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J Dobrucki

Publications and source records attributed to J Dobrucki.

13 recordsLinked to original sources
PubMed2001

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.

Antigens, CD↗
PubMed2001

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.

Acridine Orange↗
PubMed2001

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.

Actin Cytoskeleton↗
PubMed2000

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.

Apoptosis↗
PubMed2000

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.

Adenosine Triphosphate↗
PubMed2000

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.

Animals↗
PubMed1999

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.

Carcinoma, Hepatocellular↗
PubMed1999

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↗
PubMed1998

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.

Animals↗
PubMed1998

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.

4-Chloromercuribenzenesulfonate↗
PubMed1985

Cell-cell contact affects cellular sensitivity to hyperthermia.

The influence on heat sensitivity of interactions between HT29 human colon adenocarcinoma cells grown in two- or three-dimensional contact was investigated. No evidence for intercellular cooperation affecting cellular sensitivity to hyperthermia was found. Cells grown in monolayer were found to be more heat sensitive than those in aggregates, probably due to the different physical properties of the membrane of flat cells attached to the substratum. Cell-cell contact appears to affect the heat sensitivity of HT29 cells possibly by means of inducing arrest in a heat resistant phase of the cell cycle.

Adenocarcinoma↗
PubMedDate not supplied

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.

Antiviral Agents↗