Fluorescence polarization study of DNA--proflavine complexes

The binding of proflavine to DNA has been studied by measuring the polarization and intensity of emission of DNA–dye complexes. Such measurements also permit the determination of the fluorescence of the bound dye as a function of the degree of binding. Techniques of emission spectroscopy permit the study of complexing at high phosphate to dye ratios, and we have examined complexes formed at up to 12,300:1 phosphates to dye. At high phosphate to dye ratios, we find that equilibrium plots of the binding data show only one type of binding. Reports in the literature of multiple binding constants are shown to be due to the incorrect assumption that the fluorescence of the bound dye is independent of the amount bound. The emission properties can be qualitatively accounted for by assuming that nearest-neighbor interaction between bound dyes quenches the fluorescence. We report that, within experimental error, the binding constant is insensitive to the base content of the DNA. The DNA-dye complexes show a temperature dependent depolarization, the cause of which is, as yet, unknown. Heat denaturation of the DNA–dye complex may be followed on a Perrin plot.     

Dielectric behavior of DNA--dye complex. II

The dielectric relaxation of native DNA and the effect of aminoacridine dyes, such as acridine orange (AO), proflavine (PF), and ethidium bromide (EB) have been investigated at different molar DNA phosphate (P) to dye (D) ratios in the frequency range 100 Hz–100 kHz. The static dielectric constant was observed to decrease with increasing binding of aminoacridines. This was interpreted as arising from the neutralization of the surface changes of the DNA molecules as a result of dye binding. At any P/D ratio the extent of charge neutralization was greatest for AO and least for the EB–DNA complex. The relaxation time (τ) for dye-bound DNA was greater compared to that for native DNA. This increase in τ was ascribed to the increase in the length of the dye-bound DNA. The maximum value of τ occurred at P/D = 20, 10, and 2 for AO-, PF-, and EB-treated DNA, respectively. The variation of τ at various levels of binding gave a qualitative idea about the conformational changes of DNA due to its binding with the dyes.     

Fluorescence image cytometry of nuclear DNA content versus chromatin pattern: a comparative study of ten fluorochromes.

This study is intended to be the first step of an in situ exploration of the intranuclear DNA distribution by image cytometry (SAMBA) with several fluorochromes. The nuclear DNA content and the chromatin pattern, revealed by ten fluorochromes (HO, DAPI, MA, CMA3, OM, QM, AO, EB, PI, and 7-AMD), were analyzed on mouse hepatocytes fixed by the Boehm-Sprenger procedure optimal for preserving the chromatin pattern. The question was whether fluorochromes specific to DNA make it possible to accurately quantitate the total nuclear DNA content when the chromatin pattern is preserved. Only HO and MA were found to provide satisfactory quantitation of nuclear DNA content, as assumed by both a small CV and a 4c to 2c ratio equal to 2. PI, EB, 7-AMD, and OM provided higher CV values, although the 4c to 2 c ratio was still equal to 2. QM, AO, CMA3, and DAPI provided non-reproducible and non-stoichiometric nuclear DNA content measurements under the fixation conditions used. The intranuclear and the internuclear SD of the fluorescence intensities describing the fluorescence pattern of the 2c hepatocytes proved to vary according to both the basepair specificity and the binding mode of the fluorochromes. The results reported here argue in favor of an external binding of 7-AMD to DNA and an increased quantum yield of QM when bound to AT-rich DNA. For PI, EB, 7-AMD, and OM, the measured DNA content increased with the fluorescence distribution heterogeneity. This correlation was not observed with other fluorochromes and is suggested to result from decreased fluorochrome accessibility to DNA when the chromatin is condensed. This study demonstrates that under conditions that preserve chromatin organization, only HO for AT-rich DNA and MA for GC-rich DNA can be used, alone or in combination, to measure nuclear DNA content. With other fluorochromes, either the measured DNA content or the chromatin pattern is assessed in suboptimal conditions when fluorescent image cytometry is used.     

Critical evaluation of techniques to detect and measure cell death--study in a model of UV radiation of the leukaemic cell line HL60.

