Biomedical subjects

G Bruchelt

Publications and source records attributed to G Bruchelt.

At least 37 records · Page 2Linked to original sources

PubMed1997

Effect of concentration on the cytotoxic mechanism of doxorubicin--apoptosis and oxidative DNA damage.

Anthracycline derivatives such as doxorubicin are part of many chemotherapeutic regimens and reach peak plasma concentrations of 5 microM. We investigated the cytotoxic mechanisms of various doxorubicin concentrations in MOLT-4 ALL-cells. Concentrations of up to 100 microM doxorubicin achieved similar cytotoxic effects in cultures of MOLT-4 cells, but acted via different mechanisms. Doxorubicin induced apoptosis (maximum effect at 1 microM), which was dependent on RNA synthesis and involved oxidative stress. Concentrations higher than 3 microM did not induce apoptosis, but significantly inhibited RNA synthesis. DNA strand breaks in MOLT-4 cells occurred in the presence of 1 to 5 microM doxorubicin to a similar extent, but showed a dose-dependence at higher concentrations. There was no GC/MS-detectable oxidation of DNA bases in apoptotic cells and only 1 out of 13 DNA base oxidation products, 8-hydroxyguanine, increased significantly in the presence of as much as 100 microM doxorubicin. These results suggest that at pharmacologically relevant concentrations apoptosis and not oxidative DNA damage is the main killing mechanism of doxorubicin against ALL-cells.

Antibiotics, Antineoplastic↗

PubMed1996

Control of neuroblastoma cell proliferation and differentiation by human bone marrow.

BACKGROUND: Neuroblastoma (NB) is one of the few tumors known to undergo spontaneous regression. Its progression, however, often leads to bone marrow (BM) metastasis. Proliferation and differentiation of human NB cells may be regulated in vitro by a variety of biologic agents, some of which are released by low-density BM and peripheral blood (PB) cells. Little is known regarding BM cell-derived control of NB cell growth and differentiation. METHODS: The proliferative and differentiative responses of NB cells, to BM cell-, and to PB cell-derived conditioned medium (CM) were evaluated in comparison to cytokine-induced responses. RESULTS: CM from unstimulated cultures of low density BM and PB cells, from healthy donors, from newborn infants, and from NB patients, significantly and reproducibly stimulate NB cell growth in vitro. The intensity of CM-induced stimulation was not attained by recombinant human tumor necrosis factor (rhTNF), interferon (rhIFN), or granulocyte-monocyte colony stimulating factor (rhGM-CSF); and although epidermal growth factor (rhEGF) and transforming growth factor alpha (rhTGF alpha) were strongly stimulatory, neutralizing antibodies against each of these agents did not affect CM-derived activity. In contrast to growth stimulation, differentiation of CM-treated NB cells, was reproducibly suppressed, as reflected in abrogation of neuronal cell morphology as well as of neurofilament and neuron specific enolase expression. CONCLUSIONS: Spontaneous regression of NB tumors, on one hand and BM metastasis on the other may be associated with the extent and nature of the NB cell response to regulatory activity released by BM and PB cells.

Bone Marrow↗

PubMed1996

Mobilization of iron from cellular ferritin by ascorbic acid in neuroblastoma SK-N-SH cells: an EPR study.

The mobilization of iron from intracellular ferritin by ascorbic acid has been analysed in situ by electron paramagnetic resonance (EPR) spectroscopy. EPR enables a distinction between ferritins and other Fe(3+)-binding cellular components. The ordered iron core of ferritin gives rise to a resonance signal which can be observed only at temperatures above 50 K. In the present study we clearly demonstrate that ascorbic acid is capable of mobilizing iron from ferritin in the cellular system by reduction of the ferric ion core in neuroblastoma SK-N-SH cells. This mechanism may open new ways in the therapy of this hardly curable tumor in stage IV, especially in combination with some cytostatic drugs.

Ascorbic Acid↗

PubMed1996

Phagocytic activity and oxidative burst of granulocytes in persons with myeloperoxidase deficiency.

