Abstract: OBJECTIVES: Both heat shock (HS) and ionizing radiation have an impact on the cell cycle and may induce cell cycle arrest or apoptosis. Mutations of the p53 gene are observed at a high frequency in human tumours and are recognized in about half of all human cancers. Sensitivity to radiation, heat and anticancer agents has been observed in p53(+/+) cells, but not in mutated or p53-deficient cells. Moreover, enhancement of radiosensitivity by HS has been observed in wild-type p53 cells but not in p53-deficient cells. The molecular mechanism of the differential cell response to HS or ionizing radiation is not yet understood. MATERIALS AND METHODS: Differences in cellular response to radiation (200 kV X-ray, 1, 2, 5 Gy) and HS (39 degrees C, 41 degrees C and 43 degrees C for 30 min) on cell cycle progression of cultures of human p53 mutant cells were investigated by flow cytometry. In addition, the effects of stressors used on the expression of several heat shock genes (HSP27, HSP60, HSP70, HSC70, HSP75, HSP78, HSP90) were studied by reverse transcriptase-polymerase chain reaction. RESULTS AND CONCLUSIONS: Yet, with respect to HSP gene expression, different stressors produced similar effects. Combination of HS and radiation treatment significantly induced the transcription of the HSP70 gene above the level induced by each stressor alone. Cell cycle analysis, however, revealed striking differences in prolonged dynamics of cell division in response to each stressor. Thus, p53 status could be a useful indicator in predictive assays for hyperthermia cancer treatment in combination with radiation and/or chemotherapy.
Abstract: Bone marrow (BM) derived mesenchymal stem cells (MSC) are pluripotent cells which can differentiate into osteogenic, adipogenic and other lineages. In spite of the broad interest, the information about the changes in BM cell composition, in particularly about the variation of MSC number and their properties in relation to the age of the donor is still controversial. The aim of this study was to investigate the age associated changes in variations of BM cell composition, phenotype and differentiation capacities of MSC using a rat model. Cell populations were characterized by flow cytometry using light scattering parameters, DNA content and a set of monoclonal antibodies. Single cell analysis was performed by conventional fluorescent microscopy. In vitro culture of MSC was established and their phenotype and capability for in vitro differentiation into osteogenic and adipogenic cells was shown. Age related changes in tibiae and femurs, amount of BM tissue, BM cell composition, proportions of separated MSC and yield of MSC in 2 weeks of in vitro culture were found. At the same time, neither change in phenotype no in differentiation capacities of MSC was registered. Age-related changes of the number of MSC should be taken into account whenever MSC are intended to be used for investigations.
Abstract: Mesenchymal stem cells (MSCs) are pluripotent cells that can differentiate into endothelial, osteogenic, adipogenic, and other lineages. In spite of the broad interest, little is known about the variation of MSC number in relation to the age of the donor. The aim of this study was to investigate the age-associated variations of bone marrow (BM) MSCs using a rat model. Cell populations were characterized by flow cytometry using light-scattering parameters, DNA content and a set of monoclonal antibodies and detected by magnetic resonance imaging (MRI). Single-cell analysis was performed by conventional fluorescent microscopy. BM mononucleated cells (MNCs) were isolated, in vitro culture of MSCs was established, and endothelial cells differentiation and intracellular magnetic labeling was shown. The amount of BM tissue obtainable from femurs and tibiae increased with age and reached a maximum in 8- to 12-week-old rats. At the same time, the proportional number of MNCs containing MSCs decreased. As a result, after 2 weeks of culture, the maximum yield of MSC number was registered from the youngest age group (4 weeks). MSCs were differentiated into endothelial cells by administration of vascular endothelial growth factor (VEGF) and subsequently revealed immunocytochemical and morphological characteristics of endothelial cells. The results of our study are the basis for further experiments with MSCs and their endothelial descendants, which may be labeled with different agents for cell tracking and detection experiments, but age-related changes in MSCs number should be taken into account whenever these cells are considered for practical applications.
