Modulation of DNA intercalation by resveratrol and genistein

Time correlated Single Photon Counting study (TCSPC) was performed for the first time to evaluate the effect of resveratrol (RES) and genistein (GEN) at 10–100 μM and 10–150 μM respectively, in modulating the DNA conformation and the variation induced due to intercalation by the dyes, ethidium bromide (EtBr) and acridine orange (AO). It is demonstrated using UV-absorption and fluorescence spectroscopy that RES and GEN, at 50 μM and 100 μM respectively can bind to DNA resulting in significant de-intercalation of the dyes, preventing their further intercalation within DNA. Hyperchromicity with red/blue shifts in DNA when bound to dyes was reduced upon addition of RES and GEN. DNA-dependent fluorescence of EtBr and AO was quenched in the presence of RES by 87.97% and 79.13% respectively, while similar quenching effect was observed for these when interacted with GEN (85.52% and 83.85%). It is found from TCSPC analysis that the higher lifetime component or constituent of intercalated dyes (τ₂, A ₂) decreased with the subsequent increase in smaller component or constituent of free dye (τ₁, A ₁) after the interaction of drugs with the intercalated DNA. Thus these findings signify that RES and GEN can play an important role in modulating DNA intercalation, leading to the reduction in DNA-directed toxicity.     

DNA staining in agarose and polyacrylamide gels by methyl green

ABSTRACT

Methyl green (MG) is an inexpensive, nonproprietary, traditional histological stain for cell nuclei. When bound to DNA and upon excitation with orange-red light, it fluoresces brightly in the far red region. We compared MG with ethidium bromide (EtBr), the conventional stain for DNA in gels, and Serva DNA stain G? (SDsG), a proprietary stain marketed as a safer alternative to EtBr for staining of electrophoresed DNA bands in agarose and polyacrylamide gels. DNA-MG fluorescence was recorded and 2.4 μg/ml MG produced crisp images of electrophoresed DNA after incubation for 10 min. Stain solutions were stable and detection limits for faint bands as well as relative densitometric quantitation were equivalent to EtBr. MG, EtBr and SDsG cost 0.0192, 0.024 and 157.5 US cents/test, respectively. MG is an effective stain for visualizing DNA in agarose and polyacrylamide gels. Its major advantages including low cost, comparable quality of staining, storage at room temperature, photo-resistance and low mutagenic profile outweigh its disadvantages such as staining of tracking dye and requirement for a gel documentation system with a red filter.     

De-intercalation of ethidium bromide and acridine orange by xanthine derivatives and their modulatory effect on anticancer agents: a study of DNA-directed toxicity enlightened by time correlated single photon counting

Time Correlated Single Photon Counting (TCSPC) was used for the first time to analyze the effect/changes in the mode of intercalation of ethidium bromide (EtBr) and acridine orange (AO) to calf thymus DNA brought about due to interaction of naturally occurring methylxanthines such as theophylline (X1), theobromine (X2) and caffeine (X3). UV absorption and fluorescence studies were also carried to observe the behaviour of these xanthines on the modulation of the binding mode of anticancer agents (cisplatin, novantrone, and actinomycin D) and certain intercalating dyes (EtBr and AO) to DNA. In TCSPC analysis we found that when the concentration of the drugs (X1, X2 and X3) increased from 0.025 mM to 2 mM i.e. P/D 2.4 to P/D 0.03 reduction in intercalation of EtBr and AO was observed, suggesting that xanthine derivatives could play very important role in reducing the DNA-directed toxicity in a dose dependent manner. In TCSPC, the amplitude of smaller lifetime component A(1) and higher lifetime component A(2) are attributed to free and intercalated dye concentration and their variation could indicate the process of intercalation or reduced intercalation of EtBr and AO by xanthine derivatives. We found that at the maximum drug concentration the smaller lifetime component A(1) was increased by 7-8% and 17-37% in EtBr and AO intercalated complex respectively. Also the changes in lifetime and fluorescence decay profile were observed for the DNA-intercalated dyes before and after treatment with xanthines. Especially, at maximum P/D 0.03 the lifetime of DNA-intercalated EtBr and AO reduced by 1-2 ns. The present analysis reveals that xanthines are able to interact with free dyes and also with intercalated dyes, suggesting that when they interact with free dyes they might inhibit the further intercalation of dye molecules to DNA and the interaction with intercalated dyes might lead to displacement of the dyes resulting in de-intercalation. The results obtained from UV and fluorescence spectroscopy also support the present investigation of probable interaction of xanthines with the DNA damaging agents in modulating/reducing the DNA-directed toxicity.     

