Gene transfection activities of amphiphilic steroid-polyamine conjugates

The design and evaluation of a novel potent class of DNA delivery agents based on steroid-polyamine conjugates bearing a flexible linker are reported. The hydrophobic regions are based on steroids, i.e. chlolestane and lithocholic acid motifs. The linker, which couples a hydrophobic steroid and a hydrophilic polyamine, in this study can be regarded as a two-atom extension of the conventional carbamate linker. We found that the gene transfection activity of the steroid-polyamine conjugates is influenced by the polyamine chain length and steroid structure. Molecular modeling of the relevant amphiphilic molecules revealed low-energy structures in which the polyamine chains are folded rather than stretched. This work suggests a significant effect of space-filling, i.e. the shape and orientation of the hydrophilic and hydrophobic regions, upon the efficiency of gene transfection.     

A silicone polymer as a Steroid Reservoir for enzyme-catalyzed Steroid reactions

Since steroids are only slightly soluble in the aqueous solutions in which enzymatic reactions take place, it is difficult to obtain high effective concentrations per unit reactor volume when enzymes are used to catalyze steroid reactions. In order to obtain high effective concentrations in the present work, we have used small particles of a hydrophobic polymer, poly (dimethyl siloxane), as a reservoir for the steroid substrate and product. The activity of a bacterial hydroxysteroid dehydrogenase in a buffer solution declines much more slowly in the presence of those polymer particles than in the presence of a comparable amount of butyl acetate or ethyl acetate, the organic solvents used as steroid reservoirs in previous work with steroid transforming enzymes. When another substrate of the hydroxysteroid dehydrogenase is loaded into the polymer particles and the particles are suspended in an aqueous solution containing the enzyme and its cofactor, more product is formed that when a similar solution is emulsified with butyl acetate.     

Molecular basis for the binding of competitive inhibitors of maize polyamine oxidase

Maize polyamine oxidase (MPAO), the only member of the polyamine oxidase (PAO) family whose three-dimensional structure is known, is characterized by a 30 A long U-shaped catalytic tunnel located between the substrate binding domain and the FAD. To shed light on the MPAO ligand binding mode, we studied the inhibition properties of linear diamines, agmatine, prenylagmatine (G3), G3 analogues, and guazatine, and analyzed the structural determinants of their biological activity. Linear diamines competitively inhibited MPAO, with the inhibitory activity increasing as a function of the number of methylene groups. With regard to the guanidino competitive inhibitors, including agmatine, G3, and G3 analogues, the presence of a hydrophobic substituent constitutes the principal factor influencing MPAO inhibition, as the addition of a hydrophobic substituent to the guanidino group of both G3 and G3 analogues greatly increases the inhibitory activity. Moreover, results obtained by a molecular modeling procedure indicated that in their preferred orientation, G3 analogues point the ammonium group toward the narrow entrance of the tunnel, while the terminal hydrophobic group is located within the large entrance. The high binding affinity for MPAO exhibited by G3 and G3 analogues bearing a prenyl group as a substituent on the guanidino moiety is in agreement with the observation that the prenyl group binds in a well-defined hydrophobic pocket, mainly formed by aromatic residues. Finally, docking simulations performed with the charged and uncharged forms of MPAO inhibitors indicate that the stereoelectronic properties of the MPAO active site are consistent with the binding of inhibitors in the protonated form.     

Syntheses of [12]aneN₃-oligopeptide conjugates as effective DNA condensation agents

With the aim to develop effective and low toxicity DNA condensation agents, a series of oligopeptide derived macrocyclic polyamine [12]aneN(3) conjugates 7a-h·3HCl have been designed and synthesized through multi-step amidation reactions. Structure-property study through gel electrophoresis proved that the conjugates containing high hydrophobic ending amino acids exhibited effective condensation ability at concentration of 150-250 μM, which was further confirmed by dynamic light scattering and atomic force microscopy experiments. EB displacement assay, ionic salt effect, and structure-property relationship in gel electrophoresis indicated that DNA condensation resulted from both the electrostatic interaction of [12]aneN(3) unit and hydrogen-bonding/hydrophobic multi-interaction of oligopeptide moiety in the conjugates with DNA. The reversible condensation process and their low cytotoxicity suggest that the new condensing agents are potential for the development of non-viral vectors.     

Polyamine-polyamine oxidase interaction: part of maternal protective mechanism against fetal rejection.