Abstract: Cell type-specific lectin binding is a useful tool for the analysis of developing systems. We describe the binding pattern of 21 different fluorescein isothiocyanate (FITC)-labelled lectins to the testis of two model teleost species, the medaka (Oryzias latipes) and the tilapia (Oreochromis niloticus). The analysis of the binding pattern was carried out on tissue sections (medaka and tilapia) and using primary culture cells (only tilapia). Lectin binding was studied by confocal microscopy and for histological analysis some sections were, in addition, stained with bodipy to gain additional information concerning the cytological organization of the cystic mode of spermatogenesis in fish. The observed differences in lectin staining of different cell types in primary cultures were quantified by flow cytometry. Only few lectins bound specifically to haploid cells while the reaction to diploid or tetraploid cells was generally stronger. However, the extracellular material around the haploid spermatids and spermatozoa in spermatocysts showed a strong staining reaction with several lectins (e.g., Phaseolus vulgaris Erythro agglutinin). The apparent differences in the cellular lectin-binding pattern can be used to identify particular cell types, to monitor their differentiation in vitro or to enrich particular cell types from heterogeneous cultures using magnetic beads coated with anti-FITC antibodies. Using the latter approach, we show that it is possible to enrich for gonial cells and at the same time deplete the preparation for haploid cells and Sertoli cells.
Abstract: The combiened effects of different dose rates (0.625 microGy/s - 1.1 mGy/s) of gamma-irradiation and of cuprum and of cadmium ions on the haematopoietic system of rats were studied. It was found that only low dose rates (0.625-10 microGy/s, summary doses 0.5-2.0 Gy) of gamma-irradiation yields in the increasing proliferative activity of bone marrow. The number of myelocariocytos in S-phase was increased at 1.5-1.8 times. In case of the treatment with both cadmium chloride and radiation the changes in proliferative activity of bone marrow are completely due to the radiation factor. Combination of cuprum acetate and ionizing radiation induce opposite effects providing formal normalization of the haematopoietic characteristic of bone marrow up to 3, 6 and 12 months after the end of the radiation and the chemical exposure of the animal.
Abstract: The metabolism of the flavonol quercetin in human leukaemia (HL-60) cells was investigated. The fluorescence that is elicited by quercetin upon binding to a target protein was quickly attenuated in vital cells, while apoptotic cells showed persistent fluorescence. The dynamics of induction and loss of fluorescence in the cells were quantified by flow cytometry. Several potential metabolites of quercetin, apart from isorhamnetin, had weak or no fluorogenic properties with test proteins. HPLC analysis showed that quercetin was metabolised to several substances, among them glycosylated metabolites. The loss of fluorescence in vital cells offers the unique opportunity to directly observe the metabolic conversion of quercetin in human cells.
Abstract: The biological effect of flavonoids is commonly studied by assaying the activity of a protein of interest. Taking a reverse approach, we identified target proteins of the widely studied flavonol quercetin by exploiting the altered spectroscopic properties of target proteins and ligands on their molecular interaction. Nuclear extracts of human leukemia cells were fractionated by column chromatography and assayed for their ability to alter the fluorescence emission spectra, and finally the proteins present in fractions of interest were identified by mass spectrometry. Among the identified proteins, actin was shown to be a quercetin-binding nuclear protein.
Abstract: Spermatogenesis in vertebrates is controlled by endocrine and paracrine factors and involves the communication between somatic and germ line cells. To elucidate some of the relevant factors in the complicated molecular control processes, we established an in vitro test system using primary cultures of tilapia (Oreochromis niloticus) testis cells. The cultures were enriched for germ line cells and Sertoli cells and largely depleted of spermatozoa. By staining the cells with propidium iodide and carboxyfluorescein succinimidyl ester (CFSE), different cell populations could be identified cytologically and, in addition, quantified by flow cytometry. Cells that had gone through one or more divisions could be identified unequivocally based on their CFSE staining intensity. In parallel cultures maintained for up to 16 days in the presence of 11-ketotestosterone (KT), insulin-like growth factor I (IGF), and/or human chorionic gonadotropin (hCG) the initiation of meiotic and mitotic divisions was monitored. Although KT was important for the initiation of meiosis, spermatogonial mitotic divisions between 10 days and 16 days of culture were promoted by IGF and/or hCG in the presence of KT. These results illustrate the potential of the established in vitro test system for the analysis of the molecular control mechanisms of spermatogenesis.