The reliability of eight distinct methods (Giemsa staining, trypan blue exclusion, acridine orange/ethidium bromide (AO/EB) double staining for fluorescence microscopy and flow cytometry, propidium iodide (PI) staining, annexin V assay, TUNEL assay and DNA ladder) for detection and quantification of cell death (apoptosis and necrosis) was evaluated and compared. Each of these methods detects different morphological or biochemical features of these two processes. The comparative analysis of the 8 techniques revealed that AO/EB (read in fluorescence microscopy) provides a reliable method to measure cells in different compartments (or pathways) of cell death though it is very time consuming. PI staining and TUNEL assay were also sensitive in detecting very early signs of apoptosis, but do not allow precise quantification of apoptotic cells. These three methods were concordant in relation to induction of apoptosis and necrosis in HL60 cells with the various UV irradiation time periods tested. Both AO/EB (read by flow cytometry) and annexin V-FITC/PI failed to detect the same number of early apoptotic cells as the other three methods. Trypan blue is valueless for this purpose. Giemsa and DNA ladder might be useful as confirmatory tests in some situations.     

Denaturation of nucleic acids induced by intercalating agents. Biochemical and biophysical properties of acridine orange-DNA complexes

At high binding densities acridine orange (AO) forms complexes with ds DNA which are insoluble in aqueous media. These complexes are characterized by high red- and minimal green-luminescence, 1:1 (dye/P) stoichiometry and resemble complexes of AO with ss nucleic acids. Formation of these complexes can be conveniently monitored by light scatter measurements. Light scattering properties of these complexes are believed to result from the condensation of nucleic acids induced by the cationic, intercalating ligands. The spectral and thermodynamic data provide evidence that AO (and other intercalating agents) induces denaturation of ds nucleic acids; the driving force of the denaturation is high affinity and cooperativity of binding of these ligands to ss nucleic acids. The denaturing effects of AO, adriamycin and ellipticine were confirmed by biochemical studies on accessibility of DNA bases (in complexes with these ligands) to the external probes. The denaturing properties of AO vary depending on the primary structure (sugar- and base-composition) of nucleic acids.     

Fluorescence enhancement of DNA-bound TO-PRO-3 by incorporation of bromodeoxyuridine to monitor cell cycle kinetics.

BACKGROUND:The detection of DNA-incorporated bromodeoxyuridine (BrdUrd) in mammalian cells is a well-known and important technique to study cell cycle. The use of TO-PRO-3 for detection of BrdUrd substitution of DNA by dual-laser flow cytometry has been investigated. METHODS:Fluorescence enhancement of TO-PRO-3 in BrdUrd-labeled cells is registered in combination with the fluorescence emission of the intercalating dye propidium iodide (PI) as a total DNA stain to give bivariate DNA/BrdUrd histograms. By the low concentration of only 0.3 mircoM TO-PRO-3, BrdUrd detection is optimized, and undisturbed total DNA content by PI can be detected as well. TO-PRO-3 is excited by a red HeNe laser and PI by an argon ion laser. RESULTS:In order to understand the binding of TO-PRO-3, energy transfer from PI to TO-PRO-3 has been measured as well as the influence of an external DNA binding dye such as Hoechst 33258 with Adenine-Thymine (AT) binding specificity. Cell cycle studies of human SCL-2 keratinocytes and mouse 3T3 cells prove the method to be as generally applicable as the classical BrdUrd/Hoechst quenching technique, but without need for expensive ultraviolet laser excitation. No BrdUrd sensitivity could be found for the similar dyes TO-PRO-1 and YO-PRO-3, whereas TO-PRO-5 and YOYO-3 showed only very little sensitivity to BrdUrd labeling as compared with TO-PRO-3. CONCLUSIONS:Cell cycle studies of mammalian cells can be done by dual-laser flow cytometry without the need for ultraviolet lasers by using the BrdUrd-dependent fluorescence enhancement of TO-PRO-3. Total DNA content can be measured simultaneously using PI.     

