Differences in the degrees of stability of colchicine-resistant clones of murine hepatoma XXIIa

Cells resistant to colchicine in the parental line of mouse hepatoma XXIIa could be revealed with a frequency of 4-4.5 per 10(5) cells when selected at the drug concentration as high as 0.05 mkg/ml. MNNG as a mutagene was shown to increase the number of resistant cells by 5-6 times. 6 clones of independent origin differed in the level of resistance and in the stability to retain it under non-selective conditions. Multistep selection from the stable clones via 0.25, 1.0, 4.0, 5.0, 10.0 and 20.0 mkg/ml resulted in the appearance of some highly resistant subclones. Stable clones of all the steps of selection appeared to be resistant to ethidium bromide. Genetical polymorphism of colchicine-resistance is suggested in the line of mouse hepatoma XXIIa.     

Control and virus-transformed baby hamster kidney cells resistant to ethidium bromide. I. Characterization and the respiratory enzymes

Cell lines resistant to ethidium bromide have been developed from cultured mammalian BHK21/C13 cells and these same cells transformed by Rous sarcoma virus (C13/B4). Cells resistant to 2 micrograms ethidium bromide per milliliter have been cloned. One clone of the control and one of the virus-transformed cell lines has been employed for characterization. The resistant cells, in the presence of 2 micrograms ethidium bromide/ml, grow at approximately the same rate as the untreated parental cells. The control cells possess a "normal" karyotype (44 chromosomes), while the corresponding ethidium bromide mutant has a reduced chromosome number of 41 and a number of translocations. The mitochondria displayed morphological alterations compared to the parental lines during the transition phase prior to the isolation of the ethidium bromide-resistant cells. The mitochondria of the ethidium bromide-resistant mutants appear somewhat enlarged with a normal morphology. The effect of ethidium bromide on selected respiratory enzymes in normal and virus-transformed ethidium bromide-resistant baby hamster kidney cells was determined. Ethidium bromide-resistant cells exhibited a depressed level of cytochrome aa3. This depression could not be reversed by growth in ethidium bromide-free media. Ethidium bromide-resistant cells possessed the same cytochrome b, c, and c1 levels per cell as their corresponding parental lines. Purified mitochondria isolated from virus-transformed ethidium bromide-resistant cells exhibited a depression in cytochrome oxidase-specific activity, while the ethidium bromide-resistant control cells did not. All cell lines studied showed a depression in NADH-ferricyanide and NADH-cytochrome c reductase-specific activities relative to their parental BHK21/C13 cells. No increase was observed in virus-transformed ethidium bromide-resistant cells. Ethidium bromide-resistant control cells exhibited a two-fold increase in oligomycin-insensitive adenosine triphosphatase activity relative to their parental cells. All of the cell lines studied possessed equivalent oligomycin-sensitive adenosine triphosphatase-specific activity except for the virus-transformed, dye-resistant mutant, whose activity was increased.     

Dependence of mammalian DNA replication on DNA supercoiling. I. Effects of ethidium bromide on DNA synthesis in permeable Chinese hamster ovary cells.

Chinese hamster ovary cells labelled with [14C]thymidine were made permeable, incubated with various concentrations of the intercalating dye ethidium bromide, and centrifuged through neutral sucrose gradients. The gradient profiles of these cells were qualitatively similar to those obtained by centrifuging DNA from untreated, lysed permeable cells through gradients containing ethidium bromide. The sedimentation distance of DNA had a biphasic dependence on the concentration of ethidium bromide, suggesting that the dye altered the amount of DNA supercoiling in situ. The effect of ethidium bromide intercalation on incorporation of [3H]dTMP into acid-precipitable material in an in vitro DNA synthesis mixture was measured. The incorporation of [3H]dTMP was unaffected by less than 1 microgram/ml of ethidium bromide, enhanced up to two-fold by 1--10 microgram/ml, and inhibited by concentrations greater than 10 micrograms/ml. Alkaline sucrose gradient analysis revealed a higher percentage of small DNA fragments (6--20 S) in the cells treated with 2 micrograms/ml ethidium bromide than in control cells. These fragments attained parental size within the same time as the fragments in control cells. In cells treated with 2 micrograms/ml ethidium bromide, a significant fraction of newly synthesized DNA resulted from new starts, whereas in untreated cells practically none of the newly synthesized DNA resulted from new starts. These results suggest that relaxation of DNA supercoiled structures ahead of the replication fork generates spurious initiations of DNA synthesis and that in intact cells the rate of chain elongation is limited by supercoiled regions ahead of the growing point.     