Human retroplacental blood serum significantly (p less than 0.01) suppresses the in-vitro uptake of 3H-thymidine--that is, synthesis of deoxyribonucleic acid--by spontaneously growing human lymphocytes in the presence of exogenous spermine, but only in concentrations with a higher polyamine oxidase activity than that found in maternal peripheral blood serum during pregnancy. These findings together with observations that the placenta is rich in spermine and that interaction of polyamine oxidase and substrate arrests cell proliferation suggest that such interaction might represent a localised immunoregulatory mechanism in the placental bed, which might contribute to the protection of the fetoplacental unit from possible maternal immune rejection.     

Dissociation of tumour-promoter-induced effects on prostaglandin release, polyamine synthesis and cell proliferation of 3T3 cells.

The phorbol ester 12-O-tetradecanoylphorbol 13-acetate induces tumour promotion, inflammation, cell proliferation and prostaglandin release. Recent reports suggest that the prostaglandins released by 12-O-tetradecanoylphorbol 13-acetate (TPA) initiate a cascade of events leading to polyamine synthesis and cell proliferation. In experiments designed to test this contention, it was found that addition of TPA (1 microM to 1 nM) to confluent mouse 3T3 fibroblasts successively caused the release of prostaglandins E2 and I2, induction of the enzyme ornithine decarboxylase (EC 4.1.1.17), stimulation of [3H]thymidine incorporation into DNA, and cell proliferation. Pretreatment of the cells with the anti-inflammatory steroid dexamethasone (1 microM) or the non-steroidal anti-inflammatory drug indomethacin (1 microM) inhibited TPA-induced prostaglandin release. However, dexamethasone enhanced the other effects of TPA, whereas indomethacin was ineffective. Addition of prostaglandin E2 to the cultures did not induce ornithine decarboxylase activity and cell proliferation. Pretreatment of the cells with 1,3-diaminopropane (1 mM) or alpha-methylornithine (5 mM), inhibitors of polyamine synthesis, decreased TPA-induced ornithine decarboxylase activity without affecting DNA synthesis. TPA stimulated [3H]thymidine incorporation into DNA, even when the ornithine decarboxylase activity was completely blocked. These data suggest that the proliferative effect of TPA on 3T3 cells is independent of prostaglandin release and polyamine synthesis.     

Stepwise assembly of a glucocorticoid receptor.hsp90 heterocomplex resolves two sequential ATP-dependent events involving first hsp70 and then hsp90 in opening of the steroid binding pocket

A system of five purified proteins that assembles stable glucocorticoid receptor (GR)-hsp90 heterocomplexes has been reconstituted from reticulocyte lysate. Two proteins, hsp90 and hsp70, are required for the activation of steroid binding activity that occurs with heterocomplex assembly, and three proteins, Hop, hsp40, p23, act as co-chaperones that enhance activation and assembly (Morishima, Y., Kanelakis, K. C., Silverstein, A.M., Dittmar, K. D., Estrada, L., and Pratt, W. B. (2000) J. Biol. Chem. 275, 6894-6900). Here we demonstrate that the first step in assembly is the ATP-dependent and hsp40 (YDJ-1)-dependent binding of hsp70 to the GR. After elimination of free hsp70, these preformed GR.hsp70 complexes can be activated to the steroid binding state by the hsp70 free assembly system in a second ATP-dependent step. hsp90 is required for opening of the steroid binding pocket and is converted to its ATP-dependent conformation during this second step. We predict that hsp70 in its ATP-dependent conformation binds initially to the folded receptor and is then converted to the ADP-dependent form with high affinity for hydrophobic substrate. This conversion initiates the opening of the hydrophobic steroid binding pocket such that it can now accept the hydrophobic binding form of hsp90, which in turn must be converted to its ATP-dependent conformation for the pocket to be accessible by steroid.     

The role of tyrosine in the binding of steroid by progesterone binding globulin from the guinea pig

Treatment of progesterone binding globulin (PBG) with tetranitromethane (TNM) resulted in a loss of steroid binding activity (inactivation) which was dependent on both time and concentration of reagent. Scatchard analysis of binding revealed that inactivation was due to a decrease in binding site number with no effect upon the affinity of PBG for steroid. Incorporation studies demonstrated that the loss of binding activity correlated with the incorporation of 1.3 nitro groups per molecule of PBG. The involvement of the steroid binding site in the reaction was shown by the ability of progesterone, but not cortisol, to protect against inactivation. Treatment with N-acetylimidazole did not inactivate PBG nor did the conversion of nitrotyrosyl residues to amino-tyrosines regenerate binding activity, suggesting that the pheolic hydroxyl is not involved in steroid binding. These studies suggest that inactivation was due to the incorporation of a bulky group into the aromatic ring of a tyrosine present at the steroid binding site thus blocking its ability to participate in hydrophobic interactions with the ligand.     