An evaluation of DNA fluorochromes, staining techniques, and analysis for flow cytometry. I. Unperturbed cell populations

Several preparative techniques (detergent treatment, ethanol fixation, and hypotonic cell lysis), DNA fluorochromes, and methods of numerical analysis (planimetric or curve-fitting) were compared for the estimation of cell-cycle kinetic parameters (G1, S, G2 + M) by flow cytometry. In addition, coefficients of variation (CV), relative fluorescence, and G1/chicken erythrocyte (CRBC) ratios were measured and the effects of the proportion of cycling cells and cellular RNA content were examined. DNA fluorochromes were ranked by relative fluorescence: 4,6-diamidino-2-phenylindole > ethidium bromide/mithramycin > Hoechst 33342 > mithramycin > ethidium bromide > acridine orange approximately equal to propidium iodide. The first four (DNA-specific stains) gave lower CVs than the remainder (DNA intercalators). Detergent treatment also increased relative fluorescence and slightly lowered CVs. Comparable results were obtained for the kinetic parameters independently of stain or staining procedure; intercalating dyes with cells of a high RNA content not treated with RNAse and acridine orange being the exceptions. Of the two methods of numerical analysis, the planimetric technique was more consistant. Although highly consistant G1/CRBC ratios were obtained for any one stain, independently of staining procedures, variations between stains were noted. It is suggested that the detergent treatment in combination with DNA-specific stains provide optimal results.     

Study of the structural organization of RNA from phage MS2 using fluorescent dyes

The spatial organization of phage MS2 RNA by binding to ethidium bromide (EtBr) and acridine orange (AO) to RNA was studied. The analyses of dye interaction by spectrophotometric and fluorometric methods have demonstrated that only about a half of 65-70% nucleotides of double-stranded segments can interact with AO and EtBr. On the other hand all the single-stranded segments appear to be accessible to AO binding. These interactions did not practically change when ionic strength (0.01-0.3), Mg2+ and Zn2+ concentrations (10(-3) M) or pH (4.7-7.4) varied. The data permit to suppose that phage MS2 RNA has a very stable tertiary structure which makes part of double-stranded segments unaccessible to inter calating dyes. Taking these and other facts into consideration we suppose that double-stranded segments play an important role in stabilization of the RNA tertiary structure. One of the most possible structure is a compact "rod-like" intramolecular aggregate of double-stranded hairpin-like segments of RNA with parallel orientation.     

Study of the secondary and tertiary structure of phage MS2 RNA using nucleases and fluorescent dyes specific for secondary structure

The binding of ethidium bromide (EtBr) and acridine orange (AO) to RNA in native state or after hydrolysis by S1 and SV nucleases that specifically split single-stranded and double-stranded segments was studied. Nuclease S1 hydrolysis of RNA does not increase the number of EtBr strong binding sites, Tm and hyperchromic effect being also unchanged. Hydrolysis by double-stranded segments accessible to EtBr is followed by the diminishing of Tm and hyperchromism. A supposition is put forward that the main role in stabilization of the RNA tertiary structure is played by double-stranded segments arranged so that some of them are hidden and do not interact with dyes. One of the possible models may be parallel oriented intramolecular "hair-pins" forming compact "rod-like" structures.     