In the present study, phagocytosis and the oxidative metabolism of neutrophil granulocytes from five clinically healthy persons with different degrees of myeloperoxidase deficiency were investigated and compared to those of normal persons. The identification of individuals with myeloperoxidase deficiency was performed with the Bayer/Technicon H3 blood cell counter, which differentiates the leukocytes by measuring the peroxidase activity. Neutrophils of three out of five investigated myeloperoxidase deficient persons showed extremely low peroxidase indices (-53 and lower), but only the neutrophils of one person totally lacked myeloperoxidase. This was demonstrated by comparing myeloperoxidase mass concentration measured with an enzyme immunoassay, lack of HOCl production, and was further confirmed by measuring luminol- and lucigenin-enhanced chemiluminescence. Characteristically, myeloperoxidase deficient granulocytes showed a strikingly decreased luminol-enhanced chemiluminescence while the lucigenin-enhanced chemiluminescence was significantly increased compared to normal granulocytes. Although there is a DNA sequence homology of about 70%, the activity of peroxidase in eosinophils was not affected in any myeloperoxidase deficient person investigated. Moreover, a person with a very rare defect of eosinophil peroxidase had completely normal myeloperoxidase activity. The lack of myeloperoxidase activity is compensated for by an increased phagocytic activity, an increased production of superoxide anion (lucigenin-chemiluminescence) and probably by an alternative metabolism of H2O2; since persons lacking myeloperoxidase activity do not normally suffer from severe infections, H2O2 is obviously metabolized to other reactive oxygen substrates than HOCl, e.g. to OH-radicals.

Adult↗

PubMed1995

Presence of iron catalytic for free radical reactions in patients undergoing chemotherapy: implications for therapeutic management.

We investigated the kinetics of generation of iron 'catalytic' for free radical reactions in children with diagnosed acute lymphoblastic leukaemia (ALL) who received high-dose methotrexate infusions. In 76% of the chemotherapy courses studied, 'catalytic' iron appeared in plasma in the concentration range from 0.1 to 3 mumol/l. Positive correlations between maximum levels of 'catalytic' iron and plasma hepatic enzymes could be established in the majority of cases and in one subset of patients (low and medium risk ALL) mean 'catalytic' iron levels correlated well to clinically observable toxicities. The damaging potential of 'catalytic' iron was also demonstrated experimentally: oxidative damage to proteins was significantly (P < 0.05) higher in plasma samples showing the presence of 'catalytic' iron and in addition a strong correlation (r = 0.95, P < 0.02) was seen between plasma concentration of 'catalytic' iron and the ability of the plasma to stimulate lipid peroxidation. Our data show that chemotherapy releases 'catalytic' iron which may relate to toxic side effects. Hence binding this 'catalytic' iron by judicious co-administration of iron chelating agents could be beneficial in minimizing the iatrogenic adverse effects of chemotherapy of acute leukaemia.

Adolescent↗

PubMed1995

Cytotoxicity of ether phospholipid BM 41.440 on neuroblastoma cells.

The thioether lysophospholipid BM 41.440 proved to be toxic against cells of two neuroblastoma cell lines in a dose- and time-dependent manner. The ID50 estimated in three different in vitro test systems declined from about 10 micrograms/ml after 24 h to 1 microgram/ml after a 1-week treatment of the neuroblastoma cells. These values are comparable to the ID50 found for neoplastic cells derived from other tissues. In comparison, hematopoietic progenitor cells (granulocyte/monocyte-colony-forming units) proved to be less sensitive to short-term treatment with BM 41.440. After long exposure to this drug the selectivity towards neuroblastoma cells decreased. This observation makes it unlikely that BM 41.440 can be used for treatment of neoplasia such as neuroblastoma, because only short-term treatment is acceptable considering the high bone marrow toxicity.

Antineoplastic Agents↗

PubMed1995

Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of monoamine transporters in neuroblastoma cell lines: correlations to meta-iodobenzylguanidine (MIBG) uptake and tyrosine hydroxylase gene expression.

Radiolabelled meta-iodobenzylguanidine (MIBG) has been widely used in scintigraphy and targeted radiotherapy in patients with neuroblastoma. Recently, it has been demonstrated that MIBG is incorporated into neuroblastoma cells by the noradrenaline transporter. In vitro experiments on SK-N-SH human neuroblastoma cells performed in the present study showed that uptake of MIBG is inhibited by noradrenaline, more so by dopamine and to a lesser extent, by serotonin, indicating that the respective transporters may also contribute to MIBG uptake. However, neither dopamine nor serotonin transporter gene expression was detected. Noradrenaline transporter gene expression was found in 4 of 6 investigated cell lines, which correlated with specific MIBG uptake. Furthermore, an inverse correlation of noradrenaline transporter and tyrosine hydroxylase gene expression, the key regulatory enzyme of catecholamine synthesis, was observed. These data show that MIBG is specifically incorporated only in neuroblastoma cells in which there is noradrenaline transporter gene expression. Furthermore, the catecholamine status in neuroblastoma cells is regulated by a coordinate expression of the key elements of catecholamine synthesis and reuptake systems.

