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Biomedical subjects

J Kapuscinski

Publications and source records attributed to J Kapuscinski.

29 records · Page 2Linked to original sources
PubMed1985

Interactions of antitumor agents Ametantrone and Mitoxantrone (Novatrone) with double-stranded DNA.

Interactions of Mitoxantrone and Ametantrone with natural and synthetic nucleic acids in aqueous medium [0.15 NaCl, 5 mM 4-(2-hydroxyethyl)-1-piperazine-ethanesulfonic acid (Hepes), pH 7.0, 25 degrees] have been studied using computer-aided spectrophotometric techniques. Absorption spectra of the drugs in monomeric and dimeric form and their complexes with DNAs at low drug/phosphate ratios (D/P) have been established. The latter were red-shifted and had lower amplitude as compared with the spectra of the free ligand's monomer; the change is consistent with the already well-established intercalative mode of drug-nucleic acid interaction. Drug-DNA equilibria have been studied using the McGhee-von Hippel model of noncooperative ligand-polymer interaction, with the correction for dimerization of drugs. Although Mitoxantrone is two orders of magnitude more potent an antitumor drug than Ametantrone, the intrinsic association constants (Ki) of both drugs were of similar magnitude. Also, no significant DNA-base specificity for either of the drugs (measured as Ki value for various homopolymers) was observed. Therefore, no correlation was apparent between the intercalative mode of binding to DNA, regardless of base composition, and the pharmacological activity of these drugs. At higher D/P ratios, a secondary mode of binding was detected by both spectroscopy and light-scattering measurement. Homopolymer-pairs and polymers containing only dI and dC were especially susceptible to this secondary type of binding. The possibility that this secondary type of binding may be responsible for the antitumor properties of the drugs is considered.

Anthraquinones↗
PubMed1985

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

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

Acridine Orange↗
PubMed1984

Accessibility of DNA in situ to various fluorochromes: relationship to chromatin changes during erythroid differentiation of Friend leukemia cells.

Friend leukemia cells from exponentially growing or differentiated (DMSO-induced) cultures were permeabilized and their DNA was stained with 4'6-diamidino-2-phenylindole (DAPI), Hoechst 33342, acridine orange, ethidium bromide, propidium iodide, quinacrine, 7-amino-actinomycin D, mithramycin, or chromomycin A3. Accessibility of DNA to each of the above fluorochromes was compared in differentiated and nondifferentiated cells before and after nuclear proteins, mostly histones, were extracted with 0.1N HCl. A decrease in the accessibility of DNA to several dyes, especially pronounced in the case of some intercalators, was observed in differentiated cells. After extraction of nuclear proteins with HCl there was an increase in DNA accessibility, of varying degree depending on the fluorochrome and the difference between differentiated and nondifferentiated cells was abolished for most of the intercalating dyes. The increase was the lowest for DAPI (45%), the highest for 7-amino-actinomycin D (13-fold), and in general was higher for the intercalating dyes that unwind DNA than for dyes binding externally to the double helix. The results are discussed in terms of the mode of interactions between DNA and the fluorochromes and factors associated with chromatin structure that may affect accessibility of DNA in situ in exponentially growing and differentiated cells.

Animals↗
PubMed1984

Condensation of nucleic acids by intercalating aromatic cations.

Certain intercalating aromatic cations, such as the fluorochrome acridine orange or the antitumor drug Mitoxantrone, induce condensation of nucleic acids in solutions. The appearance of the condensed form during titration of nucleic acids with these intercalating ligands can be quantitatively monitored by light scatter measurements. The resulting highly reproducible light scatter transition curves are typical of the cooperative processes, and the transitions occur at different critical concentrations of the ligands depending upon both the ligand itself and the primary structure (base and sugar composition) and the secondary structure (single- or double-stranded) of the nucleic acids. The mechanism of condensation of nucleic acids by intercalating cationic ligands is discussed in light of the model of interactions occurring between certain intercalators and single-stranded nucleic acids and compared with the condensation induced by polyvalent "simple" cations such as Co3+ or spermine4+. The described phenomenon can have an application in analytical and preparative biochemistry for characterization of the primary and secondary structure of nucleic acids and for separation of the compounds. The possibility that the condensation plays a role in mutagenic and pharmacological effects of aromatic cations is considered.