Amphotericin B susceptibility testing of Candida species by flow cytometry.

We have developed an 8 hr flow cytometry (FCM) method for assessing susceptibility of yeasts to amphotericin B (AmpB). The method detects both high-level and relative-resistance to the drug. Variables found to affect fluorescence of control and AmpB treated cells included pH, presence of glucose, incubation conditions, concentration and length of exposure to both AmpB and ethidium bromide (ETBR), and the degree of resistance to AmpB. The FCM method was optimized based on increased red fluorescence intensity (RF), decreased forward angle light scatter (FALS), and a negative gating technique. A dose response was seen between 0.1 and 10 micrograms AmpB/ml for the susceptible control strain. Greater than 50% of cells from all susceptible strains tested transfer into the negative gate when exposed to 2.5 micrograms Amp B/ml while fewer than 5% of cells of the highly resistant C. tropicalis (ATCC 28707) are affected at concentrations up to 20 micrograms/ml. This method may provide a more accurate assessment of Amp B susceptibility than conventional tube dilution methods.     

Ethidium bromide resistance in a rat liver epithelial cell line: Association with enhanced drug efflux

Ethidium bromide-resistant cell strains were obtained by continuous selection of an adult rat liver-derived cell line (ARL6T) grown in the continuous presence of 200 ngl ml ethidium bromide. Comparison of resistant strains and parental (sensitive) cells was made for uptake and binding of ethidium bromide, visualized as fluorescent ethidium bromide-nucleic acid complexes. Although uptake of ethidium bromide was similar in parental and resistant cells, efflux kinetics were markedly different. Over a three-hour period, parental (sensitive) cells maintained fluorescence following a short ethidium bromide pulse (100 g/ ml ethidium bromide). In contrast, ethidium bromide-resistant cell lines eliminated photographically detectable fluorescent complexes within three hours following pulse exposure to ethidium bromide. The rapid elimination of ethidium bromide fluorescent complexes in all (5) resistant cell strains examined supports an efflux mechanism as contributing to the resistance of ethidium bromide cytotoxicity in these cells.Abbreviations EtBr ethidium bromide - HBSS Hanks' balanced salt solution     

Ethidium bromide: destruction and decontamination of solutions

Ethidium bromide in water, TBE buffer, Mops buffer, and cesium chloride solution may be completely degraded by reaction with sodium nitrite and hypophosphorous acid. Only non-mutagenic reaction mixtures were produced. Destruction was greater than 99.8% in all cases; the limit of detection was 0.5 micrograms ethidium bromide per milliliter of solution. Ethidium bromide also may be removed completely from the above solutions by using Amberlite XAD-16 resin. The limit of detection was 0.05 micrograms ethidium bromide per milliliter of solution (0.27 micrograms/ml when cesium chloride solution was used).     

Change in the structural organization of Mycobacterium rubrum cells upon the action of ethidium bromide

The action of ethidium bromide on Mycobacterium rubrum cells was studied. The culture growth was found to depend on ethidium bromide (EB) concentration in the medium. The reaction of EB with nucleoid DNA was shown to be specific and changes in the nucleoid structure were detected. Low EB doses (ca. 2 micrograms/ml) caused DNA despiralization in many cells. The process was reversible, which accounted for the elevated ability of reactivation at low EB doses. A higher EB dose (ca. 5-10 micrograms/ml and more) made the nucleoid structure coarser and denser in most cells and the nucleoid broke down to small fragments. As a result, due to the pool of enzymes present in the cells prior to EB addition, secondary changes developed. They involved all the cellular structures as well as the metabolism of lipids, polyphosphates, and glycogen. As a rule, these changes were incompatible with the cell viability.     

An ethidium bromide-agarose plate assay for the nonradioactive detection of cDNA synthesis

Ethidium bromide was used to determine the success of cDNA synthesis reactions. Since ethidium bromide in agarose can be used to quantitate RNA and DNA, conditions under which the greater fluorescence of double-stranded DNA (dsDNA) is utilized were devised to assay dsDNA synthesis from mRNA. Ethidium bromide at 5 micrograms/ml in agarose allowed quantitative detection of cDNA in the range of 0.03 to 0.0015 microgram. Sodium dodecyl sulfate had an adverse effect on the measurement of cDNA. Subsequent cDNA analysis by alkaline gel electrophoresis and staining in 5 micrograms/ml ethidium bromide allowed accurate and rapid sizing of cDNA and required only 0.1-0.05 microgram cDNA.     