Abstract: The estrogenic flavanone rac-8-prenylnaringenin (8-PN) and 3 derivatives (rac-7-(O-prenyl)naringenin-4'-acetate (7-O-PN), rac-5-(O-prenyl)naringenin-4',7-diacetate (5-O-PN), and rac-6-(1,1-dimethylallyl)naringenin (6-DMAN) were prepared by chemical synthesis and analyzed with respect to their toxicity and possible cell cycle effects in human acute myeloid leukemia (HL-60) cells. With the exception of 5-O-PN, all the other naringenins showed only weak toxic effects at concentrations below 50 micromol/l. A cell cycle analysis over several cell generations up to 4 days was carried out using the fluorescent dye carboxyfluorescein diacetate N-succinimidyl ester (CFSE) followed by propidium iodide (PI) staining at the end of the experiment. The well-studied flavonol quercetin was included in the analysis as a reference substance. All flavonoids affected cell proliferation, but the extent and the resulting changes in the proliferation pattern were specific for each substance. In contrast to the radical scavenging activity of quercetin, the tested flavanones showed no anti-oxidative properties using several different test systems. Similarly, the mitochondrial membrane potential (DeltaPsim) was hardly effected by these compounds, while both menadione and quercetin strongly reduced the potential after 1 h of treatment. The reported chemical modification of interesting lead substances (like the strongly estrogenic 8-PN) presents a promising approach to modulate the properties of a relevant substance in a pharmacologically desirable way. The low toxicity and weak cytostatic properties of the tested naringenin derivatives is encouraging for further studies on known naringenin target molecules.
Abstract: The biological effects of epicuticular substances in farinose exudates accumulated on inflorescence shafts and calyces of Primula denticulata on human acute myeloid leukemia cells (HL-60) were analyzed. The crude material possessed little antioxidative capacity but strong cytostatic properties. Some of its known components (5-hydroxyflavone, 2'-hydroxyflavone, 5,2'-dihydroxyflavone, and 5,8-dihydroxyflavone) were further tested to identify the biologically active compounds. The effects of these flavones on cell cycle progression, mitochondrial membrane potential, and reactive oxygen species have been investigated by flow cytometry. The flavonol quercetin was included in the study as reference compound because of its known cytostatic properties and its activity as radical scavenger. Compared to quercetin the flavones induced little apoptosis (up to 40 microM), but despite their low toxicity, the Primula flavonoids possessed strong cytostatic properties even at low concentrations. The cell cycle distribution showed a characteristic time-dependent shift, giving evidence of a generally short-lived effect of the test compounds in the exposed cells. The antioxidative properties quantified according to two different methods correlated with the number of hydroxyl groups. Whereas quercetin strongly affected the mitochondrial membrane potential, none of the Primula flavones showed a comparable effect.
Abstract: Cell cultures of lavender (Lavandula officinalis) were analyzed for the metabolite profile under normal growth conditions and under stress as well as after jasmonic acid treatment. The main compound synthesized was rosmarinic acid, which was also secreted into the culture medium. Different solvent extraction methods at different pH values altered the profile slightly. Anoxic stress induced the synthesis of a cinnamic acid derivative, which was identified as caffeic acid by gas chromatography-mass spectrometry. Caffeic acid was also induced after treatment of the cell cultures with jasmonic acid. Although the antioxidative activity of both compounds, rosmarinic acid and caffeic acid, was confirmed in an assay using 2,2-diphenyl-1-picrylhydrazyl (DPPH), it was demonstrated that both substances have a low cytotoxic potential in vitro using acute myeloid leukemia (HL-60) cells. The potential of the system for finding new bioactive compounds is discussed.
