DNA Interaction and photonicking properties of DNA-targeted acridine (2,2'-Bipyridine)platinum(II) complexes

Synthesis of two (2,2'-bipyridine)platinum(II) complexes tethered to one or two acridine chromophores is reported. These acridine complexes efficiently unwind and photocleave supercoiled plasmid DNA under physiological conditions of temperature and pH.     

Interaction between tryptophan‐Sm(III) complex and DNA with the use of a acridine orange dye fluorophor probe

The interaction of the Trp–Sm(III) complex with herring sperm DNA (hs‐DNA) was investigated with the use of acridine orange (AO) dye as a spectral probe for UV‐vis spectrophotometry and fluorescence spectroscopy. The results showed that the both the Trp–Sm(III) complex and the AO molecule could intercalate into the double helix of the DNA. The Sm(III)–(Trp)₃ complex was stabilized by intercalation into the DNA with binding constants: K^?_25°C = 7.14 × 10⁵ L·mol^?1 and K^?_37°C = 5.28 × 10⁴ L·mol^?1, and it could displace the AO dye from the AO–DNA complex in a competitive reaction. Computation of the thermodynamic functions demonstrates that Δ_rH_m^? is the primary driving power of the interaction between the Sm(III)(Trp)₃ complex and the DNA. The results from Scatchard and viscometry methods suggested that the interaction mode between the Sm(III)(Trp)₃ complex and the hs‐DNA is groove binding and weak intercalation binding. Copyright © 2015 John Wiley & Sons, Ltd.     

Circular dichroism and structure of the complex of acridine orange with poly(L-glutamic acid)

Circular dichroism and absorption spectra have been measured on solutions of acridine orange and poly(L -glutamic acid) mixed at two molar ratios of carboxyl group-to-dye, P/D 25 and 0.8, and at different pH's. Characteristic circular dichroism is induced at the absorption bands of acridine orange when the P/D is 25 and the solution is acidic. Another type of circular dichroism is manifest at neutral and alkaline pH when P/D is 0.8. For the induction of the former type of circular dichroism, a helical array of acridine-orange dimers bound to the α-helix is postulated, in which the dye molecular planes are almost perpendicular to the helical axis. Assuming the helical geometry and optical parameters, combined with the observed magnitude of transition electric moment, the rotatory strength of the complex is calculated to the zeroth order approximation, and the observed circular dichroism spectra have been reproduced.     

Elimination of plasmid-linked polyglutamate production by Bacillus subtilis (natto) with acridine orange

Treatment of Bacillus subtilis (natto) strains Asahikawa, F, and M with acridine orange resulted in the conversion of approximately 64.2% of the Asahikawa population, 22.4% of the F population, and 9.2% of the M population to polyglutamate-nonproducing colonies. Such curing is suggestive of the involvement of plasmid DNA. Samples of cleared lysates of both parental and their cured strains were subjected to agarose gel electrophoresis to determine the plasmid composition. Parental strains were found to possess a plasmid, but polyglutamate-nonproducing derivatives were missing the plasmid. The plasmid-linked polyglutamate production, which was originally isolated from B. subtilis (natto), could be transformed in B. subtilis.     

Dimethyl-10,12-benz(a)acridine; evidence for differential effects on the synthesis of RNA of mammalian or avian fibroblasts and some RNA viruses.

