Impact of four antimutagens on apoptosis in genotoxically damaged lymphocytes in vitro

An antimutagenic activity of fluphenazine, todralazine, anthocyanins and alkylresorcinols was established in a battery of short-term cytogenetic tests. One of the possible mechanisms of their antimutagenic action could be an increase in apoptotic elimination of heavily-damaged cells from a culture. In this paper we provide data on quantitative estimation of the antimutagens' impact on apoptosis in lymphocyte cultures exposed in the G(0)-phase to genotoxic agents: hydrogen peroxide (0.2mM, 20 min.) or benzo[a]pyrene (40 microM, 90 min.), and then cultured for 36 hrs in the presence of a lectin (PHA-M, 1% v/v) and each of the tested antimutagens. Apoptosis was estimated by means of microscopic examination of cell smears stained with a mixture of fluorochromes (ethidium bromide/acridine orange) as well as of the results of DNA separation with the field inversion gel electrophoresis. By microscopic examination we assessed that the frequencies of cells exhibiting morphological features of apoptosis considerably increased in the cultures containing the antimutagens. The FIGE separation of DNA from those cultures proved that the DNA content in the 30-50 kb domain was markedly elevated, as compared with the control cultures that did not contain antimutagens. It was established in the regression analysis that the apoptosis-enhancing effect significantly depended on the concentration of each tested antimutagen in a culture medium. However, marked differences of apoptosis-enhancing potency were noticed among the four antimutagens. The multicriterial analysis proved that the apoptosis-enhancing effects of fluphenazine and also, to a smaller extent, by alkylresorcinols, were many times stronger than those of anthocyanins and of todralazine. The results suggest that the enhancement of apoptosis by fluphenazine and by alkylresorcinols can explain a major part of their antimutagenic activity, whereas in the case of anthocyanins and of todralazine other mechanisms of antimutagenic action should be sought for.     

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.     

Induction of Apoptosis and Autophagy Using Ectopic DSCR1 Expression in Breast Cancer Cells

Down syndrome critical region 1 gene (DSCR1) is an anti-angiogenesis gene that inhibits the growth of tumor cells. In this study, the role of autophagy and apoptosis in DSCR1-induced cytotoxicity were investigated in MDA-MB-468 breast cancer cells. Lentivirus vector harboring DSCR1 (LV-DSCR1⁺) was constructed in HEK 293 cells and the optimal dosage of lentivirus vector for infection was determined by the MTT assay. After infection of cells using LV-DSCR1⁺, acridine orange and ethidium bromide staining was performed to investigation of apoptosis and autophagy. Expression of DSCR1 and marker genes for angiogenesis (VEGF), apoptosis (Bax and Bcl2) and autophagy (LC3 and Beclin) were determined by Real time PCR. The cellular morphological changes related to apoptosis and autophagy was happened after 48 hours of viral infection. Fragmented bright orange nucleuses and vacuoles were observed due to the cell apoptosis and autophagy after acridine orange and ethidium bromide staining. Upregulation of Bax, Lc3, DSCR1 and Beclin1 and downregulation of Bcl2 and VEGF was detected due to treatment with LV-DSCR1⁺. These results demonstrated that LV-DSCR1⁺ can induce apoptosis and autophagy, therefore suggesting that it may serves as an efficient tool to breast cancer treatment.     

Imaging and measuring the rituximab-induced changes of mechanical properties in B-lymphoma cells using atomic force microscopy

The topography and mechanical properties of single B-lymphoma cells have been investigated by atomic force microscopy (AFM). With the assistance of microfabricated patterned pillars, the surface topography and ultrastructure of single living B-lymphoma cell were visualized by AFM. The apoptosis of B-lymphoma cells induced by rituximab alone was observed by acridine orange/ethidium bromide (AO/EB) double fluorescent staining. The rituximab-induced changes of mechanical properties in B-lymphoma cells were measured dynamically and the results showed that B-lymphoma cells became dramatically softer after incubation with rituximab. These results can improve our understanding of rituximab’effect and will facilitate the further investigation of the underlying mechanisms.     

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.     