Staining with pyronin Y detects changes in conformation of RNA during mitosis and hyperthermia of CHO cells

Cellular RNA in Chinese hamster ovary (CHO) cells synchronized in mitosis (M) or G2 phase, as well as in interphase cells subjected to hyperthermia (42 degrees C, 10 min), was stained with acridine orange (AO), ethidium bromide (EB), or pyronin Y (PY) and the resultant fluorescence was measured by flow cytometry. Total RNA content detected after staining with AO increased in M as compared to G2-phase cells, consistent with continued RNA synthesis during G2 phase. The content of double-stranded RNA, stained with EB (after DNase treatment), was also somewhat higher in M cells. In contrast, the stainability of RNA with PY decreased by 27% in M- compared to G2-phase cells. Furthermore, a decrease in stainability of RNA with PY was observed in G2 cells compared to cells in G1 phase. In separate experiments, RNA stainability with AO or EB was generally unaffected when interphase CHO cells were exposed to 42 degrees C for 10 min, though this same treatment resulted in a 26% decrease in RNA stainability with PY. The decreased PY stainability of cellular RNA in M or heat-treated cells was observed at a relatively narrow range of dye concentration (1.0-2.0 micrograms/ml). The observed hypochromicity of RNA coincides with dissociation of polyribosomes into single ribosomes known to occur during mitosis and following exposure to hyperthermia. It is presumed that the phenomenon involves selective denaturation and condensation of ribosomal (r) RNA by PY in single ribosomes which does not occur in polyribosomes. While the molecular mechanisms responsible for stabilization of rRNA in polyribosomes preventing its denaturation and condensation by PY are unknown, PY appears to be a sensitive probe that can be used to detect and study these changes in rRNA confirmation in situ.     

Further fluorospectrophotometric studies on the binding of acridine orange with DNA. Effects of thermal denaturation of DNA and additions of spermine, kanamycin, dihydrostreptomycin, methylene blue and chlorpromazine

Fluorospectrophotometric studies on the binding of acridine orange (AO) with calf thymus DNA showed that the thermal denaturation of DNA reduced markedly the fluorescence of Complex II and the extent of this decrease depended on the temperature to which the DNA solutions were heated. The denaturation was carried out in the absence and presence of AO (methods A and B, respectively), and then fluorescence measurements of solutions were carried out at 23 °C. The fluorescence intensity-heating temperature curves obtained by methods A and B were similar in shape to the usual melting curves of DNA and AO-DNA solutions, respectively. The higher midpoint value obtained with method B indicates the stabilizing activity of AO against denaturation. These findings support an intercalation model for Complex II and an external self-association binding model for Complex I.A high concentration of ethylene diamine (EDA) restored the fluorescence of denatured Complex II to about 80% of the intensity value of native Complex II. The effects of spermine, kanamycin and dihydrostreptomycin were much stronger than that of EDA.Methylene blue (MB) and chlorpromazine (CP) reduced the fluorescence of native Complex II markedly. Since the analysis of the difference absorption spectra declared that MB and CP were intercalated without release of bound AO, the interacting MB and CP were considered to weaken the interaction between AO and DNA bases, that made AO more fluorescent. Free radical (CP·) of CP was prepared by a new method using H₂O₂, peroxidase, and ascorbic acid. Intercalated CP· showed a much stronger quenching effect on Complex II, indicating that unpaired electron spin contained in the costacking unit between CP· and DNA bases might affect the fluorescence of the adjacent AO molecule by paramagnetic perturbation.     

Effect of organic solvents on the properties of the complexes of DNA with proflavine and similar compounds

In order to obtain information on the binding forces involved in the formation of the complex proflavine–DNA by the stronger process I, the stability of the complexes was investigated in the presence of various organic solvents, methanol, ethanol, n-propanol, isopropanol, formamide, dimethyl sulfoxide, p-dioxane, glycerol, and ethylene glycol. Quantitative data on binding in terms of K/n and r were obtained by means of absorption and fluorescence spectra, as well as by a thermal denaturation technique. All organic solvents used decrease the binding ability of the dye. The effectiveness of the solvents increases with their hydrocarbon content, but can hardly be related to their dielectric constant. The complex formation is effectively suppressed by organic solvent concentrations, in which DNA still preserves its double-helical conformation. These results demonstrate the importance of hydrophobic forces in the formation of the complex proflavine–DNA in aqueous solution. The similarity in spectroscopic properties of proflavine bound to DNA by process I and the same dye dissolved in an organic solvent make it possible to interpret the observed red shift of the long-wavelength absorption peak as being due to the interaction of the dye molecules with the less polar environment. The same behavior was found for other dyes capable of intercalation like purified trypaflavine, phenosafranine and ethidium bromide. However, intercalation is not a necessary condition, as it was shown in the case of pinacyanol, which binds only at the surface of DNA.     