Electrostatic interactions of androgens and progesterone derivatives with rainbow trout estrogen receptor

In primary cultures of immature male rainbow trout (rt) hepatocytes, vitellogenin (Vg) gene expression is regulated by E(2) via the estrogen receptor (ER). However, steroids other than estrogens can also stimulate Vg gene expression. These steroids are hardly converted into E(2) during incubation and their stimulatory activity is completely inhibited by tamoxifen implying rtER involvement. These steroids have no or a slightly positive charge on the Connolly surface. In contrast, steroids that failed to stimulate Vg gene expression had a strong positive or negative charge around rings C and D due to polarization. The amino acid sequences of the ligand binding domains (LBD) of rtER and human ER alpha have 57.7% homology; only one amino acid differs in the presumed steroid binding site. We modeled the three-dimensional structure of the LBD of rtER using X-ray crystallographic data for hER alpha in order to investigate the fit (structural and electrostatic) between steroid and rtER. Two factors are essential for binding to rtER: (i) hydroxyl or carbonyl groups near C3 and C17 of the steroids (hydrophilic regions) that can form hydrogen bonds with His(489), Arg(359), and Glu(318), (ii) a hydrophobic steroid nucleus that interacts with a hydrophobic region of the rtER LBD through van der Waals forces. If polar functional groups are present, the hydrophobic interaction between steroid and the rtER LBD is considerably weakened.     

Regulation of polyamine transport in Chinese hamster ovary cells

Control Chinese hamster ovary (CHO) cells and mutant CHO cells lacking ornithine decarboxylase activity (CHODC-) were used to study the regulation of polyamine uptake. It was found that the transport system responsible for this uptake was regulated by intracellular polyamine levels and that this regulation was responsible for the maintenance of physiological intracellular levels under extreme conditions such as polyamine deprivation or exposure to exogenous polyamines. Polyamine transport activity was enhanced by decreases in polyamine content produced either by inhibition of ornithine decarboxylase with alpha-difluoromethylornithine in CHO cells or via polyamine starvation of CHODC- cells. The provision of exogenous polyamines resulted in rapid and large increases in intracellular polyamine content followed by decreased polyamine transport activity. Soon after this decrease in uptake activity, intracellular polyamine levels then fell to near control values. Cells grown in the presence of exogenous polyamines maintained intracellular polyamine levels at values similar to those of control cells. Protein synthesis was necessary for the increase in transport in response to polyamine depletion, but appeared to play no role in decreasing polyamine transport. Bis(ethyl) polyamine analogues mimicked polyamines in the regulation of polyamine transport but this process was relatively insensitive to regulation by methylglyoxal bis(guanylhydrazone), a spermidine analogue known to enter cells via this transport system and to accumulate to very high levels.     

Bis(benzyl)polyamine analogs as novel substrates for polyamine oxidase

N,N'-Bis(benzyl)polyamine analogs were found to be substrates for highly purified polyamine oxidase. Metabolism of these analogs was apparently dependent on molecular O2 and resulted in the formation of benzaldehyde, H2O2, and a polyamine analog with free terminal amines. The debenzylation reaction was optimal between pH 9 and 10, identical to the pH optimum for polyamine oxidase activity when N1-acetylspermine was used as the substrate. On a molecular sieve column the debenzylating activity co-eluted with N1-acetylspermine oxidizing activity, at an apparent molecular mass of approximately 65 kDa. The purified enzyme also appeared to have a molecular mass of approximately 65 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Debenzylation of the bis(benzyl)polyamines was competitively inhibited by N1-acetylspermine and N1-acetylspermidine. The specific irreversible inhibitor of polyamine oxidase, N1,N4-bis(buta-2,3-dienyl)butanediamine also inhibited the debenzylation, whereas inhibitors of diamine and monoamine oxidases did not. The evolution of benzaldehyde from bis(benzyl)polyamine analogs by polyamine oxidase allowed the development of a simple rapid spectrophotometric assay for use in the measurement of polyamine oxidase activity in partially purified tissue or cell extracts. Further, metabolism of a bis(benzyl)polyamine analog by polyamine oxidase was found to be an important element in the growth inhibitory properties of the compound in a mouse model of malaria.     