Mitochondrial DNA replication in petite mutants of yeast: Resistance to inhibition by ethidium bromide, berenil and euflavine

Summary Mitochondrial DNA (mtDNA) replication in petite mutants ofSaccharomyces cerevisiae is generally less sensitive to inhibition by ethidium bromide than in grande (respiratory competent) cells. In every petite that we have examined, which retain a range of different grande mtDNA sequences, this general phenomenon has been demonstrated by measurements of the loss of mtDNA from cultures grown in the presence of the drug. The resistance is also demonstrable by direct analysis of drug inhibition of mtDNA replication in isolated mitochondria. Furthermore, the resistance to ethidium bromide is accompanied, in every case tested, by cross-resistance to berenil and euflavine, although variations in the levels of resistance are observed.In one petite the level of in vivo resistance to the three drugs was very similar (4-fold over the grande parent) whilst another petite was mildly resistant to ethidium bromide and berenil (each 1.6-fold over the parent) and strongly resistant (nearly 8-fold) to inhibition of mtDNA replication by euflavine. The level of resistance to ethidium bromide in several other petite clones tested was found to vary markedly. Using genetic techniques it is possible to identify those petites which display an enhanced resistance to ethidium bromide inhibition of mtDNA replication.It is considered that the general resistance of petites arises because a product of mitochondrial protein synthesis is normally involved in facilitating the inhibitory action of these drugs on mtDNA synthesis in grande cells. The various levels of resistance in petites may be modulated by the particular mtDNA sequences retained in each petite.     

Contribution of the multidrug efflux pump LfrA to innate mycobacterial drug resistance

Multidrug resistance (MDR) in bacteria has been associated with efflux pumps that export structurally unrelated compounds and decrease cytoplasmic drug accumulation. To investigate MDR in mycobacteria, we studied the Mycobacterium smegmatis mutant mc(2)11, which is resistant to doxorubicin, tetracycline, rhodamine, ethidium bromide and the hydrophilic fluoroquinolones. A genomic library constructed from this mutant was used to select clones conferring resistance to doxorubicin. Surprisingly, the clone selected encodes the efflux pump LfrA, which has been reported to confer resistance to hydrophilic fluoroquinolones, ethidium bromide, rhodamine, and acriflavine. To define the contribution of LfrA to the innate mycobacterial drug resistance and to the MDR phenotype in mc(2)11, the lfrA gene was disrupted in both the mc(2)11 mutant and the mc(2)155 wild-type parent. LfrA disruption of the wild-type strain decreased resistance to ethidium bromide and acriflavine, and increased accumulation of ethidium bromide. However, disruption of lfrA gene results only in a 2-fold decrease in minimal inhibitory concentrations (MICs) for ciprofloxacin, doxorubicin, rhodamine, and accumulation of [(14)C]ciprofloxacin was unchanged. LfrA disruption of the MDR strain mc(2)11 produced a similar phenotype. Thus, LfrA contributes significantly to the intrinsic MICs of M. smegmatis for ethidium bromide and acriflavine, but not for ciprofloxacin, doxorubicin or rhodamine.     

Improved electrophoretic separation of supercoiled and relaxed DNA in the presence of ethidium bromide.

Supercoiled and relaxed DNA were resolved electrophoretically in the presence of 0.5 micrograms/ml ethidium bromide. Under these conditions the Gaussian distributions of topological isomers of both supercoiled and relaxed DNA migrated as discrete bands. The separation of these DNAs was optimized by varying the concentration of electrode buffer. Electrophoresis in the presence of 160 mM Tris-acetate, pH 8.3, 4 mM EDTA resulted in a 20-fold increase in the separation of relaxed and supercoiled DNA relative to electrophoresis in 40 mM Tris-acetate, pH 8.3, 1 mM EDTA.     

Control and virus-transformed baby hamster kidney cells resistant to ethidium bromide. II. Membrane properties

Aspects of membrane stucture and functions were studied in ethidium bromide resistant cells. Submitochondrial particles were solubilized and electrophoresed. The gel patterns, representing mitochondiral membrane proteins, demonstrated qualitative and quantitative alterations in mitochondrial preparations derived from virus-transformed cells and ethidium bromide resistant cells as compared to the control cells. The plasma membrane glycoproteins were labelled by the sodium borohydride method. The glycoporteins were released with Triton X-100 and electrophoresed. Fluorograms of the gels demonstratred some marked differences between the ethidium bromide resistant cells and their parental strain. The observed alterations in the membrane glycoproteins did not result in altered glucose transport properties or in the elution patterns of plasma membrane glycopeptides as analyzed by Sephadex G-50 chromatography. Dye uptake and binding studies with intact parental and drug resistant cells and their isolated mitochondria demonstrated no alteration of the membrane permeability or the number of binding sites for ethidium bromide. Similar results were also obtained with a cyanine dye. This latter finding was significant in that it permitted one to exclude dye exclusion as a mechanism for ethidium bromide resistance.     

