Divalent cations are dispensable for binding to DNA of a novel positively charged olivomycin A derivative.

The current model of binding of the antitumor antibiotic olivomycin A (1) to GC-rich DNA regions presumes that coordination of the magnesium divalent cation with drug dimers is necessary for binding of 1 into the minor groove of the DNA duplex. Previously we have synthesized the derivatives of 1 termed 'short acid' (2) and its N,N-dimethylaminoethylamide (3). The latter compound demonstrated an improved tolerance in vivo compared to 1 and good therapeutic potency in animal models. We herein report that compound 3 is able to form stable complexes with DNA in the absence of Mg2+, in striking contrast to 1 whose binding to the DNA absolutely requires Mg2+. The mode of binding of 3 to DNA is similar in the presence or absence of Mg2+ as determined by circular dichroism. The affinity to DNA of 3 in Mg2+-free solution was similar to that of 1 or 3 in the presence of Mg2+ at low ionic strength. Non-electrostatic contributions to total free energy of binding of 1 and 3 to DNA were comparable for Mg2+-free complexes. Our data strongly suggest that electrostatic interaction of the positively charged 3 can compensate for the absence of divalent ions in complexes with DNA. This new property of the olivomycin A derivative expands the mechanistic knowledge of the modes of interaction with DNA of small molecular weight drug candidates.

Cell membrane-dependent chromatin condensation.

It is shown by means of flow cytometry that during several seconds after cell membrane damage by a non-ionic detergent in physiologically relevant buffer solution, the chromatin of mouse thymocyte nuclei undergoes a drastic decondensation, which is revealed by a sharp increase of binding of DNA-specific fluorochromes (olivomycin or propidium iodide) and of DNA accessibility to DNAse I digestion. A similar change is observed in dead cells. Roughly half of this decondensation can be prevented by lowering the pH of the outside medium to the level known to be inside the cells; the other half remains thus far unexplained (divalent cations and the difference between small anion species seem not to be involved). The approach is based on a novel observation that fixation by formaldehyde conserves chromatin structure before the action of detergent. Flow cytometric assay is proposed for monitoring these condensation/decondensation events in media of different composition. In addition, a new approach to viable/dead cell determination, which has the advantage of immediately fixing the cell state and preserving it for a reasonably long time, is proposed.

Modelling basic features of specificity in DNA-aureolic acid-derived antibiotic interactions.

The nonintercalative groove binding of a simplified model of olivomycin, to sequences d(CGCGCGC)2, d(TATATAT)2, and d(CICICIC)2 is investigated. A significant preference is displayed for the minor groove of the d(CG) sequence. This is due predominantly to the formation of H-bonds between the hydroxyl groups on the aglycone of the drug and the 2-amino group of the central guanine of the oligonucleotide.

Investigations into the sequence-selective binding of mithramycin and related ligands to DNA.

The preferred binding sites for mithramycin on four different DNA fragments have been investigated by DNAase I footprinting. Sites containing at least two contiguous GC base pairs are protected by the antibiotic, the preferred binding site consisting of the dinucleotide step GpG (or CpC). Related antibiotics chromomycin and olivomycin produce similar, but not identical footprinting patterns suggesting that they can recognize other sequences as well. All three antibiotics induce enhanced rates of enzyme cleavage at regions flanking some of their binding sites. These effects are generally observed in runs of A and T and are attributed to DNA structural variations induced in the vicinity of the ligand binding site. The reaction of dimethylsulphate with N7 of guanine was modified by the presence of mithramycin so that we cannot exclude the possibility that these antibiotics bind to DNA via the major groove.

[Stereochemistry and kinetics of interaction with DNA of the antineoplastic antibiotic olivomycin]. / Stereokhimiia i kinetika vzaimodeistviia s DNK protivoopukholevogo antibiotika olivomitsina.

The kinetics of interaction of antitumor glycoside antibiotic olivomycin with DNA has been investigated. The existence of two relaxation times in the experimental kinetics curves indicates that two types of antibiotic--DNA complex are formed. We have measured the rate constants of association and dissociation processes and determined their temperature dependences. It is suggested, that one of the complex form results from nonspecific interaction between glycoside residues of the antibiotic molecule and sugar-phosphate backbone of DNA whereas the other type of complex exhibits a pronounced specificity for GC-rich regions on DNA. The binding specificity probably results from formation of a H-bond between the antibiotic chromophore ring and guanine 2-amino group. A stereochemical model for olivomycin-DNA complex is proposed. According to this model the antibiotic chromophore and glycoside residues are located in the narrow groove of DNA.

