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H Acker

Publications and source records attributed to H Acker.

At least 37 records · Page 2Linked to original sources
PubMed1998

Cytochromes and oxygen radicals as putative members of the oxygen sensing pathway.

This study applies biophysical methods like light absorption spectrophotometry of cytochromes, determination of NAD(P)H-dependent superoxide anion (O2-) formation and localisation of hydroxyl radicals (*OH) by 3-dimensional (3D) confocal laser scanning microscopy to reveal in human cells putative members of the oxygen sensing signal pathway leading to enhanced gene expression under hypoxia. A cell membrane localised non-mitochondrial cytochrome b558 seems to be involved as an oxygen sensor in the hepatoma cell line HepG2 in cooperation with the mitochondrial cytochrome b563 probably probing additionally metabolic changes. *OH the putative second messenger of the oxygen sensing pathway generated by a Fenton reaction could be visualized in the perinuclear space of the three human cell lines used. Substances like cobalt or the iron chelator desferrioxamine, which have been applied in HepG2 cells to mimic hypoxia induced gene expression, interact on various sides of the oxygen sensing pathway confirming the importance of b-type cytochromes and the Fenton reaction.

Chemoreceptor Cells↗
PubMed1997

Cytochrome b558 and hydrogen peroxide production in small intensely fluorescent cells of sympathetic ganglia.

Small intensely fluorescent (SIF) cells are paraganglionic cells derived from sympathicoblasts which may serve as interneurons, endo-/paracrine cells or arterial chemoreceptors within sympathetic ganglia. Like paraganglionic cells of other locations, e.g., carotid body glomus cells, they are responsive to hypoxia. Recent studies on glomus cells and other hypoxia-sensing cells suggested the involvement of a b558-type cytochrome and intracellular generation of H2O2 in the process of oxygen sensing. In the present study, we demonstrate the occurrence of the small subunit of cytochrome b558, p22phox, in SIF cells of guinea-pig sympathetic ganglia by immunohistochemistry using two different antisera. H2O2 production was monitored in explanted intact superior cervical ganglia of 2-day-old rats by confocal laser scanning analysis of rhodamine 123 fluorescence generated due to oxidation of dihydrorhodamine 123 by H2O2. Using this technique, SIF cell clusters appeared as sites of highest H2O2 production within the ganglia. Thus, SIF cells exhibit two key features of an oxidase system generating reactive oxygen species. This may be involved in the proposed chain of events in oxygen sensing, but alternative cellular functions of this system have also to be considered.

Animals↗
PubMed1997

Cobalt and desferrioxamine reveal crucial members of the oxygen sensing pathway in HepG2 cells.

Cobalt and desferrioxamine, like hypoxia, stimulate the production of erythropoietin in HepG2 cells. It is believed that cobalt as well as desferrioxamine interact with the central iron atom of heme proteins by changing their redox state similar to hypoxia. A subsequent decrease of the intracellular H2O2 levels under hypoxia was presumed to be the key event for stimulating erythropoietin production. We therefore investigated whether cobalt and desferrioxamine control the intracellular H2O2 levels that regulate gene expression by interacting with hemeproteins. Deconvolution of light absorption spectra revealed respiratory heme proteins such as cytochrome c, b558 and cytochrome aa3, as well as cytochrome b558, which is a nonrespiratory heme protein found in HepG2 cells. Whereas respiratory heme proteins are located in mitochondria, cytochrome b558 similar to the one described for the neutrophil NADPH oxidase can be visualized in the cell membrane of HepG2 cells by immunohistochemistry. Incubation with cobalt (100 microM/24 hr) interacts predominantly with cytochrome b558 and cytochrome b558. The interaction of cobalt with the respiratory chain results in an increased oxygen consumption of HepG2 cells as revealed by PO2 microelectrode measurements. Desferrioxamine (130 microM/24 hr), however has no influence on the cytochromes. In response to an external application of NADH (1 mM), the membrane bound cytochrome b558 produces two times more O2- than to the external NADPH (1 mM) application. Neither desferrioxamine not cobalt has any influence on the NADH stimulated O2- generation. Incubation with cobalt or with desferrioxamine, however, leads to a decrease of the intracellular H2O2 level as revealed by the dihydrorhodamine 123 technique, perhaps causing the well-known enhanced erythropoietin production. The cobalt-induced H2O2 decrease seems to be caused by an increased activity of the glutathion peroxidase that is also induced under hypoxia. Desferrioxamine, however, leads to an apparent H2O2 decrease only because it seems to inhibit the iron catalyzed reaction of H2O2 with dihydrorhodamine 123, hinting at the occurrence of the Fenton reaction in HepG2 cells. Therefore, it must be determined whether or not degradation products of H2O2 by the Fenton reaction suppress erythropoietin production under normoxia.