Acridine Orange↗
PubMed1984

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

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

Acridine Orange↗
PubMed1983

Interactions of acridine orange with nucleic acids. Properties of complexes of acridine orange with single stranded ribonucleic acid.

Interactions between acridine orange (AO) and nucleic acids (calf thymus DNA, and homoribo- and homodeoxyribo-polynucleotides) were studied in solutions containing ethanol as a cosolvent. Light absorption, scattering and luminescence were measured as a function of AO concentration at different dye/phosphate (D/P) ratios, and the data were analyzed using the McGhee-von Hippel probabilistic model of the polymer-ligand interactions. The absorption spectra of AO complexes with four homoribopolymers are presented. The intrinsic association constants and cooperativity coefficients of the formation of the complexes were calculated. The effects of ethanol (up to 35%, v/v) on these interactions were concentration dependent and may be extrapolated to zero concentration of this cosolvent. The possibility of destabilization of the double helix of nucleic acids by AO at high D/P ratios is discussed in light of the available thermodynamic data.

Acridine Orange↗
PubMed1983

Increased accessibility of bases in DNA upon binding of acridine orange.

Acridine orange (AO) forms 1:1 complexes with dsDNA which are insoluble in aqueous media, exhibit red luminescence, have minimal green luminescence and resemble complexes of AO with ss nucleic acids. During formation and/or dissociation of these complexes, accessibility of DNA bases to two conformational probes, formaldehyde and diethyl pyrocarbonate is increased, suggesting that the base pairing is destroyed and DNA at least partially denatured. Adriamycin and Ellipticine, but not Ethidium Bromide exert similar destabilizing effects. The results confirm our earlier predictions based on thermodynamic calculations that the double helix undergoes destabilization upon binding an intercalator characterized by high cooperativity in interaction with ss nucleic acids. Thus, the highly cooperative ligand binding to ss sections during the "breathing" of the polymer may progressively destabilize the adjacent ds structure.

Acridine Orange↗
PubMed1983

Denaturation of RNA and DNA in situ induced by acridine orange.

The products of interaction of acridine orange (AO) with single-stranded (ss) nucleic acids are precipitates which exhibit red luminescence. Titration of rRNA or thymus DNA with AO results in formation of such products suggesting that the dye, per se, denatures double-stranded (ds) sections of these biopolymers. This transition, measured as the increase of red luminescence, a concomitant decrease of green fluorescence, and followed by an increase of light scatter of the AO-nucleic acid complexes, is cooperative and at 0.15 N NaCl occurs at 4-20 and 10-50 microM range of AO concentration for rRNA and DNA, respectively. The changes in stainability of nucleic acids in situ, in permealized cells, occur at higher AO concentration. Thus, the transition of RNA in situ is biphasic and seen at 20-120 microM AO. In the presence of EDTA, however, the change is monophasic and shifted to the 10-30 microM range of AO concentration. The change in stainability of DNA also shows two phases: one at 30-60 microM and another at 70-120 microM of AO. Extraction of basic proteins with 0.08 N HCl shifts the transition of DNA to the 30-60 microM AO concentration and makes it monophasic. The observed differences in denaturability of RNA vs DNA explain the specificity of AO in differential staining of these bipolymers in histochemical reactions. In living cells the products of interaction of AO with nucleic acids are detected by electron microscopy. In the cytoplasm of interphase cells the formation of dense precipitates within ribosomes and polysomes, simultaneous with a specific retraction of ribosome-polysome complexes from the periphery of the cell to the nucleus is evident. The latter suggests higher order organization of these particles involving their association with each other or with the nucleus via polyanionic macromolecules which collapse upon binding with AO. The DNA in heterochromatin is more sensitive to AO-induced denaturation, as evidenced by the fact that the dense complexes are formed preferentially in the regions of condensed chromatin of the interphase nucleus, or in metaphase chromosomes.

