Stage-specific embryonic antigen-4 as a novel marker of ductal cells of human eccrine sweat glands.

BACKGROUND: Different populations of unipotent or multipotent stem cells have been identified in human epidermis and its appendages. It is well documented that these cells maintain tissue homeostasis and actively participate in epidermal regeneration after injury. However, there is no evidence of the presence of pluripotent stem cells in human epidermis. OBJECTIVES: In this study we investigated whether cells positive for embryonic stem cell marker stage-specific embryonic antigen-4 (SSEA-4) are present in adult human epidermis and, if so, whether they are pluripotent and correspond to the population of primitive stem cells. METHODS: The expressions of SSEA-4 and pluripotency transcription factors were analysed using flow cytometry. By means of immunohistochemical staining, we studied the exact localization of these cells in sections of human skin. RESULTS: We show that a population of SSEA-4+ cells is present in human epidermis. In contrast to the commonly accepted belief, the expression of SSEA-4 is not connected with the pluripotent character of isolated cells. We found that these SSEA-4+ cells are localized in the ducts of eccrine sweat glands. CONCLUSIONS: Our results indicate that SSEA-4 is a novel marker identifying the ductal cells of human sweat glands. The surface character of the antigen provides for a simple method of isolating this cell population and suggests applications of SSEA-4 for future cell therapy research.

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.

Spatial heterogeneity of dynamics of H1 linker histone.

Linker histone H1 participates in maintaining higher order chromatin structures. It is a dynamic protein that binds to DNA and exchanges rapidly with a mobile pool. Therefore, the dynamics of H1 were probed in the nuclei of intact, live cells, using an array of microscopy techniques: fluorescence recovery after photobleaching (FRAP), raster image correlation spectroscopy (RICS), fluorescence correlation spectroscopy (FCS), pair correlation functions (pCF) and fluorescence anisotropy. Combination of these techniques yielded information on H1 dynamics at small (1-100 µs: FCS, RICS, anisotropy), moderate (1-100 ms: FCS, RICS, pCF) and large (1-100 s: pCF and FRAP) time scales. These results indicate that the global movement of H1 in nuclei (at distances >1 µm) occurs at the time scale of seconds and is determined by processes other than diffusion. Moreover, a fraction of H1, which remains immobile at the time scale of tenths of seconds, is detectable. However, local (at distances <0.7 µm) H1 dynamics comprises a process occurring at a short (~3 ms) time scale and multiple processes occurring at longer (10-2,500 ms) scales. The former (fast) process (corresponding probably to H1 diffusion) is more pronounced in the nuclear regions characterized by low H1 concentration, but the latter (slow, attributable to H1 binding) in the regions of high H1 concentration. Furthermore, some regions in nuclei (possibly containing dense chromatin) may constitute barriers that impair or block movement of H1 histones within short (<1 µm) distances.

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.

Hepatoma HepG2 cells as a model for in vitro studies on mitochondrial toxicity of antiviral drugs: which correlation with the patient?

Currently, drugs have been synthesised that can significantly delay the course of several viral infections, including those provoked by HBV, HCV or HIV, but that display consistent side effects, including toxicity for organelles such as mitochondria. Several in vitro models and techniques have been developed to analyse the effects of such compounds. HepG2 cells (from human hepatoma) are an excellent model to investigate mitochondrial (mt) toxicity because of their high content of organelles and mtDNA, and actually different investigators are indeed using such cells. Studies in vitro on cell lines are relatively easy, but it is necessary to be careful in the interpretation of data, which are usually obtained on continuously growing, tumour cells, quite different from normal, resting, non-neoplastic cells collected from a patient. Direct analysis of drug-induced mt damage in patients is extremely more complex than that performed using in vitro models because of the difficulty to obtain adequate cells or to have discrete amounts of biological material, the status of the patient at the moment of cell collection, the use of an adequate assay and its correct execution, and finally the possibility to find sex- and age-matched healthy controls as source of reference parameters.

Anchorage-dependent chick embryo fibroblasts synthesise DNA and proliferate when cultured in multilayered sheets suspended among glass fibres.