Cell Line↗
PubMed1997

Cobalt chloride and desferrioxamine antagonize the inhibition of erythropoietin production by reactive oxygen species.

We have recently proposed a H2O2-generating b-type cytochrome as part of the cellular oxygen sensor that controls O2-dependent erythropoietin (Epo) production in the human hepatocellular carcinoma cell line HepG2. H2O2 could act as an intracellular signaling molecule because its production in HepG2 cells is strictly dependent on the pericellular PO2. High cellular levels of H2O2 inhibit hypoxia-induced Epo production while low levels-as under hypoxic conditions-allow full expression of the Epo gene. Since cobalt chloride (CoCl2) and the iron chelator desferrioxamine (DSF) both mimic the hypoxic induction of Epo production we studied the influence of CoCl2 and DSF on the formation and on the action of reactive O2-species with respect to Epo production. Both chemicals reduced the H2O2-dependent 123-dihydrorhodamine fluorescence in HepG2 cells. The inhibition of Epo production by exogenous H2O2 was completely antagonized by DSF. This might indicate that H2O2 exerts its inhibition through a Fenton type reaction. On the other hand, NADPH and pyrogallol which stimulate the production of O2- inhibited Epo production. CoCl2 antagonized their effects. From our results we propose different sites of interaction with the putative signaling chain for DSF and CoCl2. While DSF appears to reduce the action of the H2O2 molecule, CoCl2 might act further upstream through the induction of H2O2-scavenger systems or by interfering with its production.

Cell Line↗
PubMed1997

Tomography of cells by confocal laser scanning microscopy and computer-assisted three-dimensional image reconstruction: localization of cathepsin B in tumor cells penetrating collagen gels in vitro.

We used the nondestructive procedures of confocal laser scanning microscopy in combination with computer-assisted methods to visualize tumor cells in the process of penetrating collagen gels. Three independent sets of images were collected. The image information of all data sets was combined into one image, giving a three-dimensional (3D) impression at high light microscopic resolution and sensitivity. We collected information about the extracellular matrix using the reflection mode, the cell surface/morphology by staining with the fluorescent dye DiOC6(3), and the distribution of cathepsin B by Cy-3-labeled immunolocalization. The specific aim of our study was visualization of the spatial relationship of cell organelles as far as they contain the enzyme cathepsin B to cell morphology and motility in a 3D model of extracellular matrix. The majority of the enzyme was localized pericellularly, with no visible relationship to the direction of movement. However, substantial amounts also appeared in intramatrix pseudopodia and associated with the extracellular face of the plasma membrane, which may be indicative either of secretion and/or epicellular activity. Our approach has general applicability to study of the spatial relationships of cell compartments and their possible reorganization over time. This could open new horizons in understanding cell structure and function.

Adenocarcinoma↗
PubMed1997

[PO2-affinity of oxygen-sensitive intracellular signal cascades].

The oxygen partial pressure field in different organs ranging from 0 to 100 Torr is likely to be a mirror of oxygen sensitive intracellular signal cascades determining ion channel open probability, metabolic pathway activities and gene expression. High or low PO2 affinities of the particular signal cascade optimise the oxygen sensitive cellular response for adapting organ functions to variations of the oxygen supply conditions. The signal cascades are triggered by an oxygen sensor which is believed to be a heme protein. In some cases oxygen radicals are acting as second messengers revealing these signal cascades as an evolutionary highly conserved principle first described in bacteria.

Gene Expression↗
PubMed1996

Oxygen supply and oxygen-dependent gene expression in differentiating embryonic stem cells.

Blastocyst-derived pluripotent mouse embryonic stem cells can differentiate in vitro to form so-called embryoid bodies (EBs), which recapitulate several aspects of murine embryogenesis. We used this in vitro model to study oxygen supply and consumption as well as the response to reduced oxygenation during the earliest stages of development. EBs were found to grow equally well when cultured at 20% (normoxia) or 1% (hypoxia) oxygen during the first 5 days of differentiation. Microelectrode measurements of pericellular oxygen tension within 13- to 14-day-old EBs (diameter 510-890 micron) done at 20% oxygen revealed efficient oxygenation of the EBs' core region. Confocal laser scanning microscopy analysis of EBs incubated with fluorescent dyes that specifically stain living cells confirmed that the cells within an EB were viable. To determine the EBs' capability to sense low oxygen tension and to specifically respond to low ambient oxygen by modulating gene expression we quantified aldolase A and vascular endothelial growth factor (VEGF) mRNAs, since expression of these genes is upregulated by hypoxia in a variety of cells. Compared with the normoxic controls, we found increased aldolase A and VEGF mRNA levels after exposing 8- to 9-day-old EBs to 1% oxygen. We propose that EBs represent a powerful tool to study oxygen-regulated gene expression during the early steps of embryogenesis, where the preimplantation conceptus resides in a fluid environment with low oxygen tension until implantation and vascularization allow efficient oxygenation.

