Characteristics of an ethidium efflux system in Enterococcus hirae.

An energy-dependent efflux system for ethidium and related cations has been detected in Enterococcus hirae ATCC 9790. The system was partially expressed when the organism was grown on a complex medium but was induced by the addition of phosphonium ions and related compounds. Mutants showing constitutive expression of the efflux system have been isolated on the basis of increased resistance to ethidium.

Antagonism by propidium of petite induction by ethidium and ethidium azide in Saccharomyces cerevisiae.

Propidium, a phenanthridinium dye similar to ethidium, did not induce petite mutations in non-growing yeast cells in contrast to ethidium. Combined exposure to ethidium and an excess of propidium for periods up to 2 h resulted in the expected petite induction expressed after subsequent plating on growth medium. As incubation was continued with propidium, the numbers of petites declined on subsequent plating whether the drug had been added before, during, or after the mutagenic treatment by ethidium. Propidium decreased petite induction by the monoazide analog of ethidium when applied before but not after photolytic attachment of the drug.

A protective effect of caffeine on the ethidium induced petite mutation in yeast.

A prerequisite for petite induction by ethidium bromide (EB) is an initial covalent attachment of the drug to cytoplasmic DNA. This DNA modification is thought to initiate repair processes. The repair inhibitor, caffeine, provided a protective effect against the ethidium induced petite mutation at caffeine concentrations known to inhibit the repair of UV damage in cytoplasmic DNA (Fig. 1). Mitochondrial DNA isolated from yeast exposed to EB in vivo was not as degraded in the presence of both drugs as with EB alone (Fig. 2).

Mutagenicity, comutagenicity, and antimutagenicity of erythrosine (FD and C red 3), a food dye, in the Ames/Salmonella assay.

Erythrosine (diNa, tetraiodofluorescein) was nonmutagenic to the Ames/Salmonella typhimurium strains TA97a, TA98, TA100, TA102, and TA104, to a concentration of 2 mg/plate. No mutative intermediates were detected on metabolism by rat caecal cell-free extracts or rat liver S9 mixture; or on incubation with the comutagens, harman and norharman (+/- S9). Instead, an unexpected dose-dependent suppression in spontaneous reversion frequencies was observed (maximum approximately equal to 35% decrease). Erythrosine was antimutagenic to benzo[a]pyrene, but it did not decrease the mutagenicity of the other adduct-forming mutagen, 4-nitroquinoline N-oxide. The food dye was strongly antimutagenic to the bifunctional alkylating agent, mitomycin C, though it did not exhibit a similar effect on the mutagenicity of the corresponding monofunctional agent, methyl methanesulphonate. It partially depressed the mutagenic potentials of sodium azide. The antimutagenic effect of erythrosine on an intercalating agent, ethidium bromide, was discernible only at the highest dose (2 mg/plate). These results have been interpreted in terms of a genointeractive role of erythrosine. Erythrosine produced differential toxic effects in repair-deficient (TA97a, TA98, TA100) and repair-proficient (TA102, TA104) Salmonella tester strains; survival of the repair-deficient strains was found to be decreased. Photoinduced potentiation of erythrosine toxicity was observed, although light irradiation in the presence of erythrosine did not modify the reversion frequencies of the tester strains. The evidence strongly suggests that erythrosine, which exhibits nonmutagenicity in the Ames/Salmonella test, can interact with DNA repair enzymes and/or with DNA.

Synthesis, molecular modelling, and antiproliferative and cytotoxic effects of carbocyclic derivatives of distamycin with chlorambucil moiety.

New carbocylic analogues of distamycin and netropsin with chlorambucil moieties 5-8 have been synthesised. Data from the ethidium displacement assay showed that these compounds bind in the minor groove of DNA. The observed reduced affinity to AT pairs and increased affinity towards GC sequences of the carbocyclic lexitropsins with chlorambucil moiety 5-8 in comparison with netropsin and distamycin was observed and rationalised by means of molecular modelling techniques. All of the compounds 5-8 showed antiproliferative and cytotoxic effects in the standard cell line of the mammalian tumour MCF-7.

[Differences in the degrees of stability of colchicine-resistant clones of murine hepatoma XXIIa]. / Razlichiia stepen' stabil'nosti ustoichivykh k kolkhitsinu klonov myshinoi gepatomy 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.

[Proliferation of cultured cells with genetically induced resistance to ethidium bromide on synthetic nutrient media without serum]. / Proliferatsiia kul'tiviruemykh kletok s geneticheski obuslovlennoi ustoichivbost'iu k bromistomu étidiiu na sinteticheskikh pitatel'nykh sredakh bez syvorotki.

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).