3-Iodobenzylguanidine↗

PubMed1995

[Clinical significance of reactive oxygen species].

There is increasing evidence that reactive oxygen substances are involved in the pathogenesis and/or progression of differ diseases. A short introduction to the biochemistry of reactive oxygen substances will be given in this review. Subsequently, the role of reactive oxygen substances will be discussed exemplarily on pathophysiological aspects of neutrophil granulocytes and of neurodegenerative diseases.

Alzheimer Disease↗

PubMed1994

Determination of 2-5A synthetase and 2-5A phosphodiesterase in neuroblastoma cells by analytical capillary isotachophoresis: effects of cytokines and comparison with radioenzymatic methods.

2-5A Synthetase and 2-5A phosphodiesterase were determined by analytical capillary isotachophoresis in comparison to radioenzymatic methods. By means of isotachophoretic analysis, a frequently used radioenzymatic 2-5A synthetase assay was optimized and the results of both assays were compared. Using the isotachophoretic assay the influence of interferon-related cytokines (tumor necrosis factor-alpha and interleukin-2) on 2-5A synthetase induction in neuroblastoma cells was estimated. In contrast to mononuclear blood cells, the tumor necrosis factor induced 2-5A synthetase in these cells. 2-5A Phosphodiesterase was determined using an isotachophoretic assay and a radioenzymatic method. Degradation of A2'p5'A2'p5'A (trimeric form of 2-5A core) was measured by isotachophoresis whereas degradation of a mixture of phosphorus-32 labeled 2-5A cores was registered by radioenzymatic assay. Activity of 2-5A phosphodiesterase was only insignificantly enhanced by interferon in mononuclear blood and neuroblastoma cells. In contrast to the radioenzymatic assays, an accurate determination of 2-5A synthetase as well as of 2-5A phosphodiesterase is possible using the isotachophoretic method because the reactions are followed by measuring the substrates ATP and A2'p5'A2'p5'A, respectively.

2',5'-Oligoadenylate Synthetase↗

PubMed1994

Ascorbic-acid-mediated iron release from cellular ferritin and its relation to the formation of DNA strand breaks in neuroblastoma cells.

Ascorbic acid at pharmacologically attainable concentrations effectively inhibited the growth of the catecholamine-positive neuroblastoma cell line SK-N-SH; it inhibited LS cells to a smaller extent and catecholamine-negative SK-N-LO cell growth least effectively. In all three cell lines high concentrations of H2O2 were found. Since ascorbic acid was shown to release iron from ferritin in vitro and to keep it in the reduced state, we suggested that it acted as a pro-oxidant in ferritin-rich neuroblastoma cells in the presence of H2O2 and Fe2+ (Fenton reaction), implying iron release from cellular ferritin. We show here that iron could be mobilized from cellular ferritin by 1 mM ascorbic acid in iron-59-preloaded SK-N-SH and LS cells, but not in SK-N-LO cells. In agreement with these results, DNA strand break formation by ascorbate was only observed in SK-N-SH and LS cells. In SK-N-LO cells, DNA strand breaks could be induced by a combination of 1 mM ascorbic acid and 100 microM H2O2. Since cell-damaging effects caused by chemotherapy further facilitate iron release from ferritin, we conclude that ascorbate could be a powerful enhancer of some cytostatic drugs in neuroblastoma therapy.

Ascorbic Acid↗

PubMed1994

Chemotactic activity of substances derived from antibody-loaded tumor cells on granulocytes.

Chemotactic activity of granulocytes attracted by tumor cells loaded either with anti-ganglioside monoclonal antibodies (mAb) or with antibody-glucose oxidase conjugates (mAb-GO) was investigated. The melanoma cell line SK-Mel-28 which expresses the ganglioside GD3 at high density as well as the neuroectodermal cell line SK-N-LO which expresses GD2 were used for the experiments. In the presence of 50% human AB-serum, antibody-loaded tumor cells induced chemotactic activity on granulocytes, probably due to the generation of C3a/C5a which could be detected in serum incubated with anti-GD3 loaded SK-Mel-28 cells. Both compounds could also be detected in vivo in the plasma of patients suffering from neuroblastoma during therapy with anti-GD2 antibodies. In another set of experiments mAb-GO conjugates generating high amounts of H2O2 in the presence of glucose were bound to these tumor cells. A significant lipid peroxidation could be observed in the simultaneous presence of iron and ascorbate. The lipid peroxidation products were measured as thiobarbituric acid-reactive substances (TBARS) and were also shown to induce chemotactic effects on granulocytes.