Abstract: The aim of this study was to demonstrate the expression of heat shock (HS) genes in human cells in response to extremely low-frequency electromagnetic fields (ELF-EMF) alone and in combination with thermal stress. After exposing human myeloid leukemia (HL-60) cells to the stressor(s) for 30 min we quantified the expression of the HS genes HSP27, HSP60, HSP70 (A, B, and C), HSC70, HSP75, HSP78, and HSP90 (alpha and beta) by RT-PCR. The results clearly show that HS genes, in particular the three HSP70 genes (A, B, and C), are induced by ELF-EMF, a reaction that is enhanced by simultaneous HS (43 degrees C for 30 min). The results show similarities and some significant differences to previous experiments in which transgenic nematodes were used to monitor the induction of the HSP70 gene under similar stress conditions. We also studied the effect of different flux densities on gene expression in the range of 10-140 microT. Even the lowest dose tested (10 microT) resulted in a significant induction of the genes HSP70A, HSP70B, and HSP70C. The reaction to ELF-EMF shows a maximum at a flux density of 60-80 microT. The unusual dose-response relation reveals an interesting difference to other stressors that elicit the HS response.
Abstract: Stress-induced effects in human acute leukaemia cells (HL-60) were studied by flow cytometry using the fluorescent dye carboxyfluorescein succinimidyl ester which allows the analysis of several successive cell generations for up to 10 days. Asynchronously cycling cells subjected to heat shock (30 min at 41 degrees C) responded in two distinct ways: while one fraction of the cell population (about 15%) re-entered the cell cycle after a short delay, other cells became arrested at different phases of the cell cycle and remained arrested for up to several days and finally underwent apoptosis. Weak electromagnetic fields (60 micro T, 50 Hz) alleviated the heat-induced block and the fraction of arrested cells was significantly smaller.
Abstract: The amino-reactive derivative of tetraphenylporphine meso-tetrakis[4-(carboxy)phenyl]porphine (TCPP) was synthesized, which is characterized by a high molar absorption coefficient (epsilon 416 = 36,500 M-1.cm-1). TCPP was covalently attached to oligonucleotides d(CG)5 [d(CG)5-TCPP] and d(TA)5 [d(TA)5-TCPP]. The spectral characteristics of these complexes were studied in 0.01 M phosphate buffer, pH 7 at 23 degrees C. UV-visible absorption spectra of these complexes have a clearly pronounced Soret band at (414 +/- 1) nm for d(CG)5-TCPP and at (412 +/- 1) nm for d(TA)5-TCPP. The fluorescence spectra of these complexes have maxima at (648 +/- 2) nm for d(CG)5-TCPP and at (658 +/- 2) nm for d(TA)5-TCPP. In this study we also determined fluorescence quantum yields q and fluorescence lifetimes tau [q = 0.099 +/- 0.011, tau = (9.0 +/- 0.3) ns for d(CG)5-TCPP and q = 0.080 +/- 0.011, tau = (8.7 +/- 0.3) ns for d(TA)5-TCPP]. A temperature rise from 5 to 50 degrees C produced only slight (within 23%) emission changes in both samples studied. Taking into account: a) high fluorescence yields (q), b) weak dependence of q on temperature, c) weak q dependence of q on the oligonucleotide type, we conclude that TCPP may be used as a sensitive fluorescence label in DNA studies.