A simple technique for quantifying apoptosis in 96-well plates

Background  

Analyzing apoptosis has been an integral component of many biological studies. However, currently available methods for quantifying apoptosis have various limitations including multiple, sometimes cell-damaging steps, the inability to quantify live, necrotic and apoptotic cells at the same time, and non-specific detection (i.e. "false positive"). To overcome the shortcomings of current methods that quantify apoptosis in vitro and to take advantage of the 96-well plate format, we present here a modified ethidium bromide and acridine orange (EB/AO) staining assay, which may be performed entirely in a 96-well plate. Our method combines the advantages of the 96-well format and the conventional EB/AO method for apoptotic quantification.     

Evaluation of a new cytotoxicity assay for Clostridium perfringens type D epsilon toxin

Abstract A new cytotoxicity assay for determining the activity of epsilon toxin produced by Clostridium perfringens type D has been developed. Viability of cultured cells was determined by the ability of only live cells to convert 5-(3-carboxymethoxyphenyl)-2-(4,5-dimethylthiazolyl)-3-(4-sulfophenyl)tetrazolium to the coloured product formazan in the presence of phenazine methosulfate. Of the 12 cell lines tested, only the MDCK cell line was susceptible to epsilon toxin. Specificity was confirmed by the ability of only specific monoclonal antibodies to inhibit cytotoxicity. Good correlation was obtained with the mouse lethality assay ( r = 0.991) and over a wide range of viability (15–75%) as determined by ethidium bromide/acridine orange staining ( r = 0.995).     

Analysis of commercial samples of acridine orange using high-performance liquid chromatography

We analyzed commercial samples of acridine orange using reverse-phase high-performance liquid chromatography. The mobile phase of 90:10:2.5 acetonitrile:de-ionized water:pentane sulfonic acid gave baseline separations of components of acridine orange samples in 15 min. Many of the samples were fairly homogeneous; the absorbance due to the acridine orange component ranged from 83-97%. Among the 11 samples tested, one was not acridine orange, reputedly different samples were identical, and the principal component of one sample was not acridine orange.     

A comparison of fluorochromes for direct viable counts by image analysis

Total direct and direct viable counts of fresh and injured cultures of Escherichia coli were determined by image analysis in preparations stained with acridine orange, ethidium bromide and 4',6-diamidino-2-phenyl indole (DAPI). Cells stained with DAPI were not detected by image analysis. Fresh cultures stained with acridine orange or ethidium bromide gave comparable counts. Injured E. coli stained with ethidium bromide gave higher counts that with acridine orange. Injured cultures stained with acridine orange contain high proportions of green cells which are less easily detected than red cells in image analysis. In certain cases it may be better to use ethidium bromide, which stains all cells red, for direct viable counts by image analysis.     

维甲酸对A549细胞增殖和凋亡及Skp2、p27^kip1蛋白表达的影响

目的探讨维甲酸对A549细胞增殖和凋亡及相关基因表达的影响。方法MTT法观察ATRA对A549细胞增殖的抑制作用;流式细胞仪、AO/EB荧光双染法检测细胞凋亡;免疫细胞化学检测ATRA处理前后A549细胞Skp2、p27^kip1蛋白表达的情况。结果ATRA处理后①MTT法结果显示ATRA对A549细胞具有增殖抑制作用,在一定范围内呈时间-剂量依赖性。②AO/EB荧光双染色法观察到ATRA 25μmol/L作用A549细胞48h后,即可发现典型的凋亡形态学改变。③流式细胞仪结果出现凋亡峰,与对照组细胞相比,实验组细胞周期延长,主要表现为G0/G1期细胞比例增加,同时S期细胞比例减少。④免疫细胞化学结果显示,ATRA 25μmol/L处理细胞48h后,维甲酸处理组Skp2有明显下调,p27^kip1则明显上调。结论ATRA具有抑制肺腺癌A549细胞增殖,诱导细胞凋亡的作用,其机制可能与下调Skp2,上调p27^kip1蛋白的表达水平有关。     

Cytological Analysis of Anastomoses and Vegetative Incompatibility Reactions in <Emphasis Type="Italic">Helicobasidium monpa</Emphasis>