Proliferation of cultured cells with genetically induced resistance to ethidium bromide on synthetic nutrient media without serum

Lebr 625 and Lebr 350 cells, resistant to ethidium bromide in concentrations 25 and 50 mkg/ml, are able to grow continuously in serum- and protein-free media. Under the same conditions the parental L929 cells are not able to. Two cell lines (625 sf and 350 sf) were established capable of growing in serum- and protein free media. It is found that ethidium bromide is toxic for resistant cells grown the in serum-free medium. The addition of serum lowers the toxic action of ethidium bromide. A continuous growth of resistant cells in serum-free medium (under nonselective conditions) leads to a decreased level of resistance, which may nevertheless persist for a long period of cultivation (over 2.5 years).     

Cyclic adenosine monophosphate content and its regulatory characteristics in Chinese hamster cells with genetically determined changes in the cell surface

The intra- and extracellular concentrations of cyclic adenosine monophosphate (cAMP) in cell line CHO-K1, sensitive (clone 773) and resistant to cytotoxic action of ethidium bromide (EBr), colchicine (Cr) and actinomycin D (ADr), as well as the amount of cAMP in response to isoproterenol, 10% serum and ethidium bromide (EB) in these cells were studied. The increased level of cAMP in EBr- and, Cr-cells, and the decreased one--in ADr cells was found as compared with sensitive cells. The amount of cAMP extruded in the surrounding medium was lower for EBr- and Cr-cells and higher for ADr-cells, in comparison with sensitive cells. All the variants of resistant cells were characterized by a less intensive but a longer reaction for isoproterenol, as compared with sensitive cells. In all the investigated variants 10% serum induced a remarkable increase in the intracellular cAMP by the 2nd hour after their insignificant decrease. 1 mcg/ml concentration of EBr increased intracellular cAMP only in 773-cells. The rules changing the cAMP level to isoproterenol and EB2; are determined by differences in reaction of adenylate cyclase, as it has been demonstrated for the 773- and EBr-cells.     

Chromosomal polymorphism of mammalian cells resistant to drugs in multiple passages. I. Karyotype analysis of Chinese hamster cells resistant to ethidium bromide in the early passages of the first steps of selection

G-banded metaphase chromosomes of Chinese hamster V-79 RJK cells resistant to ethidium bromide (2.5 and 10 mcg/ml) have been analysed. The cells of the first selection step (clone IVerb-2, the 9th passage) revealed definite karyotypical instabilities. Amplifications or, in rare cases, deletions were found in chromosomes Z1 and Z6 which appear to have derived from chromosome 1. The amplified region in chromosome Z6 varied considerably in morphology. The chromosome instability, detected in Z1 and Z6, was reproducible in cells throughout the eight independent clones isolated from clone IVebr-2 under non-selective conditions. The data obtained allow to suggest a genetically conditioned mechanism of the above chromosome instability. In the population of resistant cells on the second step of selection (clone I Vebr-5, the 9th passage) the frequency of the cells with amplification increased up to 100%. The length of amplifications increased in the majority of cells. In the cells of the third step of selection (clone IVerb-10, the 12th passage) with near-tetraploid chromosome composition, besides amplifications some specific rearrangements of chromosomes 2 and 7 (markers Z16, Z17) were revealed. The above rearrangements are indicative of the karyotypical destabilization in the drug resistant cells, and may be evaluated as secondary phenomena casually connected with amplifications found at the earlier steps of selection.     

Fluorometric methods employing low concentrations of ethidium bromide for DNA topoisomerase and endonuclease assays

DNA topoisomerase activity can be rapidly assayed by measuring the change in ethidium bromide fluorescence intensity after treatment of closed duplex DNA with enzyme. The sensitivity of the fluorometric assay has been enhanced 3-fold by a 10-fold reduction in ethidium bromide concentration to 0.1 microgram/ml. The results of the fluorometric assays are in close agreement with agarose gel electrophoretic analyses of reacted DNA. A sensitive fluorometric method using 0.1 microgram/ml ethidium bromide has also been developed to determine the fraction of nicked and linear DNAs in a mixture containing closed duplex DNA by measuring the fluorescence intensities of ethidium-DNA complexes at pH 7.0 and pH 12.0. These methods make possible very rapid and sensitive measurements of DNA topoisomerase and endonuclease activities.     