DNA-DNA cross-linking mediated by bifunctional [SalenAlIII]+ complex

The aluminum (III) complex [SalenAl(III)]Cl (1), (Salen=(R,R)-N,N'-bis[5-methyl-3-(4-methylpiperazinyl)-salicylidene]-1,2-diphenylethanediamine) has been synthesized and characterized by elemental analysis, FT-IR, (1)H and (13)C NMR measurements. The interaction of complex (1) with calf thymus (CT) DNA has been studied extensively by experimental techniques. Thermal denaturation study of DNA with (1) revealed the DeltaT(m) of 5+/-0.2 degrees C. Viscosity and steady-state fluorescence measurements showed that the complex cross-links DNA and the metal center is interacting with DNA during the cross-linking. Also, the phenyl ring in the complex may intercalate between the base pairs of the DNA during the cross-linking. Competitive binding study shows that the enhanced emission intensity of ethidium bromide (EB) in the presence of DNA was quenched by the addition of the metal complex indicating that it displaces EB from its binding site in DNA and the apparent binding constant has been estimated to be (2.8+/-0.2)x10(5) M(-1). Further, time-resolved fluorescence experiments confirm the binding of (1) with DNA and its cross-linking nature. Aluminum ions shown to precipitate DNA completely above the pH 6.0, but no such precipitation was observed with complex (1). The DNA-DNA cross-linking mediated by (1) is further confirmed by gel electrophoresis.     

Cytofluorometric and cytochemical comparisons of normal and abnormal human cells from the female genital tract.

Acridine orange staining of exfoliated cells from epithelial tissues facilitates discrimination between normal and abnormal cells: abnormal cells develop highly elevated nuclear fluorescence. Comparisons of acridine orange (AO) staining with propidium iodide (PI) or Feulgen staining have shown that: (a) PI staining also provides highly elevated nuclear fluorescence from abnormal cells; (b) the distributions of nuclear fluorescence following AO or PI staining were usually not significantly different as judged by the Kolmogorov-Smirnov test; (c) fluorescence emission spectra from AO and PI stained cells are consistent with the hypothesis that both fluorochromes bind to DNA within cell nuclei; (d) DNAse treatment of AO stained normal cells eliminates the nuclear fluorescence peak from slit-scan contours; RNAse treatment has no effect on nuclear fluorescence; (e) the distribution of abnormal cell nuclear fluorescence after AO staining is usually, but not always, significantly different from the distribution of abnormal cell nuclear absorbance after Feulgen staining, with relative nuclear fluorescence being greater than relative nuclear absorbance. The hypothesis currently most consistent with these results is that elevated Feulgen DNA content can account for only part of the discrimination provided by AO staining, and that the chromatin within abnormal cells is altered so as to increase accessibility of DNA to intercalating dyes.     

NMR determination of the hetero-association of phenanthridines with daunomycin and their competitive binding to a DNA oligomer

500 MHz (1)H NMR spectroscopy has been used to determine thermodynamic and structural information on the hetero-association of daunomycin (DAU) with the phenanthridine mutagenic dyes ethidium bromide (EB) and propidium iodide (PI). The NMR complexation data have been analysed by a statistical-thermodynamic model which takes into account indefinite association for both the self-association of the drugs and their hetero-association. The results have been used to estimate the effect of the side chains of the phenanthridines on the competitive binding between DAU and the mutagens with DNA. Knowledge of the equilibrium constants for self-association of the phenanthridines and DAU, their hetero-association and their complexation with a DNA fragment, the deoxytetranucleotide 5'-d(TpGpCpA), enabled the relative content of each of the EB-DAU, PI-DAU, EB-DAU-d(TGCA) and PI-DAU-d(TGCA) complexes to be calculated as a function of drug concentration in mixed solutions. The results provide some insight into the molecular basis of the action of combinations of biologically-active molecules. When intercalating drugs are used in combination, it is found that the decrease in binding of drug or mutagen with DNA is due both to formation of drug-mutagen hetero-association complexes in the mixed solution and to competition for the binding sites by the aromatic molecules; the relative importance of each process depends on the molecular properties of the drug or mutagen molecules being considered. Thus, the longer branched side chain of PI and the electrostatic contribution of the extra positive charge of the molecule compared with the ethyl group of EB results in lower affinity for self-association of PI molecules and their hetero-association with DAU, but increases the degree of binding of PI with DNA.     

