Activation of new replication foci under conditions of replication stress.

DNA damage, binding of drugs to DNA or a shortage of nucleotides can decrease the rate or completely halt the progress of replication forks. Although the global rate of replication decreases, mammalian cells can respond to replication stress by activating new replication origins. We demonstrate that a moderate level of stress induced by inhibitors of topoisomerase I, commencing in early, mid or late S-phase, induces activation of new sites of replication located within or in the immediate vicinity of the original replication factories; only in early S some of these new sites are also activated at a distance greater than 300 nm. Under high stress levels very few new replication sites are activated; such sites are located within the original replication regions. There is a large variation in cellular response to stress - while in some cells the number of replication sites increases even threefold, it decreases almost twofold in other cells. Replication stress results in a loss of PCNA from replication factories and a twofold increase in nuclear volume. These observations suggest that activation of new replication origins from the pool of dormant origins within replication cluster under conditions of mild stress is generally restricted to the original replication clusters (factories) active at a time of stress initiation, while activation of distant origins and new replication factories is suppressed.

Relationship between DNA damage response, initiated by camptothecin or oxidative stress, and DNA replication, analyzed by quantitative 3D image analysis.

A method of quantitative analysis of spatial (3D) relationship between discrete nuclear events detected by confocal microscopy is described and applied in analysis of a dependence between sites of DNA damage signaling (γH2AX foci) and DNA replication (EdU incorporation) in cells subjected to treatments with camptothecin (Cpt) or hydrogen peroxide (H2O2). Cpt induces γH2AX foci, likely reporting formation of DNA double-strand breaks (DSBs), almost exclusively at sites of DNA replication. This finding is consistent with the known mechanism of induction of DSBs by DNA topoisomerase I (topo1) inhibitors at the sites of collisions of the moving replication forks with topo1-DNA "cleavable complexes" stabilized by Cpt. Whereas an increased level of H2AX histone phosphorylation is seen in S-phase of cells subjected to H2O2, only a minor proportion of γH2AX foci coincide with DNA replication sites. Thus, the increased level of H2AX phosphorylation induced by H2O2 is not a direct consequence of formation of DNA lesions at the sites of moving DNA replication forks. These data suggest that oxidative stress induced by H2O2 and formation of the primary H2O2-induced lesions (8-oxo-7,8-dihydroguanosine) inhibits replication globally and triggers formation of γH2AX at various distances from replication forks. Quantitative analysis of a frequency of DNA replication sites and γH2AX foci suggests also that stalling of replicating forks by Cpt leads to activation of new DNA replication origins. © 2013 International Society for Advancement of Cytometry.

[Localization and composition of renal immunodeposits in mice developing HgCl2-induced autoimmune process].

A characteristic feature of systemic autoimmune diseases along with appearance of autoantibodies targeting self-antigenes is deposition of immunoglobulins and components of the complement system in kidneys. However, mechanisms of the deposit formation and their cytotoxic effects still remain poorly studied. To elucidate these questions, we used SJL/J mice which are known to develop autoimmune process accompanied by the appearance of anti-fibrillarin antibodies following regular administrations of sublethal dozes of HgCl2. Using antibodies to the total murine ummunoglobulins we showed that immunodeposits were present in glomeruli of autoimmune and control (not-autoimmune) animals, but their intensity was directly correlated with the titer of anti-fibrillarin autoantibodies and was minimal in control mice. By confocal microscopy and conventional fluorescence microscopy it was defined that immunodeposits deeply penetrate glomeruli and are the most likely located within mesangial cells. In autoimmune animals, ummunoglobulins completely colocolized with the C3--component of complement, but not with the major autoantigen--the protein fibrillarin. We failed to determine the signs of cell proliferation or death in glomeruli. The most prominent difference between control and autoimmune mice was the presence if immunodeposits in renal blood vessels. These observations argue in favor of the idea that destructive and disfunctional renal lesions accompanying development of autoimmune diseases can be caused, in part, by accumulation of immunodeposits in blood vessels.

