concentrations an order of magnitude lower than those required for stimulation by F23.1:F23.1 mAb dimers. The induction of proliferation by mAb dimers occurred in the absence of Fc components and in rigorously APC depleted, purified T cell preparations. Effective stimulation of resting T cells could be induced also by heterodimers of monovalent Fab fragments. Heterologous cross-linking of Lyt-2 to the TCR was superior to homologous TCR cross-linking primarily with respect to proliferation in IL-2 containing media and to IL-2R expression, whereas proliferation in response to other lymphokines and the production of IL-2 itself were similar under both cross-linking regimens. Thus, when linked to the TCR, Lyt-2 contributed a strong, positive signal toward IL-2-dependent growth of resting T cells. We assume that in the case of Ag-driven T cell activation, the class I MHC molecule acts as the physiologic cross-linking ligand for Lyt-2 and the TCR.     

Induction of spermidine/spermine N1-acetyltransferase in rat tissues by polyamines.

Treatment of rats with spermidine, spermine or sym-norspermidine led to a substantial induction of spermidine/spermine N1-acetyltransferase activity in liver, kidney and lung. The increase in this enzyme, which was determined independently of other acetylases by using a specific antiserum, accounted for all of the increased acetylase activity in extracts from rats treated with these polyamines. Spermine was the most active inducer, and the greatest effect was seen in liver. Liver spermidine/spermine N1-acetyltransferase activity was increased about 300-fold within 6 h of treatment with 0.3 mmol/kg doses of spermine; activity in kidney increased 30-fold and activity in the lung 15-fold under these conditions. The increased spermidine/spermine N1-acetyltransferase activity led to a large increase in the liver putrescine content and a decline in spermidine. These changes are due to the oxidation by polyamine oxidase of the N1-acetylspermidine formed by the acetyltransferase. Our results indicated that spermidine was the preferred substrate in vivo of the acetylase/oxidase pathway for the conversion of the higher polyamines into putrescine. The induction of the spermidine/spermine N1-acetyltransferase by polyamines may provide a mechanism by which excess polyamines can be removed.