Comparison of the conformation of RNA from phage MS2 and 16S rRNA. Interaction with dyes specific for the secondary structure of native RNA and RNA subjected to hydrolysis by nuclease S1

The interaction of ethidium bromide (EtBr) with double-stranded (ds), and acridine orange (AO) with single-stranded (ss) fragments of 16S rRNA Escherichia coli in a wide range of ionic strength, at various pH, Zn2+ ion concentrations and partial hydrolysis by nuclease S1 was investigated. It was shown that about 90% of the RNA molecule is accessible to both dyes, when the ionic strength is near of 0.01 (pH 7). Approximately half of the RNA becomes inaccessible to dyes, when the ionic strength was increased up to 0.08-0.24 (pH 4.7-7), independent on the presence of Zn2+ ions (10(-3) M). About a half of the ds-, and a quarter of the ss-segments of the RNA, deduced from the secondary structure model were protected from the interaction with EtBr and AO. The hydrolysis of about a half of ss-segments upon addition of the Zn2+ (10(-3) M) ions did not affect the RNA tertiary structure. The experimental data obtained confirm the idea of the existence of some "nucleus" (or "nuclei") within the 16S rRNA molecule. The "nucleus" seems to be inaccessible to the dyes and is very stable to heat denaturation. It was supposed that this structure is organized by means of interaction of some of the parallelly oriented ds-segments, as it was suggested earlier for the phage MS2 RNA structure.     

"Liquidless" cell staining by dye diffusion from gels and analysis by laser scanning cytometry: potential application at microgravity conditions in space

BACKGROUND: Conventional staining of cells or tissue sections on microscope slides involves immersing the slides into solutions of dyes then rinsing to remove the unbound dye. There are instances, however, when use of stain solutions is undesirable-e.g., at microgravity conditions in space, where the possibility of accidental spill (many dyes are known carcinogens) introduces health hazard. Likewise, transporting bulk of liquid stains and rinses may be burdensome in certain situations such as field expeditions or combat. METHODS: The "liquidless" staining procedure is proposed in which the dyes are contained in thin strips of hydrated polyacrylamide or gelatin gels that have been presoaked in the stain solutions. Fluorochromes that have affinity to DNA (propidium iodide, PI; 4,6-diamidino-2-phenylindole, DAPI, Hoechst 33342) or to protein (sulforhodamine 101) were used to saturate the gels. The gel strips were placed over the prefixed cells or tissue sections deposited on microscope slides and relatively low (20 g/cm2) pressure was applied to ensure the contact. The cells were also stained by using commercially available mounting media into which DAPI or PI were admixed. Intensity of fluorescence of the PI stained cells was measured by laser scanning cytometry (LSC). RESULTS: Satisfactory cell and tissue staining, with minimal background, was achieved after 10-20 min contact between the cells and gels. Optimal concentrations of the dyes in the solutions used to presoak the gels was found to be 2-4-fold higher than the concentrations used routinely in cytometry. The measurements of intensity of cellular fluorescence by LSC revealed that the staining of DNA was stoichiometric as reflected by the characteristic cellular DNA content frequency histograms with distinct G1, S, and G2/M cell populations and 2:1 ratio of G2/M to G1 peak fluorescence. Individual gels can be saturated with more than a single dye-e.g., to obtain differential DNA and protein staining. Cell staining with DAPI or PI in the gelatin-based mounting media led to high fluorescence background while staining with DAPI in "aqueous" medium was satisfactory. CONCLUSIONS: Relatively fast staining of cells or tissue sections on microscope slides can be achieved by nonconvective dye diffusion using hydrated gels permeated with the dyes, applied to cells at low pressure. The quality of the staining provided by this methodology is comparable to conventional cell staining in dye solutions.     