Caffeine dissociates complexes between DNA and intercalating dyes: application for bleaching fluorochrome-stained cells for their subsequent restaining and analysis by laser scanning cytometry

BACKGROUND: Removal of the nucleic acid-bound fluorochrome is desirable when stained cells have to be reanalyzed using other fluorochromes. It is also often desirable to remove DNA-bound antitumor drugs from drug-treated cells, to improve cell staining. We have previously observed that in aqueous solutions, the methylxanthine caffeine (CFN) decreases interactions between planar aromatic molecules such as intercalating dyes or antitumor drugs and nucleic acids. The aim of this study was to explore whether this property of CFN can be utilized to remove the DNA-bound intercalating dyes propidium iodide (PI) or 7-aminoactinomycin D (7-AAD) from the cells and whether the bleached cells can be restained and reanalyzed. METHODS: HL-60 cells were fixed in 70% ethanol and their DNA was stained with PI or 7-AAD. The cells were then rinsed with a 0.05 M solution of CFN in phosphate-buffered saline (PBS) or with PBS alone. The decrease in intensity of cell fluorescence during rinsing was measured by laser scanning cytometry (LSC) to obtain the bleaching kinetics of individual cells. The bleached cells were then restained with PI, 7-AAD, or the protein-specific fluorochrome sulforhodamine 101(S101). Their fluorescence was measured again by LSC. In addition, free DNA was subjected to gel electrophoresis, DNA bands in the gels were stained with ethidium bromide (EB), and the gels were rinsed with a solution of CFN or PBS to bleach the DNA band's fluorescence. RESULTS: Rinsing the PI or 7-AAD-stained cells with solutions of CFN led to nearly complete removal of PI and a more than 75% decrease in 7-AAD fluorescence after 10 min. The rinse with PBS decreased the PI cell fluorescence intensity by less than 30% and the 7-AAD fluorescence by about 50%. The differences in kinetics of PI or 7-AAD removal by CFN from G2/M versus G1 cells suggest that these intercalators bind more strongly to DNA in chromatin of G2/M than G1 cells. The CFN-bleached cells were then successfully stained with S101 and again with PI or 7-AAD. The bivariate analysis of the LSC merged files of the cells sequentially stained with PI and S101 revealed typical DNA/protein distributions. The fluorescence of EB-stained DNA bands in gels was also nearly completely removed by rinsing gels in 0.05 M CFN; PBS alone had a distinctly lesser effect. CONCLUSION: Solutions of CFN can dissociate the DNA-bound PI, 7-AAD, EB, and possibly other intercalating fluorochromes. The bleached cells can be restained and reanalyzed by LSC. This approach can also be used to remove such fluorochromes from nucleic acids immobilized in gels and perhaps in other solid matrices. Analysis of the kinetics of fluorochrome removal from cells can possibly be used to study their binding affinities to nucleic acids in situ.     

Fluorescence anisotropy of DNA/DAPI complex: torsional dynamics and geometry of the complex.

Fluorescence depolarization of synthetic polydeoxynucleotide/4'-6-diamidino-2-phenylindole dihydrochloride complexes has been investigated as a function of dye/polymer coverage. At low coverage, fluorescence depolarization is due to local torsional motions of the DNA segment where the dye resides. At relatively high coverage, fluorescence depolarization is dominated by energy transfer to other dye molecules along the DNA. The extent of the observed depolarization due to torsional motion depends on the angle the dye molecule forms with the DNA helical axis. A large torsional motion and a small angle produce the same depolarization as a small torsional motion and a large projection angle. Furthermore, the extent of transfer critically depends on the relative orientation of dye molecules along the DNA. The effect of multiple transfer is examined using a Monte Carlo approach. The measurement of depolarization with transfer, at high coverage, allows determination of the dye orientation about the DNA helical axis. The value of the torsional spring constant is then determined, at very low coverage, for few selected polydeoxynucleotides.     