Minimizing photobleaching during confocal microscopy of fluorescent probes bound to chromatin: role of anoxia and photon flux.

Exposure to light can destroy the ability of a molecule to fluoresce. Such photobleaching limits the use of fluorescence and confocal microscopy in biological studies. Loss of fluorescence decreases the signal-to-noise ratio and so image resolution; it also prevents the acquisition of meaningful data late during repeated scanning (e.g. when collecting three-dimensional images). The aim of this work was to investigate the role of oxygen in the photobleaching of fluorophores bound to DNA in fixed cells, and to explore whether anoxia could minimize such bleaching. Anoxia significantly reduced bleaching rates and changed the order of reaction of both propidium iodide (an intercalator) and chromomycin A3 (a minor-groove binder) bound to DNA; it afforded the greatest protection at low photon fluxes. However, it had no effect on the bleaching of the green fluorescent protein (GFP) covalently attached to a histone and so bound to DNA, probably because the protein shielded the chromophore from oxygen. Bleaching of all three fluorophores depended on photon flux. Practical ways of minimizing bleaching were examined, and examples of three-dimensional images of DNA marked by propidium and GFP (collected under standard and optimized conditions) are presented.

Interaction of oxygen-sensitive luminescent probes Ru(phen)(3)(2+) and Ru(bipy)(3)(2+) with animal and plant cells in vitro. Mechanism of phototoxicity and conditions for non-invasive oxygen measurements.

Understanding the role of oxygen in the physiology, pathophysiology and radio- and chemosensitivity of animal cells requires accurate and non-invasive measurements of oxygen concentrations in the range of 0-2x10(-4) M, in cells in vitro or in vivo. High resolution 3D imaging techniques could be particularly useful in investigating tissue oxygenation in vivo and in model tissues (multicellular spheroids) in vitro. The goals of this work were to develop microscopy techniques and (i) to define conditions under which two oxygen-sensitive luminescent dyes, Ru(bipy)(3)(2+) (tris(2,2'-bipyridyl)ruthenium(II) chloride hexahydrate) and Ru(phen)(3)(2+) (tris(1,10-phenanthroline)ruthenium(II) chloride hydrate) can be used to probe oxygen concentrations within viable cells in vitro, when no phototoxic effects are evident, and (ii) to investigate the mechanism of phototoxicity once cell damage occurs. This report demonstrates that Ru(bipy)(3)(2+) and Ru(phen)(3)(2+) do not pass through intact biological membranes, do not cause measurable photodamage to plasma membranes at a concentration of 0.2 mM and, when loaded into endosomes, yield a strong luminescent signal. However, at an extracellular concentration of 1 mM, in the presence of 457-nm light, detectable amounts of both complexes accumulate at the plasma membrane and cause a loss of membrane integrity via a mechanism which may involve the generation of singlet oxygen.

The role of plasma membrane in bioreduction of two tetrazolium salts, MTT, and CTC.

Despite widespread use of various tetrazolium assays, the mechanisms of bioreduction of these compounds have not been fully elucidated. We investigated the capacity of tetrazolium salts to penetrate through intact cell plasma membranes. 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) and 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) tetrazolium salts appear to represent examples of species that are reduced by different mechanisms. We provide evidence suggesting that MTT readily crosses intact plasma membranes and is reduced intracellularly. MTT appears to be reduced by both plasma membrane and intracellular reductases; reducing cells are not damaged and remain metabolically active for at least 45 min. In contrast, CTC remains extracellular with respect to viable cells and thus requires plasma membrane permeable electron carrier to be reduced efficiently. However, reduction of CTC in the presence of an electron carrier inflicts damage on plasma membranes. The intracellular vs extracellular sites of reduction of tetrazolium salts were established on the basis of deposition of formazans. Crystals of formazan were detected using fluorescence or backscattered light confocal laser microscopy. We postulate that the capacity of a tetrazolium salt to cross intact plasma membranes constitutes an important experimental variable which needs to be controlled in order to correctly interpret the outcome of tetrazolium assays designed to measure cellular production of oxygen radicals, activity of mitochondrial, cytosolic, or outer membrane reductases, etc.