[The isolation and genetic characteristics of the new cell line of mouse myeloma spEBR-5 selected for ethidium bromide resistance and characterized by multiple drug resistance]. / Poluchenie i geneticheskaia kharakteristika novoi linii kletok mielomy myshi spEBR-5, otselektirovannykh na ustoichivosti k bromistomu étidiiu i kharakterizuiushchikhsia mnozhestvennoi lekarstvennoi ustoichivost'iu.

Stable mutant cells spEBR-5, resistant to ethidium bromide in concentration of 5 micrograms/ml, have been isolated by multistep selection in mouse myeloma cells sp2/0-Ag14. The spEBR-5 cells, selected with ethidium bromide, appeared to be cross resistant to unrelated drugs in connection with mdr/lb gene amplification and overexpression. Five specific chromosomal markers were detected in the karyotype of spEBR-5 cells as a result of chromosome structural rearrangement. No cytological manifestation of gene amplification such as HCR of chromosomes or DMs was found. A diffuse location of amplified sequences in chromosome(s) is suggested. The new mutant cell lines spEBR-5 can be used as a model for investigation of multidrug resistance mechanisms.

[The karyotypic variability of Chinese hamster CHLV-79 RJK cells characterized by multiple drug resistance resulting from the amplification of the mdr gene family]. / Izmenchivost' kariotipa kletok kitaiskogo khomiachka CHLV-79 RJK, kharakterizuiushchikhsia mnozhestvennoi lekarstvennoi ustoichivost'iu, obuslovlennaia amplifikatsiei genov semeistva mdr.

Variability in karyotype structure of Chinese hamster lung V-79 RJK cells and of their six cell sublines, selected for increasing concentrations of ethidium bromide (EB), was investigated in addition to the number of mdr gene copies in cells, both EB sensitive and resistant. It is shown that EB resistant cells exhibit cross-resistance to different drugs resulting from mdr genes amplification. Southern DNA blot hybridization has shown that in Vebr-2 cells (the 1st step of selection) the number of mdr gene copies increased by 10 times, whereas in Vebr-30 cells (the 6th step of selection) the number of mdr gene copies remained the same as in Vebr-2 cells. The level of mdr genes expression in Vebr-30 cells being higher than in Vebr-2 cells. In Vebr-2 cells, homogeneously and differentially stained regions (HSRs) were detected in loci 1p31 and 1q26 of chromosome 1 material (markers Z1 and Z6, respectively). On the following selection steps (prolonged cultivation or increased drug concentration) additional HSRs appeared in chromosome 2 (locus 2qter), in derivatives of chromosome 5 (marker Z7, locus Z7pter) and chromosome X (marker Z2, locus Z2qter). In the course of prolonged cultivation, chromosome 2 and derivatives of chromosomes 1, 2, 5 and X, in which HSRs were found, participated in the formation of new markers resulting from deletions, inversions, insertions and translocations of the chromosomal material. It is supposed that mdr genes amplification in V-79 RJK cells resistant to EB may be regarded as a factor inducing subsequent genome destabilization and eventual progressive changes in the karyotype structure.

[Changes in the alkaline cation transport across the plasma membrane of CHO-K1 cell lines resistant to ethidium bromide]. / izmenenie transport shchelochnykh kationov cherez plazmaticheskuiu membranu kletok linii CHO-K1, ustoichivykh k bromistomu étidiiu.

Sodium and potassium intracellular concentration, rate constants of sodium and potassium and rubidium influx were determined for cultured CHO-K1 cell lines, both sensitive and resistant to ethidium bromide. The emission technique was used to measure cation fluxes into potassium and sodium-free magnesium medium. Ethidium bromide sensitive and resistant cell have similar potassium concentrations, whereas sodium concentration in the latter is by almost 2.5 times higher than that in the former. A wide range of changes both in ouabain-sensitive and ouabain-resistant fluxes was found in resistant cells: the ouabain-inhibited rubidium influx is by 3-5 times lower, the ouabain-sensitive sodium efflux is practically absent, and ouabain-inhibited potassium efflux appears. In resistant cells the ouabain-insensitive influx of rubidium is decreased by almost 2 times. It is assumed that the selection of cells for resistance to ethidium bromide is accompanied by ":pleiothypic" changes of cell membrane, involving both active and passive cation transport.

[Genetic characteristics of Chinese hamster cell resistance to colchicine, actinomycin D and ethidium bromide]. / Geneticheskaia kharakteristika ustoichivosti kletok kitaiskogo khomiachka k kolkhitsinu, aktinomitsinu D i bromistomu étidiu.