ABO Blood-Group System↗

PubMed1994

Activation of cellular cytotoxicity and complement-mediated lysis of melanoma and neuroblastoma cells in vitro by murine antiganglioside antibodies MB 3.6 and 14.G2a.

Mouse monoclonal antibodies against tumour-associated gangliosides GD2 (14.G2a) and GD3 (MB 3.6) were tested to mediate antibody-dependent cellular cytotoxicity (ADCC) with various effector cells or complement-dependent cytolysis (CDC). We also evaluated the immunomodulating potential of interferons in combination with cellular cytotoxicity. Using effector:target (E/T) ratios of 40:1, ADCC with effector cells such as granulocytes or mononuclear blood cells was not detectable against melanoma cell lines GR, SK-MEL-28 and G-361 which preferentially express GD3 and bind antibody MB 3.6. Neuroblastoma cell line SK-N-LO, which was used for comparative purposes, mainly expressed GD2 and the tumour cells were killed effectively after labelling with antibody 14.G2a. Granulocytes did not show significant killing of melanoma cells by ADCC, but neuroblastoma cells were killed very efficiently. Peripheral blood mononuclear cells (PBMC) also failed to kill melanoma cells. Interferon-beta slightly stimulated PBMC and increased killing of neuroblastoma cells, but no additive effects with ADCC were detectable. Incubation of target cells with interferons produced no significant differences in susceptibility of the target cells to interferon-activated PBMC cytotoxicity. Despite the lack of effectiveness in mediating cellular cytotoxicity, GD3 antibody MB 3.6 showed strong complement-dependent cytolysis in the presence of human plasma. There were remarkable differences in individual activity and different susceptibility of the melanoma cell lines. We assume that CDC may have more activity against melanoma cells than cytotoxicity associated with various effector cells.

Animals↗

PubMed1993

Isotachophoresis of nucleic acid constituents.

Determination methods for purine and pyrimidine bases, nucleosides, nucleotides and related compounds using analytical capillary isotachophoresis are reviewed. First, the isotachophoretic characterization of these compounds, as well as methods for sample preparation prior to analysis, and the different ways of detecting unknown substances in complex biological systems are described. Then applications of isotachophoretic analysis in medical diagnosis and biomedical research are reviewed. In particular, the analysis of purines, pyrimidines, nucleosides and related compounds in blood and serum for the diagnosis and treatment of inherited diseases and cancer is described. Selected applications of nucleotide analysis in biomedical research using different tissue extracts are also reviewed, and some examples of nucleotide-dependent enzymic reactions, which were performed by means of analytical isotachophoresis, are presented.

Animals↗

PubMed1993

Determination of ascorbic acid by isotachophoresis with regard to its potential in neuroblastoma therapy.

Analytical capillary isotachophoresis was used to determine ascorbic acid (AA) in different matrices (cell-free system, neuroblastoma cell extracts and urine). The system for purging bone marrow of neuroblastoma cells, including 6-hydroxydopamine (6-OHDA) and AA, was analysed with regard to the interaction of AA with 6-OHDA and its autoxidation product, hydrogen peroxide. Furthermore, analyses concerning the uptake of AA into neuroblastoma cells as well as its excretion in urine after uptake of large amounts were carried out.

Ascorbic Acid↗

PubMed1992

A simple assay for determination of iron release from ferritin in neuroblastoma cells.

A commercially available enzyme immunoassay was used to determine ferritin content and subsequently the loading and release of iron from ferritin in neuroblastoma cells. LS cells were incubated with 59Fe for 24 h, lysed, and the cytoplasmic ferritin was bound to monoclonal antibodies coupled to globules. After determination of the ferritin content the same globules with bound radioactive ferritin were measured in a gamma-counter. To illustrate the applicability of this test system, increased iron loading of cellular ferritin could be demonstrated in cycloheximide-treated cells; furthermore, release of iron was documented after incubation of LS cells with a combination of 6-hydroxydopamine and ascorbate. The assay turned out to be a simple method for determination of changes in 59Fe content of ferritin in neuroblastoma cells.