Nonperturbing test for cytotoxicity in isolated cells and spheroids, using electron paramagnetic resonance.

Electron paramagnetic resonance imaging (EPRI) and EPR spectroscopy of nitroxides have been applied to detect and quantify cytotoxic effects in mammalian cells in vitro. Images depicting microscopic viable and nonviable areas in the same multicellular spheroid (a model of solid tumors) have been acquired prior to and following the treatment with an antitumor drug, Adriamycin. The loss of viability in the inner region of the viable rim was detected. Such mapping of the time-course changes of the localization of viable and nonviable cells in the same intact biological object is not possible with routinely used methods to measure viability, such as histological examination. The experimental conditions required for high accuracy and sensitivity of the EPRI of spheroids have been evaluated and directions for further development of this approach are indicated.

Distribution of 5-doxylstearic acid in the membranes of mammalian cells.

Concentration-dependent spin broadening of ESR spectra of the nitroxide 5-doxylstearic acid has been used to evaluate the distribution of 5-doxylstearic acid in the membranes of intact mouse thymus-bone marrow (TB) and Chinese hamster ovary (CHO) cells. TB cells, CHO cells, erythrocytes, and isolated plasma membranes from CHO cells were labelled with 5-doxylstearic acid and the peak to peak linewidths of the central line of the resulting ESR spectra were measured. The measured line widths were linearly dependent on the amount of 5-doxylstearic acid incorporated into the sample over the range of 0-0.18 mol nitroxide per mol lipid. In erythrocytes, the relationship between linewidths approximated a linear function at lower concentrations of 5-doxylstearic acid, up to 0.07 mol nitroxide per mol lipid. The amount of broadening of the central line for a given amount of 5-doxylstearic acid was far less for intact cells than for either erythrocytes or plasma membrane, indicating that the 5-doxylstearic acid samples a much larger lipid pool in the intact cells. With the broad assumption that the mobility of the 5-doxylstearic acid is similar in different membranes, the size of the lipid pool sampled by 5-doxylstearic acid is approximately equal to the total cellular lipid in intact cells. If a given concentration of 5-doxylstearic acid sampled only the plasma membrane of TB or CHO cells, we would expect to see a linewidth corresponding to a 12-20-fold greater local concentration of 5-doxylstearic acid than was observed, since the plasma membranes of CHO and TB cells represent only 5-8 percent of the total cellular lipid. Therefore, the 5-doxylstearic acid must distribute into most or all cellular membranes of intact cells and is not localized in the plasma membrane alone.

The stoichiometry of tyrosinase-catalyzed oxidation of 4-hydroxyanisole.

Oxygen utilisation during tyrosinase-catalysed oxidation of 4-hydroxyanisole was investigated using an electron spin resonance technique which employs quantitative changes in the characteristics of the electron spin resonance spectrum of the spin label 3-carbamoyl-2,5-dihydro-2,2,5-5-tetramethyl-1-H-pyridoyl-1-ylox y (CTPO) to follow changes in the oxygen concentration. Reaction mixtures containing mushroom tyrosinase (15 micrograms ml-1) and differing initial concentrations of 4-hydroxyanisole in aerated phosphate buffer at pH 6.8 were incubated at room temperature. The ratio of utilisation of oxygen was found to be in approximately 1:1 molar ratio with the initial 4-hydroxyanisole concentration in the reaction mixture between 50 and 200 mumol/l 4-hydroxyanisole. The results are consistent with the stoichiometry of oxygen utilisation being accounted for by the oxidation of 4-hydroxyanisole to anisyl quinone.