Aerobiosis↗
PubMed1996

PO2 affinities, heme proteins, and reactive oxygen intermediates involved in intracellular signal cascades for sensing oxygen.

The oxygen partial pressure field in different organs ranging from 0 to 100 Torr is likely to be a mirror of oxygen sensitive intracellular signal cascades determining ion channel open probability, metabolic pathway activities and gene expression. High or low PO2 affinities of the particular signal cascade optimize the oxygen sensitive cellular response for adapting organ functions to variations of the oxygen supply conditions. The signal cascades are triggered by an oxygen sensor which is believed to be a heme protein. In some cases reactive oxygen intermediates (ROI) are acting as second messengers revealing these signal cascades as an evolutionary highly conserved principle first described in bacteria.

Animals↗
PubMed1996

Retention and processing of the monoclonal anti-prostate E4 antibody after binding to prostatic adenocarcinoma DU 145 cells.

Cellular retention and processing of a radiolabelled monoclonal anti-prostate antibody were evaluated after binding to prostatic adenocarcinoma DU 145 cells. An endocytosis assay revealed that the rate of release of radioactivity from the cells had an initially rapid phase within the first hours after antibody incubation, which was presumably due to release of monovalently bound antibodies. This was followed by a slower phase, with the possible release of intact bivalently bound antibodies and excretion of degraded internalized antibodies. The relative amount of released radioactivity of high molecular mass was high, indicating that the major part of the antibodies were released without being internalized and degraded. However, when only the radiolabelled antibody that remained cell-associated after 2 h and longer was considered, a substantial part was found to be internalized and radioactive degradation products were excreted. About 30% of the initially cell-associated radioactivity still remained associated with the cells after 48 h, indicating a rather slow antibody processing, which is favourable if the antibody is to be used for targeted radiotherapy. The retention of cell-associated radioiodine was very similar irrespective of whether the antibodies were radiolabelled with the direct chloramine-T method or the indirect ATE (succinimidyl based reagent) method. Since the ATE method can be used to form stable antibody constructs with the therapeutically relevant alpha-emitting radionuclide, astatine-211, this was an interesting finding that will be further evaluated in the future.

Adenocarcinoma↗
PubMed1996

Three-dimensional imaging of rhodamine 123 fluorescence distribution in human melanoma cells by means of confocal laser scanning microscopy.

Three-dimensional (3D) imaging of intracellular rhodamine 123 fluorescence distribution was performed by means of confocal laser scanning microscopy (CLSM). Human IGR melanoma cells grown in monolayer or multicellular spheroid culture were studied for elucidating mitochondrial membrane potential characteristics, and cell and nucleus volume dimensions. Microspheres 6 microns in diameter loaded with rhodamine B were used to calibrate our instruments for performing 3D imaging of optical sections as obtained by CLSM. Accurate optical slicing is only possible taking into consideration the physical characteristics of the objectives used like chromatic and spherical aberrations, depth discrimination or cover slip correction and the temperature dependence of the immersion medium. While 3D imaging of optical slices can be carried out showing the original shape of the object being tested without physical distortion, 3D images of microspheres show well-reproducible structures of rhodamine B fluorescence. These can be explained by a superposition of two effects, namely scattering of the fluorescence light and a gradient of the electromagnetic field strength of the laser beam due to the shape of the object. 3D imaging of optical slices of IGR cells in monolayer or multicellular spheroid culture, which have been loaded with rhodamine 123, show the location of the dye predominantly within the cytoplasm of the cells with a remarkable heterogeneity of fluorescence intensity within and between single cells, indicating differences in the mitochondrial membrane potential and thus in the metabolic activity. Due to the heterogeneity of the cell shape the cell nucleus occupies between 4 and 14% of the total cell volume. These data reveal calibrated 3D imaging as a valuable noninvasive tool to visualize the heterogeneity of cell parameters under different cell culture conditions.

Fluorescent Dyes↗
PubMed1995

Quantitative recording of vitality patterns in living multicellular spheroids by confocal microscopy.