Down-regulation of Bcl2 and Survivin,and up-regulation of Bax involved in copper (II) phenylthiosemicarbazone complex-induced apoptosis in leukemia stem-like KG1a cells

Previous studies suggested that phenylthiosemicarbazones are considered as a new apoptosis-inducing agent. In this study, anti-proliferative and apoptotic effects of the copper (II) phenylthiosemicarbazone complex (Cu-PTSC) were investigated in human acute myeloid leukemia KG1a cell line. The KG1a cells were treated with various concentrations (20−140 μM) of the Cu-PTSC, and cell viability was determined by MTT assay. The IC₅₀ value of 80 ± 2.5 μM was selected for further evaluations. Apoptosis, as the antitumor strategy in the cells, was investigated morphologically by acridine orange/ethidium bromide (AO/EtBr) double staining, and surface expression assay of phosphatidylserine by Annexin V/PI technique was studied via flow cytometry. Results indicated that the cells undergo morphologic changes with chromatin condensation and G₀/G₁ cell cycle arrest after treatment with Cu-PTSC. The presence of phosphatidylserine on the outer surface of the cell membrane confirmed the apoptosis occurrence in the KG1a cells. Real-time PCR and western blot analyses revealed that the incubation of KG1a cells with Cu-PTSC down-regulated the expression of Bcl-2 (anti-apoptotic protein) and Survivin (as an IAP protein) while induced the expression of Bax (pro-apoptotic protein). Based on present data, it seems that Cu-PTSC may provide a novel therapeutic approach for the treatment of acute myeloid leukemia.     

Antiproliferative and apoptosis-induction studies of 5-hydroxy 3′,4′,7-trimethoxyflavone in human breast cancer cells MCF-7: an in vitro and in silico approach

The aim of this study was to find the efficacy of 5-hydroxy 3′,4′,7-trimethoxyflavone (HTMF), a flavonoid compound isolated from the medicinal plant Lippia nodiflora, in inhibiting the proliferation and inducing apoptosis in human breast cancer cell line MCF-7. The anti-proliferative effect of the compound HTMF was confirmed using MTT cytotoxicity assay. Increased apoptotic induction by HTMF was demonstrated by acridine orange/ethidium bromide (AO/EtBr) and Hoechst 33258 staining studies. The phosphatidylserine translocation, an early feature of apoptosis and DNA damage were revealed through AnnexinV-Cy3 staining and comet assay. Moreover, the significant elevation of cellular ROS was observed in the treated cells, as measured by 2,7-diacetyl dichlorofluorescein (DCFH-DA). The mRNA expression studies also supported the effectiveness of HTMF by shifting the Bax:Bcl-2 ratio. The treatment of MCF-7 cells with HTMF encouraged apoptosis through the modulation of apoptotic markers, such as p53, Bcl-2, Bax, and cleaved PARP. In silico molecular docking and dynamics studies with MDM2-p53 protein revealed that HTMF was more potent compound that could inhibit the binding of MDM2 with p53 and, therefore, could trigger apoptosis in cancer cell. Overall, this study brings up scientific evidence for the efficacy of HTMF against MCF-7 breast cancer cells.     

Ultrasensitive staining of nucleic acids with silver

A method for ultrasensitive detection of proteins on polyacrylamide gels by staining with silver, recently described by C. R. Merril, D. Goldman, S. A. Sedman, and M. H. Ebert (Science211, 1437–1438 (1981)), was applied with slight modifications to staining nucleic acids. Silver staining of double-stranded DNA was at least 100 times as sensitive as fluorescence staining with ethidium bromide, and at least 20 times as sensitive as staining with ammoniacal silver. The limit of detection of double-stranded DNA was approximately 25–50 pg/band with a cross-sectional area of 5 mm². The intensities of silver staining of double-stranded fragments 271 bp or longer from HaeIII endonuclease digests of φX174 RF DNA were linear over a concentration range of 0.25 to 4 ng DNA/band. RNA and single-stranded DNA species as short as 10 to 20 nucleotides were detected with high sensitivity after electrophoresis on denaturing gels containing urea, suggesting that silver staining may be applicable to the sequencing of a few micrograms of unlabeled DNA. Methods for staining DNA using ammoniacal silver were relatively insensitive for small DNA fragments.     