High-performance hydrophobic interaction chromatography of proteins

A new, weakly hydrophobic, high-performance liquid chromatography column has been developed for the separation of native proteins based on their relative hydrophobicities. Starting with a covalently bound, hydrophilic polyamine matrix, packing materials were synthesized through acylation with anhydrides and acid chlorides of increasing chain length to obtain increasingly hydrophobic surfaces. Proteins in aqueous buffers were induced to bind hydrophobically to the columns by the use of high salt concentrations in the mobile phase. Elution was achieved by decreasing the ionic strength of the solvent in a linear gradient. A mixture of cytochrome c, conalbumin, and beta-glucosidase was used as a standard to test the resolving power of newly synthesized columns. On a 4-cm butyrate column, baseline resolution was achieved in 20 min with a gradient of 3.0 mu sodium sulfate in 0.1 M potassium phosphate buffer, pH 7.0, to water. The static loading capacity for each column was determined using a hemoglobin binding assay. Capacities normally ranged between 150 and 180 mg of hemoglobin per gram of support. Since proteins are not denatured in hydrophobic interaction chromatography, enzymes eluted from the column retained enzymatic activity. Samples of alpha-amylase and beta-glucosidase ranging in size from 10 to 200 micrograms were recovered from the butyrate column with greater than 92% enzymatic activity in all cases. In a single trial, the enzyme citrate synthase was recovered from the benzoate column with 92% retention of enzymatic activity.     

Differences in polyamine metabolism between carcinomatous and uninvolved human breast tissues

The polyamine biosynthetic enzymes ODC and SAMDC show higher activity in carcinomatous human breast tissue than in uninvolved tissue of the same breast; the interconversion enzyme PAO shows significantly lower activity in carcinomatous than uninvolved tissue. The polyamine metabolism in carcinomatous human breast tissue thus appears to differ from that in uninvolved tissue. Intracellular polyamine concentrations, particularly spermine, are high in carcinomatous tissue. This increase and that of the biosynthetic enzymes suggest that a higher polyamine concentration is needed for carcinomatous cell growth. If this is the case, the lower PAO activity in carcinomatous tissue may be explained as a mechanism that conserves the high intracellular polyamine concentration.     

Changes in free polyamines and related enzymes during stipule and pod wall development in Pisum sativum

Level of free polyamines, their key metabolic enzymes, and other features related to ageing were examined during stipule and pod wall development in pea (Pisum sativum). Free polyamine titre (per unit fresh mass) in both the organs, the specific activities of arginine decarboxylase and ornithine decarboxylase in the pod wall, gradually decreased with maturation. In stipule, these enzymes attained peak activity at 15 days after pod emergence and declined thereafter. Ornithine decarboxylase activity was greater in pod wall than in stipule; while, arginine decarboxylase activity was higher in stipule. Activity of degradative enzyme diamine oxidase increased with the onset of senescence in both the organs. Chlorophyll and electrical conductance had a inverse relationship throughout the experimental period, whereas, the chlorophyll content was directly related with polyamine levels in both stipule and pod wall during aging. On the other hand, protein and RNA contents were positively correlated with free polyamines throughout the test period in stipule, but in the pod wall this was true only for the later stages of development.     

TATA-binding protein-associated factor 7 regulates polyamine transport activity and polyamine analog-induced apoptosis

Identification of the polyamine transporter gene will be useful for modulating polyamine accumulation in cells and should be a good target for controlling cell proliferation. Polyamine transport activity in mammalian cells is critical for accumulation of the polyamine analog methylglyoxal bis(guanylhydrazone) (MGBG) that induces apoptosis, although a gene responsible for transport activity has not been identified. Using a retroviral gene trap screen, we generated MGBG-resistant Chinese hamster ovary (CHO) cells to identify genes involved in polyamine transport activity. One gene identified by the method encodes TATA-binding protein-associated factor 7 (TAF7), which functions not only as one of the TAFs, but also a coactivator for c-Jun. TAF7-deficient cells had decreased capacity for polyamine uptake (20% of CHO cells), decreased AP-1 activation, as well as resistance to MGBG-induced apoptosis. Stable expression of TAF7 in TAF7-deficient cells restored transport activity (55% of CHO cells), AP-1 gene transactivation (100% of CHO cells), and sensitivity to MGBG-induced apoptosis. Overexpression of TAF7 in CHO cells did not increase transport activity, suggesting that TAF7 may be involved in the maintenance of basal activity. c-Jun NH2-terminal kinase inhibitors blocked MGBG-induced apoptosis without alteration of polyamine transport. Decreased TAF7 expression, by RNA interference, in androgen-independent human prostate cancer LN-CaP104-R1 cells resulted in lower polyamine transport activity (25% of control) and resistance to MGBG-induced growth arrest. Taken together, these results reveal a physiological function of TAF7 as a basal regulator for mammalian polyamine transport activity and MGBG-induced apoptosis.     