Fluorescent dyes were used in conjunction with confocal microscopy to record the vitality status of cells in multicellular glioma spheroids. Multicellular spheroids are in vitro models for micrometastases or intravascular microregions of large tumors. With progressing growth three distinct concentric annular shells develop. A rim of proliferating cells in the periphery is followed towards the center by layers of quiescent cells and at a defined spheroid diameter cell death occurs in the central core. Fluorescein diacetate (FDA) and Calcein/AM were used as vital stains and Lucifer Yellow/VS (LYVS) was used as a marker for dead cells. For loading multicellular spheroids with the esterase substrate dyes we used a two step cold incubation technique to avoid dye accumulation in the most peripheral cell layers. Homogenously stained tissue allowed to describe the fluorescence attenuation in depth as a monoexponential decay. An attenuation coefficient C was calculated from calibration experiments to be 12.5 x 10(-3) in vital stained tissue and 17.9 x 10(-3) in lethal stained tissue. Using the respective attenuation coefficient the raw data were corrected for light absorption and scattering in depth. In radial recordings of the vitality status of multicellular glioma spheroids using CLSM-technique we showed that spheroids up to a diameter of 250 microns were homogenously stained with Calcein/AM and FDA. Spheroids larger than 250 microns consist of vital stained cells and unstained cells. They do not show dead cell staining until they reach a diameter of about 400 microns. The thickness of the rim of vital stained cells decreased with increasing diameter of the spheroids to 64 +/- 7 microns in spheroids of a diameter of 550 +/- 25 microns. Thereafter the thickness of the Calcein/AM or FDA stained rim augmented again, reaching 93 +/- 9 microns in spheroids of 700 microns in diameter. The first signs of dead cell staining in the central core occurred at a diameter of 400 +/- 25 microns. The radius of the core increased in an exponential way. The cell layer which was stained neither by vital nor by lethal dyes showed a thickness of 150 microns in spheroids of 550 +/- 25 microns in diameter. Our staining technique and the radial recording of mean field fluorescence signals in living multicellular spheroids will be a valuable tool for experimental cancer research providing a non invasive quantification of cell vitality in living multicellular spheroids.

Cell Survival↗
PubMed1995

Immunohistochemical demonstration of four subunits of neutrophil NAD(P)H oxidase in type I cells of carotid body.

We demonstrate, by means of immunohistochemistry, that type I cells of human, guinea pig, and rat carotid bodies react with antisera raised against the subunits p22phox, gp91phox, p47phox, and p67phox of the NAD(P)H oxidase isolated from human neutrophil granulocytes. The findings support previous photometric studies that indicate that carotid body type I cells possess a putative oxygen sensor protein that is similar to the neutrophil NAD(P)H oxidase and consists of a hydrogen peroxide generating low-potential cytochrome b558 with cofactors regulating the electron transfer to oxygen.

Amino Acid Sequence↗
PubMed1994

Effects of cobalt on haem proteins of erythropoietin-producing HepG2 cells in multicellular spheroid culture.

The hypoxia-induced increase of spectrophotometrically measured light absorption at 560 nm, considered as reduced cytochrome b, in HepG2 cells is diminished after exposure to cobalt chloride (50 or 100 microM) for 18-36 h. The redox state of cytochrome c and cytochrome aa3, however, remains stable, indicating a particular affinity of cytochrome b for cobalt. Erythropoietin production of HepG2 cells increases after application of cobalt chloride, whereas H2O2 production, as measured by the dihydrorhodamine technique, decreases. It is concluded that cobalt stimulates a signal cascade with cytochrome b as receptor and H2O2 as second messenger for regulating erythropoietin production.

Cells, Cultured↗
PubMed1994

Effects of nIFN beta and rIFN gamma on growth and morphology of two human melanoma cell lines: comparison between two- and three-dimensional culture.

We compared the anti-proliferative effects of natural interferon beta (nIFN beta) and recombinant interferon gamma (rIFN gamma) on 2 human melanoma cell lines, IGRI and SK-Mel28, grown in 2-dimensional monolayer and in 3-dimensional spheroid culture. In monolayer culture, growth of both lines was inhibited in a dose-dependent manner by 5-day treatments with IFN in concentrations ranging between 1 and 5,000 IU/ml. Incubations with 120 IU/ml nIFN beta or 25 IU/ml rIFN gamma led to a 50% growth inhibition of IGRI cells. A 50% growth inhibition of SK-Me128 cells was obtained with 60 IU/ml nIFN beta, whereas even 5,000 IU/ml rIFN gamma inhibited the growth of this line by only 30%. Growing these melanoma cell lines in 3-dimensional spheroid culture for 5 days reduced their sensitivity to interferon. Growth inhibition values of 50% were achieved with 3,000 IU/ml rIFN gamma or 9,000 IU/ml nIFN beta for IGRI spheroids and 10,000 IU/ml nIFN beta for SK-Me128 spheroids, while 10,000 IU/ml rIFN gamma reduced the growth of SK-Me128 spheroids by only 25%. Outgrowth tests showed that the proliferative capacity after 5-day incubations with IFN was only reduced in IGRI spheroids treated with high doses of nIFN beta. The macroscopically observed increased density of interferon-treated spheroids could be confirmed by light microscopy as corresponding to reduced intercellular space in these spheroids. Scanning electron microscopy furthermore showed variations on the surface of IFN-treated spheroids as well as in cellular organization and structures between cells, hinting at a possible involvement of extracellular matrix substances in the reaction to interferons.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell Division↗