We have studied the differential effect of dimethyl-10,12-benz(a)acridine (DBMAcr) on the synthesis of RNA of chicken or mouse fibroblasts in culture and that of some RNA-containing viruses such as Rous sarcoma virus and Mengovirus. DMBAcr at low concentrations blocks the cell multiplication of both normal and Rous sarcoma virus-transformed chicken fibroblasts in culture; it affects transformed cells more than normal ones. The cell growth inhibiting effect of DMBAcr is reversible after short periods of incubation. DMBAcr depresses the synthesis of cellular DNA and RNA in parallel. Concurrently the synthesis of protein proceedes at a relatively high rate in DMBAcr-treated cultures. Its inhibitory effect on cellular RNA synthesis is mostly due to a block in the formation of 28 S and 18 S ribosomal RNA species; in contrast, the synthesis of 45 S ribosomal RNA precursor is proceeding at almost control rate. Also, the synthesis of heterogeneous nuclear RNA is not blocked by DMBAcr. The production of Rous sarcoma virus in transformed fibroblasts is not affected by DMBAcr. Since this is correlated with persisting high rates of protein and heterogenous nuclear RNA synthesis, the effects of DMBAcr suggest that the synthesis of Rous sarcoma virus-RNA shares the specificity of messenger and heterogeneous nuclear RNA. DMBAcr inhibits the synthesis of viral RNA of Mengovirus under conditions where the synthesis of total cellular RNA is not appreciably depressed, suggesting its differential effect on the DNA-directed and the RNA-directed RNA synthesis.     

Effects of an acridine half-mustard (ICR 191) on growth and ploidy of frog cells in culture

The effects of an acridine half-mustard, ICR 191, on the growth rate and ploidy of four haploid and two diploid lines of Rana pipiens cells in culture were studied. Growth curves indicate that the haploid and diploid cell lines were equally resistant to a 4-hour exposure of this drug (0.1 micrometer to 10 micrometer. ICR 191 treatment induced the haploid cell cultures to become diploid. The proportion of diploid cells increased progressively with respect to time after the 4-hour exposure period. The greater the concentration of ICR 191 applied, the more rapid the rate of conversion. Autoradiographic determinations of percent labelled nuclei indicate that DNA synthesis was not inhibited in haploid or in diploid cells. Therefore, the increased proportion of diploid cells did not originate from the small percentage of diploid cells in the initial population. Instead the haploid cells were converted to diploid cells. Time lapse cinematography indicated that the conversion mechanism was other than cell fusion. Conversion to higher ploidy did not occur when diploid cell cultures were exposed to ICR 191.     

The effect of atebrine and an acridine analog (BCMA) on the coenzyme fluorescence spectra of cultured melanoma and Ehrlich ascites (EL2) cells

Summary Coenzyme fluorescence spectra of single living cells are due to free pyridine nucleotides (folded configuration), bound pyridine nucleotides (unfolded configuration) and a third component, possibly a mixture of flavins. Such spectra can be used to recognize possible differences in coenzyme composition between cell lines or changes of metabolic pathways due to chemicals acting at levels below or above cytotoxicity, by high resolution spectrofluorometry.A study of spectra recorded from cultured Ehrlich ascites (EL2), and Harding Passey melanom a cells (HPM-67 and HPM-73 line) grown under comparable conditions, shows that free NAD(P)H predominates in HPM-67 and EL2, while this coenzyme is bound in HPM-73. The free/bound ratio may be profoundly modified by chemicals, e.g. in the HPM-73 increase of free and decrease of bound NAD(P)H occurred upon treatment with 10^–6 oligomycin.When atebrine at levels (10^–6 M) below cytotoxicity was added, there was a decrease of the free NAD(P)H spectrum possibly through energy transfer from NAD(P)H to atebrine. Consideration of long range energy transfer i.e., excitation of atebrine by fluorescence of NAD(P)H vs. short range transfer of excitation energy from free NAD(P)H to atebrine, favors the latter mechanism. A transient (reversible) increase in atebrine fluorescence is seen following intracellular microinjection of substrate (e.g. glucose-6-P) leading to an increase in free NAD(P)H. At cytotoxic levels of atebrine (e.g. 2×10^–5 M) an irreversible increase of atebrine fluorescence is seen.The microspectrofluorometric technique appears therefore well suited to study physiological processes at the level of intracellular coenzymes, as well as possible processes of intermolecular energy transfer in the microenvironment.     