Chick embryo fibroblasts (CEFs) spontaneously form multicellular and multilayered sheets suspended on the network of glass fibres which are stabilized by fibronectin containing protein deposits located at cell-to-cell contacts. The cells situated within the sheets are surrounded by the neighbouring cells and their mechanical equilibrium is stabilised by intercellular "parabaric" effects. It was found that CEFs in the sheets retain relatively high mitotic activity corresponding to that observed in sparse monolayer cultures. These cells grew up to much higher local density than in confluent and contact-inhibited monolayer cultures and developed an abundance of microfilament bundles that terminated at vinculin-containing protein complexes. The results presented demonstrate that direct contact with solid substratum, cell-to-cell contacts, local cell density, and intercellular exchange of humoral factors are not directly involved in the density-dependent inhibition of growth observed in monolayer cultures. They also support the concepts concerning the role of mechanical equilibrium of cell membrane and sub-membranous cytoskeleton in the regulation of proliferation of non-transformed cells.

Chromatin condensation and sensitivity of DNA in situ to denaturation during cell cycle and apoptosis--a confocal microscopy study.

The goal of this study was to construct high resolution 3D confocal images of regions of condensed and extended chromatin in cell nuclei and individual chromosomes. It has been shown previously that sensitivity of DNA in situ to denaturation correlates with chromatin condensation and varies during cell cycle and apoptosis. Thus, detection of DNA which was partially denatured in situ provided a means to image areas of condensed chromatin. DNA denaturation was detected using a metachromatic dye acridine orange (AO) which differentially stains single stranded (ss) and double-stranded (ds) DNA sections. Early studies of denaturability of cellular DNA utilized flow cytometry and standard fluorescence microscopy. These techniques could not reveal small local differences in DNA denaturability within cell nucleus or in individual chromosomes. For instance, it was not possible to detect the initial points of chromosome condensation in G2-phase of the division cycle or in apoptosis. In order to achieve this goal we have recently extended these studies by applying confocal microscopy. We investigated DNA denaturability in normal human fibroblasts and HL-60 leukemic cells, at different stages of cell cycle and apoptosis. Following removal of RNA and partial denaturation of DNA with acid cells were stained with AO. Green (530 nm) and red (640 nm) fluorescence (exc. 457 nm) of non-denatured and denatured DNA was imaged by confocal microscopy. Blind deconvolution was used to further improve the quality of 3D images. Photobleaching of AO fluorescence was minimized and a correction for chromatic aberration and register shift was implemented. Nuclei of interphase cells exhibited predominantly green fluorescence representing AO binding to ds DNA. Punctuate areas of red fluorescence representing AO binding to denatured DNA and most likely associated with local regions of condensed chromatin were also present in all interphase nuclei. The proportion of denatured DNA increased in cells entering mitosis. In prophase individual condensing chromosomes exhibited varied proportions of green and red fluorescence indicating different content of denatured chromatin. In some chromosomes bands of denatured and denaturation-resistant chromatin were clearly resolved. In metaphase and anaphase chromosomes exhibited red fluorescence along all length of their arms indicating the highest and uniform susceptibility to denaturation. In telophase chromosomes contained predominantly denaturation-resistant DNA again and denaturated regions were significantly less abundant. At cytokinesis some decondensing chromosomes were still resolved. At this stage almost all regions of denatured DNA were located close to nuclear envelope. These regions may correspond to pockets of heterochromatin reforming at nuclear periphery. In early apoptosis condensation of chromatin appeared to commence in several distinct regions within nucleus. Some apoptotic bodies contained condensed chromatin surrounding central regions of extended chromatin. At late stages of apoptosis the whole volume of apoptotic bodies was occupied by condensed chromatin.

Differential effects of oncostatin M and leukaemia inhibitory factor expression in astrocytoma cells.