Our examination of the cytological characteristics of the vegetative incompatibility reaction in a filamentous basidiomycete, Helicobasidium monpa, by analyzing the fluorescence emitted by ethidium bromide and acridine orange stained nuclei is described. Hyphal anastomoses between strains belonging to different mycelium compatibility groups (MCG) were observed with cell death in fused hyphae, whose nuclei were intensified by ethidium bromide. In contrast, the nuclei in a living cell were not intensified by staining with ethidium bromide, but were intensified by staining with acridine orange. These results indicate that in H. monpa, ethidium bromide staining is a useful method for detecting dead cells. We also examined the relationships between the alternation of ploidy and hyphal anastomosis formation using the newly developed method on filamentous fungi. The tetraploid monokaryon strain derived from the original dikaryon strain by continuous subculture could not be fused to any wild type strains, but the original dikaryon strain could be fused without cell death to only the same MCG strain. In contrast, the haploid dikaryon strain derived from the original monokaryon strain fuses to several strains belonging to different MCGs without cell death. These results suggested that the cellular ploidy of this fungus is closely related to its mating system and, H. monpa may be a self-fertilizing fungus. Received: 13 June 2001 / Accepted: 8 August 2001     

Increased DNA polymerase and ATP-dependent deoxyribonuclease activities following DNA damage in Mycobacterium smegmatis

Summary Treatment of growing cultures of Mycobacterium smegmatis with alkylating agents (methyl methaneusulphonate, ethyl methanesulphonate, nitrogen mustard, or mitomycin C) or with ultraviolet light resulted in enhanced specific activities of a DNA polymerase and of an ATP-dependent deoxyribonuclease. Similar results had previously been obtained with hydroxyurea and with iron limitation. The three of these treatments which were tested (methyl methane-sulphonate, mitomycin C and hydroxyurea) produced strand breaks or alkali-labile regions in the DNA of this organism. The increased enzyme activities could be prevented by simultaneous treatment with inhibitors of protein synthesis.In contrast, treatment of the cultures with intercalating agents (ethidium bromide, acridine orange, or proflavine), 5-fluorouracil, caffeine, or nalidixic acid, inhibited DNA synthesis without increasing the enzyme activities. These treatments did not produce strand breaks in the DNA of this organism.The results support the hypothesis that, in M. smegmatis, damage to DNA induces increased synthesis of enzymes associated with DNA repair.     

Synthesis and antitumor activities of novel α-aminophosphonates dehydroabietic acid derivatives

A series of novel α-aminophosphonate derivatives containing DHA structure were designed and synthesized as antitumor agents. In vitro antitumor activities of these compounds against the NCI-H460 (human lung cancer cell), A549 (human lung adenocarcinoma cell), HepG2 (human liver cancer cell) and SKOV3 (human ovarian cancer cell) human cancer cell lines were evaluated and compared with commercial anticancer drug 5-fluorouracil (5-FU), employing standard MTT assay. The pharmacological screening results revealed that many compounds exhibited moderate to high levels of antitumor activities against the tested cancer cell lines and that most demonstrated more potent inhibitory activities compared with the commercial anticancer drug 5-FU. The action mechanism of representative compound 7c was preliminarily investigated by acridine orange/ethidium bromide staining, Hoechst 33258 staining, JC-1 mitochondrial membrane potential staining and flow cytometry, which indicated that the compound can induce cell apoptosis in NCI-H460 cells. Cell cycle analysis showed that compound 7c mainly arrested NCI-H460 cells in G1 stage.     

Analysis of cycloheximide-induced apoptosis in human leukocytes: fluorescence microscopy using annexin V/propidium iodide versus acridin orange/ethidium bromide

Apoptosis is a highly regulated and programmed cell breakdown process characterized by numerous changes. Since it is implicated in many pathological as well as physiological processes, it is vital to have reliable methods for detecting cell death. In this study, we compared several methods for detecting apoptosis and necrosis in human leukocytes. Apoptosis was induced either by incubating the cells with various doses of cycloheximide (CHX) or by 312 nm UVB irradiation. The methods used for detecting apoptosis were light microscopy (May Grunwald-Giemsa and trypan blue staining), fluorescence microscopy (acridin orange/ethidium bromide and annexin V/propidium iodide staining) and agarose gel electrophoresis of fragmented genomic DNA. Our study showed that CHX-induced apoptosis in cultured peripheral blood mononuclear cells but had no effect on apoptosis in polymorphonuclear cells, so its effect depends on cell type. Evaluation and comparison of the methods for detecting apoptosis showed the following. A Giemsa-stained cytospin allows the main morphological characteristics of necrotic and apoptotic death to be recognized. Trypan blue staining, widely used for estimating cell viability, is valueless for detecting apoptosis. Both fluorescence methods provided reliable and reproducible results and distinguished clearly between subpopulations of apoptotic cells, and were closely intercorrelated. Although applicable to a wide spectrum of cell types, agar electrophoresis of extracted DNA cannot be applied to all cell types and apoptotic conditions. Generally, microscopic examination of acridin orange/ethidium bromide stained cells can be recommended as the most reliable of the methods tested.     