Induced circular dichroism and mode of binding of acridine orange adsorbed on β-form poly(S-carboxyethyl-L-cysteine) in aqueous solutions

The absorption spectra and circular dichroism (CD) have been measured for aqueous solutions of acridine orange of a constant concentration, [D] = 5 × 10^?5M, mixed with poly(S-carboxyethyl-L -cysteine) in various mixing ratios, [P]/[D], ranging from 330 to 11, at different pH. The absorption spectra of the dye–polymer solutions are hypochromic, and the main band is located at 470 nm, accompanying a shoulder at 500 nm. At alkaline pH, no CD is induced in the visible region. At neutral and acidic pH, where the polymer is in the β-conformation, CD is induced in the visible and near-uv regions. A pair of CD bands is located at the region around 450 nm, when the pH is around the neutrality, while it appears at the region around 500 nm at acidic pH. Thus, the optically active species of bound dye changes from dimer to monomer on lowering the pH. These species form dissymmetric arrays along a polypeptide chain. The fraction of bound dye forming dissymmetric sequences is not high, but most of bound dye is adsorbed randomly on the ionized carboxyl groups of polypeptide chain and gives rise to hypochromism only. A dissymmetric structure of dye–polymer complexes is presented, in which the polymer has the β-conformation and the dye cations, either dimeric or monomeric, bind to its side chains, in such a way that the longer axes of molecular planes of bound dye form a two-fold, right-handed helix along the extended polypeptide chain. A zeroth-order calculation of CD based on the coupled oscillator model leads to the result that each dissymmetric array of dye consists, on the average, of two dimeric or monomeric cations. This low number of bound cations in a dissymmetric array and the large fraction of randomly adsorbed dye suggest that the hydrophobic interaction of dye with the polymer is strong, so that dye cations are adsorbed sparsely on both sides of the extended polypeptide chain.     

Induction of cytoplasmically inherited respiration-deficient ('petite') mutants by photodynamic action of acridine compounds

All acridines used (acriflavine, proflavine, acridine orange and 3-azido-10-methylacridinium chloride) produced killing in yeast cells when activated with visible light. Acriflavine, proflavine and 3-azido-10-methylacridinium chloride, but not acridine orange, produced petite and sectored colonies. Both cell killing and petite induction by light activation of acriflavine resulted apparently from photodynamic action mediated by singlet oxygen (1O2) since the effect were prevented by either sodium azide or anaerobiosis. The biological effects of 3-azido-10-methylacridinium chloride, which was developed as a potential photoaffinity probe for studying the binding and biological effects of acridines, appeared to be due to a photodynamic action analogous to that of acriflavine. Sodium azide or anaerobiosis prevented the light-activated effects of 3-azido-10-methylacridinium chloride despite the fact that the initial chemical breakdown of the azido derivative induced by light was not affected. Cells suspended in D2O demonstrated an enhanced response to 3-azido-10-methylacridinium chloride with irradiation. These results indicate that singlet oxygen mediates the light-activated biological effects of both acriflavine and 3-azido-10-methylacridinium chloride.     

Elimination of plasmid-linked polyglutamate production by Bacillus subtilis (natto) with acridine orange.

Treatment of Bacillus subtilis (natto) strains Asahikawa, F, and M with acridine orange resulted in the conversion of approximately 64.2% of the Asahikawa population, 22.4% of the F population, and 9.2% of the M population to polyglutamate-nonproducing colonies. Such curing is suggestive of the involvement of plasmid DNA. Samples of cleared lysates of both parental and their cured strains were subjected to agarose gel electrophoresis to determine the plasmid composition. Parental strains were found to possess a plasmid, but polyglutamate-nonproducing derivatives were missing the plasmid. The plasmid-linked polyglutamate production, which was originally isolated from B. subtilis (natto), could be transformed in B. subtilis.     

The mechanism of action of 3,6-bis(2-piperidinoethoxy)acridine trihydrochloride (CL 246,738) in the potentiation of natural killer cells