Acridine Orange↗
PubMed1982

Luminescence of the solid complexes of acridine orange with RNA.

The products of interaction between acridine orange (AO) and natural RNA, or the synthetic RNA homopolymers are precipitates insoluble over a wide range of ionic strength. These complexes have a composition of 1 AO molecule per 1 phosphate. The reaction is highly cooperative and the complex exhibits metachromatic luminescence. Significant differences in the luminescence spectra, related to base composition of RNA, characterize the insoluble complexes. This observation suggests that dye-base interactions take place in the AO-RNA complexes. During titration of poly(rA) with AO and simultaneous measurement of the luminescence and light scatter a two-step formation of the particles could be detected. The cooperative binding of the ligand at D/P less than 0.6 coincides with formation of small particles (molecular aggregates or micelles) and is followed at D/P greater than 0.6 by a cooperative agglomeration; the product of this agglomeration is particles of the size 0.35 micrometer and larger. Evidence is presented that suggests that the long wave length luminescence (approximately 650 nm) is a consequence of the solute-solid state transition of the AO-RNA interaction product rather than the classic dye-dye interactions previously visualized in the stacking model. Since these novel observations cannot be fully explained by the previously postulated molecular mechanisms of AO binding, an alternative model is advanced. Its implications in quantitative cytochemistry of nucleic acid as applied to flow cytometry are discussed.

Acridine Orange↗
PubMed1982

Interaction of rhodamine 123 with living cells studied by flow cytometry.

The cationic fluorochrome rhodamine 123 (R123), reported to bind specifically to mitochondria of living cells, was presently investigated with respect to its uptake by a variety of cell types in various functional states and the subsequent effect of the dye on cell growth. The emission spectrum of R123 taken up by cells undergoes a 12-nm red shift, suggesting formation of a complex. Cells accumulate R123 rapidly; near maximum binding is reached after 5 to 10 min, regardless of the temperature (0-37 degrees) of incubation. There is a dose-dependent relationship between R123 concentration in the medium and the dye accumulation in the cell that covers the range of 0.1 to 10.0 and 0.1 to 5.0 microgram of R123 per ml under equilibrium and nonequilibrium conditions, respectively. Some leakage of the dye from cells occurs, following their transfer into dye-free medium. Despite the leakage, the intracellular dye can be detected after at least two cell divisions, thus indicating that: (a) the R123-labeled cells divide; (b) during division, labeled mitochondria are distributed into the daughter cells; and (c) R123 may be used as a cell tracer. Cell death often is accompanied by a transient increase in R123 fluorescence. Dead cells exhibit either uniform, strong fluorescence or show a patchy labeling pattern suggesting swollen mitochondria. With time (4 to 8 hr), dead cells lose ability to retain R123 and lyse. Uptake of R123 by living cells is increased during the transition from quiescence into the cycle, and a decrease is seen when Friend leukemia cells undergo erythroid differentiation; in all cases, changes in R123 uptake are correlated with changes in cellular RNA content. Simultaneous cell staining with R123 and ethidium or propidium provides a rapid assay of the viability of the cells and their metabolic state, i.e., as related to proliferation or motility. Pulse-labeling of cells with up to 10 microgram of R123 per ml has no significant effect on their immediate growth and cloning efficiency. In the continuous presence of R123, however, cells become specifically arrested in the G1A compartment, i.e., in early G1 phase. Detailed analysis of the cell cycle kinetics reveals that cell progression through all phases is slowed 4 hr after addition of R123. Cell exit from G1A, however, is affected as early as 2 hr following addition of R123, and with time the cells are unable to leave this compartment at all. Uncharged rhodamine dyes (rhodamine 110 and rhodamine B) do not accumulate in mitochondria and are without effect on the cell cycle. The cytostatic effect of R123 is discussed in light of the dye specificity for mitochondrial membranes and the disruption of cell energy metabolism, resulting in the inability of the cells to attain a critical content of essential components (i.e., ribosomal RNA), necessary for cell entrance into the prereplicative (G1B) compartment of G1 phase.

Animals↗