Induction of apoptosis and necrosis in A549 cells by the cis-Pt(II) complex of 3-aminoflavone in comparison with cis-DDP

Non-small cell lung cancer (NSCLC) includes a group of tumors that respond poorly to drugs. cis-Diamminedichloroplatinum(II) (cis-DDP) toxicity still remains problematic, and not completely solved by the improvement of supportive care. Therefore, the cis-Pt(II) complex of 3-aminoflavone was selected from cis-DDP analogues with a more favourable toxic profile towards normal cells and at least similar or better anti-tumor activity in comparison with cis-DDP. The aim of this research is to compare the ability of the cis-Pt(II) complex of 3-aminoflavone and cis-DDP to induce apoptosis and necrosis in the human non-small cancer cell line A549. Trypan blue dye exclusion, fluorochrome staining (acridine orange/ethidium bromide double staining), MTT and TUNEL (TdT-mediated dUTP Nick-End Labeling) assays were used. The results obtained show that the cis-Pt(II) complex of 3-aminoflavone is more active in inducing apoptosis and necrosis and in decreasing viability in A549 cells than cis-DDP, which suggests that it could be a potential chemotherapeutic drug.     

Spectroscopic and molecular modeling studies of caffeine complexes with DNA intercalators.

Recent studies have demonstrated that caffeine can act as an antimutagen and inhibit the cytoxic and/or cytostatic effects of some DNA intercalating agents. It has been suggested that this inhibitory effect may be due to complexation of the DNA intercalator with caffeine. In this study we employ optical absorption, fluorescence, and molecular modeling techniques to probe specific interactions between caffeine and various DNA intercalators. Optical absorption and steady-state fluorescence data demonstrate complexation between caffeine and the planar DNA intercalator acridine orange. The association constant of this complex is determined to be 258.4 +/- 5.1 M-1. In contrast, solutions containing caffeine and the nonplanar DNA intercalator ethidium bromide show optical shifts and steady-state fluorescence spectra indicative of a weaker complex with an association constant of 84.5 +/- 3.5 M-1. Time-resolved fluorescence data indicate that complex formation between caffeine and acridine orange or ethidium bromide results in singlet-state lifetime increases consistent with the observed increase in the steady-state fluorescence yield. In addition, dynamic polarization data indicate that these complexes form with a 1:1 stoichiometry. Molecular modeling studies are also included to examine structural factors that may influence complexation.     

Effect of Plumbago zeylanica extract and certain curing agents on multidrug resistant bacteria of clinical origin

Alcoholic extract of Plumbago zeylanica (root) was tested against multidrug-resistant clinical isolates of bacteria (Salmonella paratyphi, Staphylococcus aureus, Escherichia coli, Shigella dysenteriae and a R-plasmid-harbouring standard strain, E.coli x⁺). The extract exhibited strong antibacterial activity against all test bacteria irrespective of their antibiotic resistance behaviour. Phytochemical analysis of crude extract revealed the presence of flavonoids, saponins and naphthoquinone. A comparative evaluation of R-plasmid elimination from E. coli x⁺ (pUK 651) by the plant extract, DNA intercalating dyes (acridine orange and ethidium bromide) and a DNA gyrase antagonizing drug (pefloxacin) were made. All these agents could cure R-plasmid effectively at their respective sub-MIC concentrations. Maximum plasmid curing was observed by pefloxacin (88%), followed by ethidium bromide (36%), acridine orange (14%) and alcoholic extract of P. zeylanica (14%). Curing of plasmid pUK651 from E. coli x⁺ was confirmed by determining the loss of resistance markers in the cured derivative culture. This revised version was published online in November 2006 with corrections to the Cover Date.     