We have tried to study genetics of Chinese hamster cellular drug resistance to colchicine, actinomycin D and ethidium bromide. Resistant variants arising at a frequency 10(-5) to 10(-7) were selected from a sensitive population. Pretreatment with MNNG increased this frequency 106, 9-15, 12-26 times for colchicine, actinomycin D and ethidium bromide, respectively. Fluctuation test analysis allows to suppose that phenotypic expression of drug resistance is genetically determined and resulted from random genetical events. Significant values of particular correlation coefficients for selecting resistant variants in a set of independently originated subpopulations suggest the genetical identity of the majority of variants selected by colchicine and actinomycin D. At the same time, the significant portion of colchicine-resistant cells may be selected using ethidium bromide. It is not unlikely that under above experimental conditions, each selective agent reveals genetically different resistant variants. The possible genetic mechanisms of resistance to the drugs used are discussed.

[Resistance to ethidium bromide in L929 cells is accompanied by regular changes in karyotypic structure]. / Ustoichivost' k bromistomu étidiiu kletok L929 soprovozhdaetsia zakonomernymi izmeneniiami struktury kariotipa.

The method of differential staining of chromosomes (G- and C-banding) has been used for comparative karyological analysis of mouse fibroblasts of L929 cells selected for resistance to ethidium bromide (EB) at concentrations 1, 10, 25, 50 mkg/ml. All variants have been shown to maintain resistance to EB for a long period of cultivation in nonselective conditions. Only 13 of 36 marker chromosomes of the initial EB-sensitive cells persist, 16 markers being specific for resistant variants. The karyotype changes found occur as early as at the first step of selection and are maintained in variants resistant to the higher drug concentrations. Detection of similar karyotypic changes in clones of independent origin, resistant to EB at concentrations 3 and 25 mkg/ml, allows to conclude that they are quite uniform for L929 cells selected for resistance to EB.

[Differences in the protein kinase C activity in CHO-K1 cells and in clone cells of this line resistant to ethidium bromide]. / Razlichiia v aktivnosti proteinkinazy C v kletkakh CHO-K1 i v kletkakh klonov étoi linii, ustoichivykh k bromistomu étidiiu.

Protein kinase C (PK C) activity in cells of two ethidium bromide (EB) resistant clones with different proliferating rates, derived from CHO-K1 cell line, was assayed. After selection in the presence of 1 mkg/ml EB cells of isolated clones acquired cross-resistance also to some unrelated drugs of different structure. It is shown that resistant cells after the first step of selection elevated PK C activity level in membrane fractions. Subsequent increase in resistance (from 1 to 10 mkg/ml EB) led to a further elevated enzyme activity both in cytosolic and membrane fractions in cells of both variants. The effect of EB presence in cultural media on the enzyme activity was tested. EB at a subtoxic concentration was shown to cause an increased PK C activity both in cytosolic and membrane fractions from resistant cells sublines.

[The amplification and overexpression of mdr-family genes in ethidium bromide-resistant Chinese hamster CHO-K1 cells and in the hybrids of sensitive and resistant cells]. / Amplifikatsiia i sverkhékspressiia genov semeistva mdr v ustoichivykh k bromistomu étidiiu kletkakh kitaiskogo khomiachka CHO-K1 i v gibridakh chuvstvitel'nykh i ustoichivykh kletok.

Stable mutant cells Cebr-1 and Cebr-2, resistant to ethidium bromide (EB) in concentration of 2 micrograms/ml, have been isolated by a multistep selection in Chinese hamster ovary cells. It was shown that Cebr-1 and Cebr-2 cells acquired a cross-resistance to unrelated drugs. Stable changes in the structure of chromosomes 1, 2, 5 and 8 were revealed by karyological analysis. Overexpression and amplification of mdr genes were detected in Cebr-2 cells using Northern RNA and Southern DNA blot hybridization. Two independent hybrids Hybr-1 and Hybr-2 were obtained by fusion of Cebr-2 cells with Chinese hamster lung V-79 RJK cells, sensitive to EB. Hybr-2 cells were characterized by the same level of EB-resistance as Cebr-2 cells. Hybr-1 cells have a lower level of EB-resistance than Cebr-2 cells. Hybr-2 cells have demonstrated amplification and overexpression of mdr gene, the same as in Cebr-2 cells, whereas in Hybr-1 cells no mdr gene amplification was observed, but the level of mdr gene expression was higher than in sensitive cells. The data suggest that resistance of Chinese hamster cells to EB is mediated by amplification and overexpression of mdr genes.

[Cholesterol and phospholipid content of cells sensitive and resistant to ethidium bromide and its alteration in resistant cells as affected by methyltestosterone]. / Soderzhanie kholesterina i fosfolipidov v kletkakh, chuvstvitel'nykh i ustoichivykh k bromistomu étidiiu, i ego izmenenie v ustoichivykh kletkakh pri deistvii metiltestosterona.