Abstract: Excessive amounts of heavy metals (e.g. Zn, Cu, Mn, Cr) are accumulated in river bottom sediments (RBS), being available to humans and animals along food chains. Increased exposure of mammals to certain metals (Cr, Cu) induces immunosuppresion, due to DNA damage and decreased survival of lymphoid cells. By contrast, excess of Zn and Cd causes inhibition of apoptosis thus suggesting increased survival of genetically mutated cells and higher cancer risks in exposed populations. Rat thymic lymphocytes represent a well-established model for apoptosis testing. The primary goal of our study was to assess the degree of apoptosis modulation with a number of RBS extracts differing in their metal contents. A series of freshly deposited RBS was collected at nine sampling stations along the Elbe River. All sediments were rich in Fe, Mn and Zn. The contents of Cu, Cr, Ni, Cd, Hg, Pb and As were much lower and interrelated. The short-term cytotoxicity of aqueous sediment extracts was assessed, using the following criteria: total cell counts; incidence of apoptosis and necrosis (morphological detection by fluorescent microscopy); and nuclear chromatin decay (by DNA flow cytometry). RBS extracts produced both apoptosis and necrosis of thymocytes. High contents of zinc and other heavy metals in the samples correlated with decreased thymocyte apoptosis (r= -0.543 to -0.608, P <0.01). The rates of thymocyte damage showed a distinct dependence on the time and region of sampling. Apoptosis modulation was also tested with pure salts of Mn(II), Zn(II), Cu(II), Cr(III) and Cd(II), at the test concentrations of 1, 10 and 100 microM. Cu(II) and Cr(III) proved to induce marked dose-related apoptosis, whereas Zn(II) ions caused significant suppression of apoptosis. These effects were similar to those trends observed with metal-rich sediments. In the present study. DNA flow cytometry proved to be a less sensitive index of cell death than morphological assay of apoptosis and/or necrosis. In summary, inhibition of lymphocyte apoptosis by RBS extracts and pure metals is associated with excess of zinc and, probably, cadmium. The proposed model of lymphoid cell apoptosis is a promising tool for screening cytotoxic effects of complex environmental samples.
Abstract: The sorption of cerebrospinal fluid (CSF) was attempted as a special detoxifying procedure in a group of sixty heroine addicts. CSF contents of cells, total protein, nucleic acids and interleukin-1 (IL-1), as well as acridine orange (AO) binding to CSF cells were determined before and after the procedure. The treatment provided immediate clinical improvement for 70% of the patients. Clinical effect was accompanied by decreased of CSF cells, diminished nucleic acids and protein amounts, along with increased DNA-AO binding and accumulation of IL-l. These data are interpreted in terms of intensive apoptosis of CSF cells and increased acute phase of aseptic inflammation-like events induced by the procedure of liquor sorption.
Abstract: Short-term incubation of rat thymocytes, bone marrow cells, and macrophages with aqueous extracts of soil demonstrated positive correlations between damage to the cells and increased levels of copper, chromium, and manganese in the soil, while increased levels of zinc and lanthanum were associated with less pronounced changes in the cells.
Abstract: Rats were exposed to gamma-radiation within dose range from 0.12 to 4.0 Gy at dose rates from 0.625 microGy/s to 1.1 mGy/s. The changes in myelokaryocyte distribution within the cell cycle phases during the period of 1 to 18 months after irradiation were studied by the method of flow cytometry. A dose-dependent increase of cell percentage in S-phase after irradiation at low dose rates (from 0.625 to 10.0 microGy/s) was found. The effect was kept till the end of irradiated rats life.
Abstract: The method of flow cytofluorimetry was used to study some population characteristics of bone marrow cells of control and irradiated rats. The simultaneous recording of cellularity and distribution of myelokaryocytes among the cell cycle phases was shown to give reliable results for determining the extent to which the organism was exposed at early times following irradiation.
Abstract: Flow cytofluorimetry and statmokinetic method were used to study the circadian rhythm of bone marrow proliferation in Pliss' lymphosarcoma-bearing and intact rats. These data were compared to those obtained in the study of the mitotic activity of the bone marrow in cancer patients. It was found that, already at early stage, tumor affected the circadian rhythm of bone marrow proliferation, reducing the amplitude of oscillations. A model simulating formation of the circadian rhythm of the bone marrow was suggested basing on the possibility to arrest cells at the end of G1 phase. The rate of transition of G1 cells to S phase was determined not only by endogenous "set-points" of the rhythm which formed the basic wave of proliferation but also by conditions of animal upkeep.