A simple method for the fluorescence analysis of nucleic acid-dye complexes in cytological preparations

Summary This communication describes a simple method for recording fluorescence emission spectra of cytological preparations using a conventional fluorescence spectrophotometer. The emission characteristics of in situ complexes between some basic fluorochromes (DAPI, 33258 Hoechst, acridine orange, pyronin Y, and ethidium bromide) and nucleic acid containing structures from smears of chicken blood and Ehrlich tumor cells (chromatin, basophilic cytoplasm) are briefly described.     

Diffusion-enhanced lanthanide energy-transfer study of DNA-bound cobalt(III) bleomycins: comparisons of accessibility and electrostatic potential with DNA complexes of ethidium and acridine orange

Energy transfer in the "rapid-diffusion" limit reflects the equilibrium properties of a donor-acceptor system. Rates of energy transfer from freely diffusing terbium chelates to DNA-binding chromophores change dramatically when DNA is added; energy transfer from an electrically neutral chelate is reduced because the energy acceptor becomes partially buried in DNA, while energy transfer from a positive chelate is increased because of electrostatic attraction. The rate constants for energy transfer to DNA-bound chromophores from a positively charged terbium chelate, relative to those from a neutral chelate, were used to estimate the following values for the electrostatic potential near the surface of each DNA-bound acceptor at 298 K in the presence of 1.0 mM added salt (in units of -e/kT): acridine orange, 4.54 +/- 0.11; ethidium, 4.66 +/- 0.07; green Co(III) bleomycin A2, 4.06 +/- 0.11; orange Co(III) bleomycin A2, 3.11 +/- 0.10. Smaller numbers indicate less negative potentials; these can be due to a combination of (1) positive charge on the chromophore, (2) location of the chromophore [particularly Co(III) bleomycin] away from the DNA phosphates, and/or (3) separation of DNA phosphate negative charges by an intercalator. The magnitudes of the individual rate constants indicate that all the DNA-bound chromophores can be directly encountered by the terbium probes. Energy-transfer rate constants from a neutral terbium chelate to DNA-bound and free acceptors can provide a measure of the accessibility of the terbium probe to each bound chromophore. The ratios of these rate constants were as follows: acridine orange, 0.17 +/- 0.01; ethidium, 0.27 +/- 0.02; green form of Co(III) bleomycin A2, 0.48 +/- 0.06; orange form of Co(III) bleomycin A2, 0.71 +/- 0.06. These results are consistent with the probable differences in binding mechanisms for the intercalating chromophores (ethidium and acridine orange) as compared to the Co(III) bleomycins (in which the relevant chromophores are nonintercalating metal centers). In addition, all the results imply that the green Co(III) bleomycin chromophore binds closer to DNA than the orange; this provides a first step toward understanding the structural basis for the different biological properties of these metallobleomycins. Control experiments and theoretical considerations necessary to establish the validity of the results are also presented.     

Evaluation of P53 and BAX gene expression and induction of apoptosis and necrosis by the cis-Pt(II) complex of 3-aminoflavone in comparison with cis-diamminedichloroplatinum(II) (cis-DDP) in human lymphocytes

Currently cis-diamminedichloroplatinum(II) (cis-DDP) is one of the most commonly applied compounds in chemotherapy of many types of cancer. However, a drawback is that its effectiveness presents with many side effects. Therefore, human normal lymphocytes were chosen as a model system to study cis-bis(3-aminoflavone)dichloroplatinum(II) (the cis-Pt(II) complex of 3-aminoflavone) in comparison with cis-DDP. We examined the effect of both tested compounds on cell viability and induction of apoptosis and necrosis. Trypan blue and acridine orange/ethidium bromide staining were carried out, as well as quantitative analysis of the apoptotic signal of P53 and BAX induction caused by the cis-Pt(II) complex of 3-aminoflavone in comparison with cis-DDP. cis-DDP induced a decrease of cell viability and led to a higher increase in necrosis and apoptosis than did the cis-Pt(II) complex of 3-aminoflavone. Moreover, at the molecular level cis-DDP increased P53 and BAX expression in comparison with the other tested compound. The cis-Pt(II) complex of 3-aminoflavone showed a weaker genotoxic effect in normal lymphocytes in comparison with cis-DDP, which was a stronger inducer of apoptosis and necrosis.