The effects of the production of two closely related cytokines, oncostatin M (OSM) and leukaemia inhibitory factor (LIF), by astrocytoma cells were investigated using the stable cell line human U373-MG, which expressed and secreted both biologically active polypeptides. The expression of LIF by these cells caused resistance to this cytokine due to loss of the LIF receptor (LIFR), from the cell surface, suggesting its retention. In contrast, cells expressing OSM were stimulated by this cytokine, utilizing an autocrine mechanism, and possessed receptors for OSM, but not LIF, on the cell surface. In these cells the continuous up-regulation of OSM-induced gene expression was found even though the Janus kinase-signal transducer and activator of transcription ('JAK/STAT') pathway was almost exhausted due to long-term autocrine stimulation of the cells by OSM. The amount of LIFR was down-regulated in both LIF- and OSM-producing cells and this effect was not found in wild-type U373-MG cells treated with externally added cytokines. To investigate the mechanism of autocrine stimulation by OSM we constructed a stable cell line expressing a form of OSM that is retained in the endoplasmic reticulum (ER). This biologically active cytokine was not secreted, but was localized in the ER. In addition, it did not stimulate the astrocytoma cells in an autocrine manner. We conclude that expression of LIF causes resistance of astrocytoma cells to this cytokine, whereas expression of OSM leads to autocrine stimulation.

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.

Evaluation of biocompatibility of apatite-wollastonite ceramics in fibroblast cultures.

The aim of this work has been to test the biocompatibility of four bioactive, gel derived glass-ceramic materials of CaO-PO2-SiO2 system, modified by addition of boron, aluminum and magnesium compounds. We have examined the growth, collagen synthesis, adhesion and morphology of NRK rat fibroblasts cultured in direct and indirect contact with biomaterials. The growth of cells cultures has been quantified by two methods: [3H]thymidine incorporation and direct counting of cells. The level of collagen synthesis has been used as a parameter describing metabolic activity of cells. Cellular morphology has been assessed following 24 h and 4 days of culturing cells on biomaterials by using SEM and confocal microscopy, respectively. Additionally, in order to obtain information about the attachment of cells to substratum the presence of focal contacts has been examined. The results of all the experiments have demonstrated that none of the materials under study significantly altered cellular functions that were tested. This indicates that additions of MgO, Al2O3 and B2O3 have not induced cytotoxicity of the materials under study. This qualifies them for further in vivo experiments.

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.

Mitochondrial heterogeneity during staurosporine-induced apoptosis in HL60 cells: analysis at the single cell and single organelle level.

BACKGROUND: Apoptosis is a complex phenomenon during which several events occur. A growing interest exists on the role and functionality of mitochondria during this type of cell death. The responsibility of modifications in mitochondrial membrane potential (Delta Psi) in triggering apoptosis is under investigation. METHODS: We evaluated Delta Psi changes in HL60 cells treated with staurosporine (STS). Flow cytometry and confocal microscopy have been used to analyze samples stained with two Delta Psi-sensitive probes, JC-1 and MitoTrackertrade mark Red CMXRos. RESULTS: At the cellular level, we found heterogeneic behavior. Indeed, after STS treatment, some cells displayed typical markers of apoptosis and a collapse in Delta Psi. Others were apoptotic with no changes in Delta Psi, others changed Delta Psi without being apoptotic, and others were healthy. The same heterogeneic response to STS was found at the single organelle level. In a given cell, some mitochondria were depolarized whereas others were not. CONCLUSION: In this model of apoptosis, changes in Delta Psi can be different among cells of the same type and among different organelles of the same cell. The collapse in Delta Psi is thus a heterogeneic phenomenon that seems to be an ancillary event following the irreversible phase of the apoptotic process.

Opposite role of changes in mitochondrial membrane potential in different apoptotic processes.

We have studied the role of changes in mitochondrial membrane potential (DeltaPsi) in two widely-used models of apoptosis, such as dexamethasone-treated rat thymocytes and U937 human cells treated with tumor necrosis factor-alpha and cycloheximide. To dissipate DeltaPsi, we used low concentrations of valinomycin, unable per se to induce apoptosis, and demonstrated that the decline in DeltaPsi exerts opposite effects in the two models. Indeed, in U937 cells, depolarization of mitochondria increased apoptosis, which decreased in rat thymocytes. This leads to the suggestion that disruption of DeltaPsi plays opposite roles depending on the experimental model. In U937 cells, the drop of DeltaPsi is a possible contributory cause for the apoptotic process; in rat thymocytes, it could be a limiting factor. We propose that these opposite effects could be due to the different ATP requirement of each apoptotic pathway.