High expression of antizyme inhibitor 2, an activator of ornithine decarboxylase in steroidogenic cells of human gonads

High activity of ornithine decarboxylase (ODC), the rate-limiting enzyme of polyamine synthesis, is typically present in rapidly proliferating normal and malignant cells. The mitotically inactive steroidogenic cells in rodent testis and ovaries, however, also display high ODC activity. The activity of ODC in these cells responds to luteinizing hormone, and inhibition of ODC reduces the production of steroid hormones. Polyamines and ODC also control proliferation of germ cells and spermiogenesis. The activity of ODC, especially in proliferating cells, is regulated by antizyme inhibitor (AZIN). This protein displaces ODC from a complex with its inhibitor, antizyme. We have previously identified and cloned a second AZIN, i.e. antizyme inhibitor 2 (AZIN2), which has the highest levels of expression in brain and in testis. In the present study, we have used immunohistochemistry and in situ hybridization to localize the expression of AZIN2 in human gonads. We found a robust expression of AZIN2 in steroidogenic cells: testicular Leydig cells and Leydig cell tumors, in ovarian luteinized cells lining corpus luteum cysts, and in hilus cells. The results suggest that AZIN2 is not primarily involved in regulating the proliferation of the germinal epithelium, indicating a different role for AZIN1 and AZIN2 in the regulation of ODC. The localization of AZIN2 implies possible involvement in the gonadal synthesis and/or release of steroid hormones.     

Polyamine Oxidase Activity in Sera of Depressed and Schizophrenic Patients after ECT Treatment

High level of polyamine oxidase activity is detected in sera of depressed as well as in schizophrenic patients. ECT treatment of depressed and schizophrenic patients reduced significantly the level of polyamine oxidase activity in their sera. After ECT treatment, clinically improved depressed and schizophrenic subjects were found to have sera polyamine oxidase activity not significantly differ from that of normal subjects. Possible biochemical mechanisms, which link polyamine oxidase activity, schizophrenia, depression and ECT effect are discussed here.     

Polyamine conjugates of meso-tritolylporphyrin and protoporphyrin IX: potential agents for photodynamic therapy of cancers

An efficient five-step synthesis method was developed to obtain tritolylporphyrin and protoporphyrin IX polyamine conjugates. These compounds were composed of either one polyamine unit (spermidine or spermine) covalently tethered to monocarboxyphenyl tritolylporphyrin or two molecules of polyamines borne by protoporphyrin IX. In each compound, an aliphatic spacer arm is linked to the N(4) polyamine position. Photocytotoxicity of these new compounds was evaluated against K562 human chronic myelogenous leukemia cells and compared to Photofrin II; protoporphyrin IX polyamine conjugates exhibited much stronger photocytocicity than Photofrin II and were shown to readily induce necrosis in treated cells.     

The role of polyamine reutilization in depletion of cellular stores of polyamines in non-proliferating tissues

It was known from previous work that specific inhibition of neither ornithine decarboxylase activity nor polyamine oxidase activity produces spermidine depletion by more than 20% in non-growing organs, which are in a steady state with regard to polyamine metabolism. Combined treatment with inactivators of both ornithine decarboxylase and polyamine oxidase for a prolonged time caused, however, a gradual decrease of spermidine levels in liver, kidney and brain of mice by 50% and more. The method is in accordance with the previously suggested role of polyamine interconversion. Inhibition of polyamine oxidase prevents the reutilization for de novo polyamine biosynthesis of putrescine and spermidine, which are formed by oxidative splitting of N1-acetylspermine and N1-acetylspermidine, respectively, and the ornithine decarboxylase inhibitor prevents the compensatory increase of putrescine from ornithine. The findings are further evidence for the physiological significance of polyamine reutilization.