Sensitivity of Prestaining RNA with Ethidium Bromide Before Electrophoresis and Performance of Subsequent Northern Blots Using Heterologous DNA Probes

Adding ethidium bromide (EtBr) at low concentrations to RNA samples before running formaldehyde–agarose gels affords the advantages of checking RNA integrity and evaluating the quality of size-separation at any time during electrophoresis or immediately after either electrophoresis or blotted the separated RNA onto the membrane without significantly compromising mobility, transfer, or hybridization. In this study, we systematically examined the factors that affect the sensitivity of RNA prestaining by heating RNA samples that include EtBr before electrophoresis under different denaturation conditions. We also examined the efficiency of the hybridization of EtBr-prestained RNA with heterologous DNA probes. The results showed that the fluorescent intensity of EtBr-prestained RNA was affected not only by the EtBr concentration as previously reported but also by the RNA amount, denaturation time, and denaturation temperature. Prior staining of RNA with 40 μg/mL EtBr significantly decreased the efficiency of Northern blot hybridization with heterologous DNA probes. We propose that to best combine staining sensitivity and the efficiency of Northern blot hybridization with heterologous DNA probes, the concentration of EtBr used to prestain RNA should not exceed 30 μg/mL. The efficiency of the hybridization of EtBr-prestained RNA was affected not only by factors that affect staining sensitivity but also by the type of probe used.     

A New Organic Dye-Based Staining for The Detection of Plant DNA in Agarose Gels

Ethidium bromide (EtBr) is used to stain DNA in agarose gel electrophoresis, but this dye is mutagenic and carcinogenic. We investigated N-719, which is a visible, reliable and organic Ruthenium-based dye, and five fluorescent alternatives for staining plant DNA. For prestaining and poststaining, N-719, GelRed, and SYBR Safe stained both DNA and PCR product bands as clearly as EtBr. SYBR Green I, methylene blue, and crystal violet were effective for poststaining only. The organic dye N-719 stained DNA bands as sensitively and as clearly as EtBr. Consequently, organic dyes can be used as alternatives to EtBr in plant biotechnology studies.     

Study on the interaction of aromatic dyes with nucleic acids by means of UV, CD and NMR spectroscopies.

The interaction of several aromatic cationic dyes such as, ethidium bromide (EB), methylene blue (MB), acridine orange (AO), and Hoechst 33258 with calf-thymus DNA and poly(A)-poly(U) duplex was investigated. The different induced extrinsic Cotton effects (greater than 300 nm) were observed for DNA- and RNA-dye complexes. The binding properties of these complexes were examined by UV, CD, and NMR spectroscopies.     

Two-Step Cell-Death Kinetics In Vitro During Cis-Platinum, Hydroxyurea and Mitomycin Incubation

A human leukaemic cell line (REH) growing in suspension was incubated with cis-platinum, hydroxyurea and mitomycin C at various concentrations causing complete cell-cycle arrest. At different times the cell suspensions were harvested, diluted 1:1 with a buffer solution, stained without further treatment with a mixture of acridine orange (AO) and ethidium bromide (EB) and analysed with a biparametrical flow cytometer. Fluorescent plastic beads were introduced into the suspensions to provide an internal numerical reference for the control of cell loss. The fluorescence distributions showed three groups of cells: vital cells (V) which were only stained with AO; dead cells in which EB stained cytoplasmic components but not the nuclear DNA (D1), and dead cells which allowed EB to stain both cytoplasm and nuclear DNA (D2). the kinetics of cells entering D1 depended on drug concentration and showed equal characteristics for cis-platinum and mitomycin, but were different for hydroxyurea. the subsequent entry into D2 occurred about 15 hr later and showed no pronounced dependence on drug concentration. Parallel trypan-blue (TB) exclusion tests revealed that TB only stained D2 cells and therefore is not useful for investigating cell-death kinetics during exposure to cell-killing agents.     