Ascorbic Acid↗

PubMed1992

Effect of constituents of burned skin and in vivo skin burning on the respiratory activity of rat liver mitochondria.

The formation of toxic substances in the skin, due to the influence of thermal energy, is supposed to be one possible mechanism responsible for the high mortality rate after severe burns covering large areas of the body surface. The effects of low-molecular-weight volatile substances (dry distillates) generated after burning mouse skin were investigated. They led to reductions of the respiratory activity of rat liver mitochondria. Among other substances carbon disulphide, methylethylketone and pyrrole were isolated and characterized using gas chromatography. All of them had strong uncoupling effects on mitochondrial respiration. In a second study respiratory measurements were made on mitochondria from rats with 20-30 per cent TBSA full skin thickness burns. A decreased respiratory control ratio and thus uncoupling of mitochondrial respiration was found on days 1, 2 and 5 postburn. The basal mitochondrial respiration was significantly decreased on day 5, which could be important for the mortality mentioned above. Finally after cross-transplantations rats having burned skin grafted showed a reduction of the RCR when compared with rats with the unburned graft. Thus it can be concluded that toxic substances must have been present in burned skin.

Animals↗

PubMed1992

Determination of 2'-5'-oligoadenylate synthetase in serum and peripheral blood mononuclear cells before and after subcutaneous application of recombinant interferon beta and gamma.

The interferon-inducible enzyme, 2'-5'-oligoadenylate synthetase, was estimated in healthy donors and in patients before and after subcutaneous application of recombinant interferon beta and gamma. Tests were carried out with lysates of peripheral blood mononuclear cells, using an established radioenzymatic assay, and in serum samples, using a new radioimmunoassay. Both test systems substantially yielded the same results: after a single injection of interferon beta (1-5 x 10(6) IU), 2'-5'-oligoadenylate synthetase increased in a dose-dependent manner reaching maximal catalytic concentrations in most patients after 24-48 hours (leukocytes) and 48-72 hours (serum). In contrast, interferon gamma (2-4 x 10(6) IU) caused only a small induction of 2'-5'-oligoadenylate synthetase. However, daily application of interferon gamma for 7 days led to a distinct time-dependent increase of 2'-5'-oligoadenylate synthetase activity concentration during this observation period. Characteristically, even during daily application, the 2'-5'-oligoadenylate synthetase activity concentration dropped just 48-72 hours after the first injection of interferon beta. The determination of 2'-5'-oligoadenylate synthetase proved to be useful for optimizing and monitoring subcutaneous therapy with interferon. The new radioimmunoassay which allows the determination of this enzyme in serum is superior to other methods used in the past.

2',5'-Oligoadenylate Synthetase↗

PubMed1991

Ascorbic acid enhances the effects of 6-hydroxydopamine and H2O2 on iron-dependent DNA strand breaks and related processes in the neuroblastoma cell line SK-N-SH.

Neuroblastoma cells accumulate ascorbic acid and iron. It was hypothesized that these features could be exploited for sensitizing neuroblastoma cells for therapy in combination with reactive oxygen intermediates. In the present study the effects of 6-hydroxydopamine (6-OHDA) and H2O2 on metabolic parameters critical for cell survival were investigated in cells with low and high ferritin content in the presence and absence of ascorbate. Human neuroblastoma SK-N-SH cells were pretreated with 100 microM FeSO4 and 10 microM desferrioxamine, respectively, for 24 h yielding cells with different ferritin contents. The effects of 6-OHDA and H2O2 (25 microM-250 microM) in the absence and presence of 1 mM ascorbic acid on DNA strand break formation, activation of poly(ADP-ribose) polymerase, and finally decrease in NAD+ and ATP concentration were investigated. All these parameters were influenced by 6-OHDA and H2O2 in a concentration-dependent manner in a similar way. The effects were most pronounced in ferritin-rich cells and in the presence of ascorbic acid. Using isolated CCC PM2 DNA, 6-OHDA and ascorbic acid caused strand breaks that were prevented in the presence of mannitol or desferrithiocine. H2O2-mediated strand breaks were observed only in the presence of ascorbic acid. Based on these data and data published by others a model explaining the deleterious effects of ascorbic acid on neuroblastoma cells is presented. It is suggested that continuous application of a high dosage of ascorbic acid might be a useful approach in neuroblastoma therapy.

Adenosine Triphosphate↗