An effort has been made to determine the mechanism by which the immunomodulator 3,6-bis(2-piperidinoethoxy)acridine trihydrochloride (CL 246,738) enhances the cytotoxic activity of natural killer (NK) cells. Orally administered CL 246,738 produced augmentation of NK cell activity in mice in a dose-related fashion over a dose range of 10 to 160 mg/kg, with a peak stimulation occurring at 40 mg/kg. The stimulatory effect was short-lived and only persisted for 3 days after a single oral dose of the drug. However, it could be boosted by a subsequent treatment. With anti-asialo GM-1 (anti-ASGM-1) antibody used as an NK cell marker, it was determined that the compound increased the number of ASGM-1-positive cells in mice, as indicated by radioimmunoassay and immunofluorescence staining. NK cells of beige mice were also activated by CL 246,738. Furthermore, the compound at concentrations of 0.02 to 0.2 microgram/ml induced NK cell activity in vitro, with a minimum 3-day incubation being required for optimal activation. This effect was dependent on the presence of macrophages and was inhibited by anti-IFN-alpha + beta but not anti-IFN-beta antibody. Taken together, it is postulated that the compound functions by stimulating macrophages to release IFN-alpha, which subsequently activates NK cells. As an effective stimulator of IFN and NK cells, CL 246,738 may prove clinically useful in the immunotherapy of certain types of malignancy.     

The effect of atebrine and an acridine analog (BCMA) on the coenzyme fluorescence spectra of cultured melanoma and Ehrlich ascites (EL2) cells.

Coenzyme fluorescence spectra of single living cells are due to free pyridine nucleotides (folded configuration), bound pyridine nucleotides (unfolded configuration) and a third component, possibly a mixture or flavins. Such spectra can be used to recognize possible differences in coenzyme composition between cell lines or changes of metabolic pathways due to chemicals acting at levels below or above cytotoxicity, by high resolution spectrofluorometry. A study of spectra recorded from cultured Ehrlich ascites (EL2), and Harding Passey melanoma cells (HPM-67 and HPM-73 line) grown under comparable conditions, shows that free NAD(P)H predominates in HPM-67 and EL2, while this coenzyme is bound in HPM-73. The free/bound ratio may be profoundly modifed by chemicals, e.g. in the HPM-73 increase of free and decrease of bound NAD(P)H occurred upon treatment with 10(-6) oligomycin. When atebrine at levels (10(-6) M) below cytotoxicity was added, there was a decrease of the free NAD(P)H spectrum possibly through energy transfer from NAD(P)H to atebrine. Consideration of long range energy transfer i.e., excitation of atebrine by fluorescence of NAD(P)H vs. short range transfer of excitation energy from free NAD(P)H to atebrine, favors the latter mechanism. A transient (reversible) increase in atebrine fluorescence is seen following intracellular microinjection of substrate (e.g. glucose-6-P) leading to an increase in free NAD(P)H. At cytotoxic levels of atebrine (e.g 2 x 10(-5) M) an irreversible increase of atebrine fluorescence is seen. The microspectrofluorometric technique appears therefore well suited to study physiological processes at the level of intracellular coenzymes, as well as possible processes of intermolecular energy transfer in the microenvironment.     

Enumeration of chlorine-stressed organisms with acridine orange 2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyl tetrazolium chloride (AOINT)

Direct microscopic enumeration ofEnterobacter cloacae with the acridine orange 2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyl tetrazolium chloride technique (AOINT) was compared with spread plate counts on nonselective media to establish the usefulness of the former technique in the enumeration of chlorine-stressed cells. Results indicate that the techniques are comparable when the organisms are not stressed. However, AOINT is more sensitive than are plate counts in the detection of chlorine-stressed cells.     

Induction of tumor-inhibitory macrophages with a novel synthetic immunomodulator, 3,6-bis(2-piperidinoethoxy)acridine trihydrochloride (CL 246,738)