Mechanism of the Heat Sensitization of Bacillus subtilis Spores by Ethidium Bromide

Pretreatment with ethidium bromide (5 μg/ml) followed by a water wash had no effect on unheated Bacillus subtilis spores, but the viability of these spores after heating was much lower than that of similarly heated spores exposed to water alone. The fate of water- or ethidium bromide-treated spores, unheated or heated, was followed by allowing them to germinate and outgrow in a minimal or a complex liquid medium. Spores exposed to ethidium bromide and then heated (85°C, 10 min) exhibited a developmental block during germination and outgrowth. Many of them were blocked at the stage when the bacterium emerged from the germinated spore. When 0.35 μg of ethidium bromide per ml was added to heated spores in the germination-growth medium, the outgrowth of heated spores was inhibited to the same extent as were pretreated spores. Ethidium bromide acted in the first hour of germination of heated spores since addition after this time was ineffective in inhibiting recovery events. Repair of heat-damaged spore DNA was detected during the first 2 h of germination. The addition of ethidium bromide (final concentration, 0.35 μg/ml) inhibited DNA repair during early outgrowth. Increased sensitivity of spores to heat after pretreatment with sublethal concentrations of ethidium bromide was due to the inhibition of the repair of heat-damaged DNA.     

Quantitating small volumes of dilute DNA samples containing sodium dodecyl sulfate

A technique to quantitate small volumes of dilute solutions of different-sized DNA fragments has been developed. The detection limit was 0.7 micrograms/ml and the technique could be used even in the presence of diffusable substances, including those such as sodium dodecyl sulfate which affect surface tension and also exhibit fluorescence when stained with ethidium bromide and excited by ultraviolet light. The DNA was mixed with low-melting-point agarose and pipetted into preformed wells in an agarose plate, where it solidified. After diffusion of small molecules, the amount of DNA was estimated by comparing ethidium bromide-mediated fluorescence of samples with that of standards.     

The toxicity of sanguinarine compared to a number of other DNA-tropic compounds for ethidium bromide-sensitive and -resistant transformed murine fibroblasts in culture

A natural DNA-intercalator plant benzo-c-phenanthridine alkaloid sanguinarine is more toxic for mouse transformed fibroblast L-cells in culture than synthetic DNA-intercalator ethidium bromide (EtB) and alkaloid berberine. Dimidium bromide is also an inhibitor of the L-cell growth. In assay conditions, growth of L-cells is stopped by 1.5 x 10(-5) M of sanguinarine. Lebr-625 cells, resistant to 25 micrograms/ml of EtB, have sanguinarine sensitivity close to that of L-cells, but Lebr-625 cells are resistant to dimidium bromide. Sanguinarine is more toxic for L-cells in culture than the anticancer drug cis-PtNH3)2Cl2. Trans-Pt(NH3)2Cl2 is less toxic for these cells. The strong toxicity of sanguinarine for L- and Lebr-625 cells in culture, as compared to other DNA-complexing drugs, seems to be associated with the wide range of potential cell targets for sanguinarine influence. Besides the inhibition of nucleic acid metabolism reactions, characteristic of DNA-intercalators, and disruption the mitochondrial ATP synthesis, also characteristic of organic heterocyclic cationic molecules of DNA-intercalators, sanguinarine can modify the thiol groups of enzymes including SH-sensitive membrane-bound Na+, K(+)-ATPase of cerebral cortex and Ca2(+)-ATPase of skeletal muscle sarcoplasmic reticulum fragments.     

Influence of ethidium bromide on hyperthermic modification of bleomycin-DNA interaction

The effect of hyperthermic treatment on the binding of 59Fe-labeled bleomycin to DNA has been studied. Enhanced binding was observed at elevated temperatures. The influence of the DNA-intercalating agent, ethidium bromide, on bleomycin-DNA interaction was also studied and revealed a considerable decrease in this interaction at ethidium bromide levels below 1 microgram/ml. Ethidium bromide was observed to remove the enhanced bleomycin-DNA interaction recorded previously following incubation at hyperthermic temperatures. Synergistic action of bleomycin and hyperthermia on loss of clonogenic ability of HT29R cells is reported. Incubation of cells under hyperthermic conditions with bleomycin in the presence of ethidium bromide removes this synergism, producing a less than additive effect for the action of bleomycin and heat after ethidium bromide effects are taken into account.