The content of cholesterol (Chl) and phospholipids (Phl) in cell lines L and CHO-K1, sensitive and resistant to the toxic action of ethidium bromide, has been studied. EB-resistant cells were shown to have lower amounts of Chl and Phl, and the ratio Chl/Phl in these cells was decreased too. EB-resistant cells, grown in serum-free medium, have dramatically decreased contents of Chl, while compared with the cells growing with serum. Treatment of EB-resistant cells with methyltestosterone in concentration of 3 X 10(-7) M caused the increase in Chl and Phl contents, thus approximating these indices to those of EB-sensitive ones.

[Genotypic and phenotypic characteristics of L-line cells resistant to ethidium bromide]. / Genotipicheskie i fenotipicheskie osobennosti keltok linii L, ustoichivykh k bromistomu étidiu.

Stable mutants resistant to ethidium bromide in concentrations of 1 and 3 micrograms/ml have been selected in a single step in L cells. The frequency of spontaneously occurring ethidium bromide resistant clones after the exposure to 1 microgram/ml of the drug has been established as 5.10(-5). Resistant variants were induced following treatment with mutagen N-methyl-N-nitro-N-nitrosoguanidine. The resistant clones were shown to be resistant to higher concentration of the agent then which was used for selection. In multistep selection, a number of clones resistant to ethidium bromide in concentration up to 50 micrograms/ml was obtained. The alteration in the permeability of plasma membrane to the drug is the clue mechanism of the resistance.

[The genome structure and phenotypic characteristics of murine hybridoma 1F7 cells selected in the presence of adriamycin and ethidium bromide]. / Issledovanie struktury genoma i fenotipicheskikh osobennostei kletok myshinoi gibridomy 1F7, otselektirovannykh v prisutstvii adriamitsina i bromistogo étidiia.

A study was made of geno- and phenotypic changes, associated with of multidrug resistance development in murine 1F7 hybridoma cells, selected for adriamycin and ethidium bromide resistance (1F7-EBR and 1F7-ADR). In both cell lines overexpression of mdr1 gene was observed, while amplification of mdr1 gene was detected only in 1F7-ADR cells. Karyotypic analysis revealed in resistant cells the presence of a specific marker M45 absent from parental cells, thus suggesting its selective importance. The M45 length instability, as well as the presence of a distal typical homogeneously stained region in it provide an evidence for a link between M45 and mdr1 gene amplification. The frequency of double minute chromosomes in parental lines was the same as in multidrug resistant lines. In 1F7-ADR cells, in contrast with 1F7-EBR cells, the enhancement of immunoglobulin production and the increase in immunoglobulin gamma 2b heavy chain gene expression were observed, which correlated with a decline in DNA-topoisomerase II activity.

[Cellular energy functions of a subline of mouse fibroblasts resistent to the action of ethidium bromide]. / Izuchenie nekotorykh énergeticheskikh funktsii kletok sublinii myshinykh fibroblastov, rezistentnoi k deistviiu bromistogo étidiia.

The respiration of subline Leb-25 cells, resistant to ethidium bromide (EB, 25 g/ml), is 2.5 times slower than the respiration of parental L cells of mouse fibroblasts. The EB resistant cells have a normal level of ATP. Disturbances of mitochondrial functions can be observed such as a defect of the succinate dehydrogenase complex and the uncoupling of respiration and oxidative phosphorylation in mitochondria of Leb-25 cells.

[Correlation between the proliferative activity and protein kinase C activities in ethidium bromide-sensitive and -resistant cells]. / Korreliatsiia mezhdu proliferativnoi aktivnost'iu i aktivnost'iu proteinkinazy C v kletkakh, chuvstvitel'nykh i ustoichivykh k bromistomu étidiiu.

The protein kinase C (PK C) activity was determined in the cytosolic and membrane fractions of L- and CHO-K1-cells, both sensitive and resistant to ethidium bromide (EB). In the resistant cells (Lebr-25 and Cebr) a decreased enzyme activity was found in addition to alteration of the enzyme elution profile in the membrane preparations purified by DE-52 cellulose column chromatography. Methyltestosterone treated cells had a decreased enzyme activity in nonpurified membrane preparations in Lebr-25 cells, whereas the enzyme quantity in purified preparations remained the same. The decreased PK C activity on membranes correlates with the rapid proliferation of the resistant cells. The differences found between Lebr-25 and Cebr-cell lines in proliferation response to methyltestosterone correspond to the change of PK C developed due to hormone treatment.

[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]. / Khromosomnyi polimorfizm kletok mlekopitaiushchikh s mnozhestvennoi lekarstvennoi ustoichivost'iu. I. Analiz kariotipa kletok kitaiskogo khomiachka, ustoichivykh k bromistomu étidiiu, na rannikh passazhakh pervykh stupenei selektsii.

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.