3,6-bis(2-piperidinoethoxy)acridine trihydrochloride (CL 246,738) has been investigated for its immunomodulatory effect on murine macrophages. Incubation of macrophages harvested from the peritoneal cavities of normal mice with the compound for 48 to 72 hr rendered these cells inhibitory to the growth of tumor cells in vitro. Activation of tumor-inhibitory macrophages occurred over a range of concentrations (0.025 to 0.1 micrograms/ml) producing no direct inhibitory effects on tumor cells. Treatment of effector cells with carrageenan abrogated the effect, whereas treatment with anti-Thy-1.2 antibody and C did not, suggesting that the primary effectors were macrophages rather than T lymphocytes. These activated macrophages also manifested in vitro tumor cytolysis. In vivo studies indicated that peritoneal macrophages from mice treated with single oral doses of 100 to 400 mg/kg of the compound were also inhibitory to tumor cell growth in vitro. Effector macrophages became demonstrable in mice as early as 1 day after drug administration, reached peak activity at day 12, and disappeared by day 31, indicating a rapid onset but long-persisting effect. The tumor cytostatic activity of these macrophages was augmented by endotoxin at the dose of endotoxin that, in itself, had no effect. The addition of protease inhibitors, N-alpha-p-tosyl-L-lysine chloromethyl ketone and aprotinin, to cultures markedly diminished the cytostatic effect, suggesting that the release of neutral protease(s) could account for the inhibitory effects of the macrophages. On the other hand, hydrogen peroxide and arginase seemed excluded as the mechanism of action because the effect was not sensitive to treatment with catalase and exogenous arginine. The present findings indicate that CL 246,738 is an orally active immunopotentiator capable of inducing tumor-inhibitory macrophages both in vitro and in vivo.     

Quinacrine and 9-amino acridine inhibit B-Z and B-H(l) form DNA conformational transitions

The interaction of quinacrine and 9-amino acridine with right-handed B-form, left-handed Z-form, and left-handed protonated (H(L))-form structures of polydG-me(5)dC was investigated by circular dichroism and absorption spectral analysis. Both the compounds bind strongly to the B-form structure and convert the Z-form and H(L)-form back to the bound right-handed form. Circular dichroic data revealed that the conformation at the binding site is right-handed even though adjacent regions of the polynucleotide may have left-handed conformation. The rate and extent of B-form-to-Z-form transition were decreased in the presence of these compounds. Scatchard analysis revealed that both quinacrine and 9-amino acridine bind strongly to the polynucleotide in the B-form in a noncooperative manner, in sharp contrast to the highly cooperative binding to the Z-form and H(L)-form. Results indicated that the cooperative binding of these drugs with the Z-form and the H(L)-forms was associated with a sequential conversion of the polynucleotide from a left-handed to a bound right-handed conformation. Experimental data enabled the calculation of the number of base pairs of Z-form (7-8 with quinacrine and 9-amino acridine) and H(L)-form (4 and 25, respectively, with quinacrine and 9-amino acridine) that adopt a right-handed conformation for each bound ligand. As these compounds are known to bind preferentially to alternating guanine--cytosine sequences, which are capable of easily undergoing the B-to-Z or B-to-H(L) transition, these effects may be important in understanding their biological activities.     

The effect of acridine orange on the structure of ribosomes

Exposure of Escherichia coli MRE-600 ribosomes to acridine orange (AO) at low ionic strength (1mM Tris-acetate pH 7.4) results in quantitative binding of the dye. Under our experimental conditions about a few hundred dye molecules can be bound to any one of the 30, 50, or 70-S particles. AO causes the 30 and the 50-S subunits to form ribosomal aggregates of approximate sedimentation constants of 70 and 100-S.     

Titration of cardiolipin by either 10-<Emphasis Type="Italic">N</Emphasis>-nonyl acridine orange or acridine orange sensitizes the adenine nucleotide carrier to permeability transition

Under the action of carboxyatractyloside or fatty acids, adenine nucleotide translocase switches its function from nucleotide carrier to modulator of the opening of a non-specific pore. In addition to the effect of these agents, this modification in activity is, in some way, dependent on the influence of the lipid milieu of the membrane. Cardiolipin is, among other membrane phospholipids, the one that interacts the most with the translocase. This work shows that 10-N-nonyl acridine orange and acridine orange, probes for this phospholipid, modify the sensitivity of the translocase to carboxyatractyloside, oleate, and palmitate to induce permeability transition. The results also show that these probes stimulate the release of mitochondrial cytochrome c, and increase labeling of the carrier by eosin 5-maleimide. Based on the aforementioned it is proposed that the increase in sensitivity is due to a conformational change in the translocase, induced by the binding of the probe to cardiolipin.