Thermodynamic and structural study of phenanthroline derivative ruthenium complex/DNA interactions: probing partial intercalation and binding properties

The binding of [Ru(PDTA-H₂)(phen)]Cl (PDTA = propylene-1,2-diaminetetra-acetic acid; phen = 1,10 phenanthroline) with ctDNA (=calf thymus DNA) has been investigated through intrinsic and induced circular dichroism, UV-visible absorption and fluorescence spectroscopies, steady-state fluorescence, thermal denaturation technique, viscosity and electrochemical measurements. The latter indicate that the cathodic and anodic peak potentials of the ruthenium complex shift to more positive values on increasing the DNA concentration, this behavior being a direct consequence of the interaction of both the reduced and oxidized form with DNA binding. From spectrophotometric titration experiments, the equilibrium binding constant and the number of monomer units of the polymer involved in the binding of one ruthenium molecule (site size) have been quantified. The intrinsic circular dichroism (CD) spectra show an unwinding and a conformational change of the DNA helix upon interaction of the ruthenium complex. Quenching process, thermal denaturation experiments and induced circular dichroism (ICD) are consistent with a partial intercalative binding mode.     

The use of fluorescence correlations spectroscopy to probe chromatin in the cell nucleus

All systems in thermodynamic equilibrium are subject to spontaneous fluctuations from equilibrium. For very small system, the fluctuations can be made apparent, and can be used to study the behavior of the system without introducing any external perturbations. The mean squared amplitude of these fluctuations contains information about the absolute size of the system. The characteristic time of the fluctuation autocorrelation function contains kinetic information. In the experiments reported here, these concepts are applied to the binding equilibrium between ethidium bromide and DNA, a system where the fluorescence properties of the dye greatly enhance the effect of spontaneous fluctuations in the binding equilibrium. Preliminary experiments employ well-characterized DNA preparations, including calif thymus DNa, SV40 DNA, and calf thymus nucleohistone particles. Additional measurements are described which have been made in small regions of individual nuclei, isolated from green monkey kidney cells, observing as few as 5000 dye molecules. The data indicate that the strength of dye binding increases in nuclei isolated from cells which have been stimulated to enter the cell growth cycle. The viscosity of nuclear material is inferred to be between one and two orders of magnitude greater than that of water, and it decreases as the cells leave the resting state and enter the cell growth cycle. Washing the nuclei also lowers the viscosity. These experiments demonstrate that fluorescence correlation spectroscopy can provide information at the subnuclear level that is otherwise unavailable.     

Denaturation and condensation of DNA in situ induced by acridine orange in relation to chromatin changes during growth and differentiation of Friend erythroleukemia cells

DNA in situ is progressively denatured when the cells or nuclei are treated with increasing concentration of acridine orange (AO). This transition can be monitored by flow cytometry as a decrease in green fluorescence. The complexes of denatured DNA and AO undergo immediate condensation and aggregation; this step is manifested by appearance of red luminescence and formation of precipitates that can be detected by electron microscopy. The precipitates form preferentially in heterochromatin as well as in ribosomes and polysomes. Their formation and further aggregation affects cellular light scatter properties in both the forward and right-angle direction. The AO-induced DNA denaturation and condensation was studied in nuclei of Friend erythroleukemia cells from exponentially growing, differentiated or quiescent cells. The DNA in nuclei of quiescent cells, from plateau-phase cultures, was the most sensitive to denaturation; it denatured (measured by changes in luminescence) at an AO concentration between 50 and 80 microM with the midpoint of the transition (Cd) at 70 microM. DNA in nuclei of differentiated cells (dimethyl-sulfoxide-induced erythroid differentiation) was more resistant (Cd = 77-83 microM), whereas DNA in exponentially growing cells was the most resistant (Cd = 86 microM). Extraction of proteins with 0.1 M HCl at 0 degree C abolished the differences between the cells and shifted the transition to a lower AO concentration (Cd = 46 microM). For comparison, the midpoint transitions representing condensation of free, nucleic acids measured as light scatter changes occurred at 13, 22, 31 and 53 microM of AO, for rRNA, tRNA, and denatured and native-calf thymus DNA, respectively. Denaturation and condensation of DNA, which can be induced by AO either in isolated nuclei or viable permeabilized or fixed cells provides a new approach to discriminate cell subpopulations with different chromatin structure by flow cytometry. The molecular mechanisms of this phenomenon are discussed.     