[Cytochemical properties of prematurely condensed chromosomes]. / Tsitokhimicheskie svoistva prezhdevremenno kondensirovannykh khromosom.

Prematurely condensed chromosomes (PCC) have been obtained by polyethylene glycol (PEG) induced fusion in suspension of the Chinese hamster metaphase cultured cells with those in interphase. As alternative approach the PEG-fusion of the Chinese hamster asynchronous culture cells in monolayer with subsequent incubation in free medium was used. A comparative cytofluorimetric investigation of PCC and chromatin of the interphase nuclei of corresponding ploidy has shown some increase (up to 10%) of acridine orange and olivomycin binding with PCC chromatin. A similar slight increase in low molecular weight ligands binding with chromatin was also found in mitotic chromosomes. The data obtained confirm the opinion about the similarity of events taking place in chromatin during physiological mitosis and premature chromosome condensation. The cytochemical study of chromatin availability to low molecular weight ligands can be used as a criterion for judging on the properties of the artificially condensed chromatin.

Interactions of antineoplastic antibiotics and DNA.

Basic concepts of interaction of antineoplastic antibiotics with DNA are discussed and information concerning the effects of actinomycins, olivomycin and related antibiotics, anthracyclines, sibiromycin, mitomycin C, bruneomycin, and bleomycin is summarized.

[Use of the antibiotic, olivomycin, for the cytochemical study of chromatin]. / Ispol'zovanie antibiotika olivomitsina dlia tsitokhimicheskogo izucheniia khromatina.

The possibility to use the antibiotic olivomycin for fluorescence cytochemistry of chromatin properties has been shown. It is found that the binding of olivomycin to nuclei reveals the functional state of chromatin and changes in the course of its activation. The essential condition for the application of the method described is the use of ethanol-aceton fixative. When other fixatives are used, in particular 70% ethanol, olivomycin binding reflects differences in nuclear DNA amount, rather than those in chromatin properties. The advantages of the method described, in comparison with the commonly used technique, are associated with the high affinity of olivomycin to DNA, absence of olivomycin binding with RNA, simplicity of the staining procedures , and with rather a high stability of complexes formed between olivomycin and DNA.

The use of base pair specific DNA binding agents as affinity labels for the study of mammalian chromosomes.

The fluorochromes Hoechst 33258 and olivomycin are base pair specific DNA binding agents. The fluorescence enhancement of Hoechst 33258 and olivomycin in the presence of DNA can be directly related to the A--T and G--C content of the interacting DNA respectively. Cytological observations of metaphase chromosomes treated with these two compounds suggest that the fluorescent banding patterns produced are the reverse of one another.--Non-fluorescent base pair specific DNA binding agents have been used as counterstains in chromosome preparations to enhance the contrast of the banding patterns produced by the base specific fluorochromes. The non-fluorescent G--C specific antibiotic actinomycin-D enhanced the resolution of fluorescent bands produced by the A--T specific fluorochrome Hoechst 33258. Similarly the non-fluorescent A--T specific antibiotic netropsin was found to enhance resolution of the bands produced by the G--C -specific fluorochrome olivomycin. Netropsin was also found to increase the differential fluorescent enhancement of complexes of olivomycin with DNAs of various base composition in solution. These findings suggest that counterstaining agents act through a base sequence dependent inhibition of subsequent binding by base pair specific fluorochromes.--The base specific DNA binding agents have been used to differentiate different types of constitutive heterochromatin in mammalian species, and to facilitate chromosome identification in somatic cell hybrids.

Biotransformation of antitumor agents by a strain of Whetzelinia sclerotiorum.

Antitumor antibiotics of the olivomycin and chromomycin class were transformed when incubated with a culture of Whetzelinia sclerotiorum. The products, when purified by counter-current distribution and column chromatography, were shown, by their physical properties, to be the deacylated analogues.

[Olivomycin-C14 distribution in the body of mice with Ehrlich's carcinoma resistant to the preparation]. / Raspredelenie olivomitsina-C14 v organizme myshei s kartsinomoi Erlikha, ustoichivoi k preparatu

Tests on mice with Ehrlich's carcinoma (solid and ascitic forms) evidenced that following a single intravenous injection of glycomycin-C14 its maximal concentrations are definable in 30 minutes--1 hour in the kidneys, liver, spleen and the blood (plasma). Six to seven percent of the total radioactivity of the tumor cells fell to the share of the nuclear fraction, while the rest of 93-94 per cent was made up by the remaining cellular components.