Retention of vital dyes correlates inversely with the multidrug-resistant phenotype of adriamycin-selected murine fibrosarcoma variants

Retention of the vital dyes rhodamine 123 (R-123) and hydroethidine (HET) correlates inversely with the multidrug resistant phenotypes of the adriamycin (ADM)-selected variants of a uv-induced murine fibrosarcoma cell line (UV-2237M). The differential affinity of these dyes for specific cellular organelles makes them unique compounds for studies of cellular transport. HET enters viable cells freely, is dehydrogenated to ethidium bromide (EtBr), and is subsequently accumulated in the nucleus. Viable cells are impermeable to extracellular EtBr, facilitating kinetic analysis of the efflux of intracellular EtBr. We found that the metabolite EtBr was rapidly cleared by ADM-resistant but not by ADM-sensitive cells. R-123 has a high affinity to mitochondria. Our results show that ADM-sensitive cells retain R-123 whereas the ADM-resistant cells do not. The clearance of both R-123 and EtBr from these cells was inhibited by verapamil. Therefore, R-123 and HET may be considered MDR-associated compounds useful in studying the MDR phenotype of cancer cells. Previously we reported a direct correlation between the level of activity of the calcium- and phospholipid-dependent protein kinase (protein kinases C) and ADM resistance in UV-2237M variant lines. In this report, we demonstrate a direct correlation between cellular calcium and MDR in these cells. Although chelation of extracellular calcium by EDTA did not alter the fluorescence profile of R-123 of the various cell lines, treating the ADM-resistant variants with verapamil restored cellular calcium to the same level as that of the parental cells and, at the same time, retarded the facilitated efflux of R-123 and EtBr and partially reversed cancer cell resistance to ADM.     

New amino and acetamido monomethine cyanine dyes for the detection of DNA in agarose gels

Some new monomethine cyanine dyes derived from quinoline and benzothiazole have been prepared and characterized by (1)H and (13)C NMR, FTIR, FABHRMS, and visible spectroscopy. The dyes containing amino and acetamido groups were conveniently synthesized by the condensation of two p-toluenesulfonate heterocyclic quaternary salts and were obtained in the forms of iodide, bromide, and tosylate counteranions. These dyes were compared to ethidium bromide as stains for DNA in electrophoretic gels. The overall results obtained for the sensitivity of these dyes suggest the suitability of acetamido moiety over the amine one and bromide as the counteranion when compared with iodide and tosylate, with a similar capacity of DNA detection in relation to the ethidium bromide stain over the concentration range of 1-3ng.     

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.     

Use of ethidium for the cytochemical study of chromatin]

A cytochemical method of chromatin study by means of nuclear staining with ethidium (bromide) is described. Dependence of stain binding by chromatin on ethidium concentration, ionic composition and buffer pH value has been analyzed. It is suggested that cells be stained in 2.10(-5) M solution of ethidium in 0.1 M tris-HCl buffer at pH 8.0 during 30 min. The fluorescence of nuclei stained with ethidium under conditions described is shown to reflect changes in physico-chemical properties of chromatin taking place in the course of its chemical modification and physiological activation in regenerating liver. The use of ethidium for chromatin cytochemistry allows to study chromatin properties in wide ranges of pH. Some other advantages of the method suggested over the commonly used method of acridine orange staining are discussed.     

Denaturation and condensation of intracellular nucleic acids monitored by fluorescence depolarization of intercalating dyes in individual cells

The intercalating binding of planar aromatic dye molecules to nucleic acids can be analyzed using fluorescence depolarization measurements of the dye molecules excited by linearly polarized light. In this study, we investigated the conformational changes of the intracellular DNA-dye complex in single cells. Flow cytometry, combined with a newly developed double-beam autocompensation technique, permitted rapid high-precision fluorescence depolarization measurements on a large number of individual cells. The dyes ethidium bromide (EB), propidium iodide (PI), and acridine orange (AO) were used in this study. Depending on the dye-to-phosphate ratio of the nuclear acid-dye complex, as well as on the spatial dye structure itself, internal and external binding sites can be monitored by fluorescence depolarization analysis. Both energy transfer and rotation and vibration of the dye molecules cause depolarization of the fluorescence emission. Differences in the concentration-dependent dye fluorescence depolarization values between PI and EB on one side and AO on the other side can be interpreted as a denaturation and condensation of double-stranded DNA regions by AO. We further show that the fluorescence polarization measurement technique can be used in an alternative way to monitor thermal denaturation of cellular DNA.