From GC-rich DNA binding to the repression of survivin gene for quercetin nickel (II) complex: implications for cancer therapy

The DNA binding and cleavage properties of quercetin nickel (II) complex have been studied, but little attention has been devoted to the relationship between antitumor activity of this complex and DNA-binding properties. In the present study, we report that quercetin nickel (II) complex showed significant cytotoxicity against three tumor cell lines (HepG2, SMMC7721 and A549). Hoechst33258 and AO/EB staining showed HepG2 cells underwent the typical morphologic changes of apoptosis characterized by nuclear shrinkage, chromatin condensation, or fragmentation after exposure to quercetin nickel (II) complex. We also demonstrate that the levels of survivin and bcl-2 protein expression in HepG2 cells decreased concurrently, and the levels of p53 protein increased significantly after treatment with quercetin nickel (II) complex by immunocytochemistry analysis. The relative activity of caspase-3 and caspase-9 increased significantly after treatment with the complex. Furthermore, fluorescence measurements and molecular modeling were performed to learn that the complex could be preferentially bound to DNA in GC region. These results imply that quercetin nickel (II) complex may intercalate into the GC-rich core promoter region of survivin, down-regulating survivin gene expression and promoting tumor cells apoptosis. So our results suggest that antitumor activity of quercetin nickel (II) complex might be related to its intercalation into DNA and DNA-binding selectivity, and that the complex may be a promising agent for cancer therapy.     

Fluorescent dyes for cell viability: an application on prefixed conditions

In recent years increasing attention has been given to apoptosis for its role in pathologic, organogenetic and homeostatic phenomena. Acridine orange (AO), Hoechst 33342 (HO) and propidium iodide (PI) are among the most used fluorescent dyes used to analyse cell culture viability. In fact, they respectively show specificity for living, apoptotic and late apoptosis/necrosis states. We explored whether HO, AO and PI can be used on prefixed monolayers of three commonly used cell lines. Here we mainly describe the metachromatic effects obtained by fluorescence microscopy with double and triple dye combinations. Furthermore, we propose an easy staining method in which a balanced sequential treatment with HO, AO and PI allows identification of different viability states onto fixed cells by using a long-pass FITC filter. This method extends the spectrum of suitable applications for these dyes in fluorescence viability detection onto previously fixed (prefixed) samples.     

Acridine orange interaction with DNA: Effect of ionic strength

BackgroundThe study of acridine orange (AO) spectral characteristics and the quenching of its singlet and triplet excited states by TEMPO radical at its binding to DNA in the function of the DNA concentration and in the absence and presence of NaCl is reported.MethodsThe study was performed using steady-state and time resolved optical absorption and florescence, fluorescence correlation spectroscopy and resonant light scattering techniques.ResultsThe presence of different species in equilibrium: AO monomers and aggregates bound to DNA, has been demonstrated, their relative content depending on the DNA and the AO concentrations. At high DNA concentration the AO monomers are protected against the contact with other molecules, thus reducing the AO excited state quenching. The addition of NaCl reduces the AO binding constant to DNA, thus reducing the AO and DNA aggregation.ConclusionsThe interaction of AO with DNA is a complex process, including aggregation and disaggregation of both components. This modifies the AO excited state characteristics and AO accessibility to other molecules. The salt reduces the DNA effects on the AO excited state characteristics thus attenuating its effects on the AO efficacy in applications.General significanceThis study demonstrates that the interaction of photosensitizers with DNA, depending on their relative concentrations, can both decrease and increase the photosensitizer efficacy in applications. The salt is able to attenuate these effects.