Reduction of a tetrazolium salt, CTC, by intact HepG2 human hepatoma cells: subcellular localisation of reducing systems.

Cell-mediated reduction of tetrazolium salts, including MTT, XTT, MTS, NBT, NTV, INT, in the presence or absence of intermediate electron carriers is used as a convenient test for animal or bacterial cell viability. Bioreduction of tetrazolium is considered an alternative to a clonogenic assay and a thymidine incorporation assay. However, correlation between clonogenic potential and capacity to reduce tetrazolium has not been demonstrated convincingly. Moreover, despite a wide use of tetrazolium viability assays, the mechanism and subcellular localisation of reducing systems or species in viable intact cells have not been fully elucidated. We report evidence indicating that a tetrazolium salt CTC can be reduced in the presence as well as in the absence of an electron carrier by viable HepG2 human hepatoma cells. CTC-formazan is formed within or at the outer surface of plasma membranes. We hypothesise that in the presence of an electron carrier the electron donors active in the reduction of CTC are located in the intracellular compartment, as well as in plasma membranes. However, in the absence of an electron carrier, the reduction occurs primarily via a plasma membrane-associated enzymatic system or species.

Heat-shocked monocytes are resistant to Staphylococcus aureus-induced apoptotic DNA fragmentation due to expression of HSP72.

Human peripheral blood monocytes became apoptotic following phagocytosis of Staphylococcus aureus. The consequences of heat stress for monocytes were studied with regard to the effect on S. aureus-induced apoptosis. Exposure of monocytes to 41.5 degrees C for 1 h resulted in HSP72 expression and had no influence on phagocytosis of bacteria; moreover, phagocytosis of S. aureus immediately or shortly after heat shock had no effect on the S. aureus-induced monocyte apoptosis, as evidenced by DNA fragmentation assay. In contrast, cells which recovered from heat shock for 18 to 24 h, although active as phagocytes, were resistant to the S. aureus-induced apoptosis. The observed protective effect was related to the induction of HSP72, since blocking of HSP72 synthesis by an antisense oligomer abolished the protective effect of heat shock on bacterium-induced monocyte apoptosis.

Apoptosis-like, reversible changes in plasma membrane asymmetry and permeability, and transient modifications in mitochondrial membrane potential induced by curcumin in rat thymocytes.

Curcumin (diferuoylmethane) is a natural compound with anticarcinogenic activities which is able to exert either proapoptotic or antiapoptotic effects in different cell types. This paper focuses on the sequence and extent of primary events induced by curcumin, in comparison with those occurring during dexamethasone-induced apoptosis in rat thymocytes. It also presents annexin VI-FITC as a new probe for studying membrane asymmetry. Curcumin readily penetrates into the cytoplasm, and is able to accumulate in membranous structures such as plasma membrane, endoplasmic reticulum and nuclear envelope. Curcumin-treated cells exhibit typical features of apoptotic cell death, including shrinkage, transient phosphatidylserine exposure, increased membrane permeability and decrease in mitochondrial membrane potential. However, nuclei morphology, DNA fragmentation, the extent and time-course of membrane changes are different from those observed during dexamethasone-induced apoptosis, suggesting that, despite many similarities, the mode of action and the events triggered by curcumin are different from those occurring during typical apoptosis.

Extracellular reduction of Cat1 free radical by transformed human hepatocytes.

Redox activities associated with plasma membranes of nonphagocytic animal and plant cells have been reported by several authors. However, the natural substrates, structure and biological role of these putative enzyme systems are not known. Data indicating extracellular reduction of a nitroxide free radical Cat1 (1-oxy-4-trimethylamine-2,2,6,6,tetramethyl-piperidine) by hepatocytes were thought to be artefactual. We report evidence in support of a notion that Cat1 as well as a tetrazolium salt, CTC (5-cyano-2,3-ditolyl tetrazolium chloride), are reduced extracellularly, probably at the cell surface, by human HepG2 hepatoma cells. These data provide evidence confirming the existence of a yet unidentified reducing activity associated with outer surface of plasma membranes of transformed human hepatocytes.