RESUMO
This study was purposed to investigate the change of biological characteristics of HL-60 cells with high tumorigenicity transplanted through repeated passages into nude mice and to explore the tumorigenic mechanisms of the cell line. The human highly tumorigenic leukemia cell line HL-60 model in nude mice were established by serial passages in vivo and in vitro, and their biological features were compared. The trypan blue staining assay was used to detect the cell growth, the flow cytometry was used to analyze the cell cycle, the transmission electron microscopy and laser scanning confocal microscopy were used to observe the cell ultrastructures and cell fluorescence level respectively. The results indicated that the cell growth velocity was quickened, cell doubling time was shortened in high tumorigenic leukemia cell line HL-60; the cell count in S phase increased; the amount of mitochondria in HL-60 cells obviously decreased, furthermore the dilation of interspace, decrease of the number of ridges, vacuolation of mitochondria, significant reduction of fluorescence level in microfilament and enhancement of cell cloning efficacy were observed. It is concluded that the high tumorigenicity of HL-60 cells multiple-passaged in nude mice is associated with enhancement of proliferative ability, changes of number and structure of mitochondria in HL-60 cells, and alteration of microfilament in cytoskeleton.
Assuntos
Células HL-60/citologia , Células HL-60/ultraestrutura , Transplante de Neoplasias , Citoesqueleto de Actina/ultraestrutura , Animais , Ciclo Celular , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Mitocôndrias/ultraestruturaRESUMO
Reconstituted discoidal high-density lipoprotein (rHDL) has potent vascular protective actions. Native HDL suppresses cellular generation of reactive oxygen species, whereas this antioxidant effect of rHDL is less clear. This study examined the effects of rHDL on NADPH oxidase, a major source of cellular superoxide generation, in both leukocytes and human umbilical vein endothelial cells. Superoxide was measured with lucigenin-enhanced chemiluminescence. Expression of NADPH oxidase sub-units was determined by real-time PCR. Pre-treatment of HL-60 cells with rHDL (10 and 25 microM) for 1 h significantly reduced phorbol 12-myristate 13-acetate-stimulated superoxide production. Treatment with rHDL for up to 24 h did not change the mRNA expression of NADPH oxidase sub-units. In HL-60 cells, depletion of cholesterol from the plasma membrane by methyl-beta-cyclodextrin mimicked the effect of rHDL, whereas cholesterol repletion blunted the effects of rHDL. Treatment with rHDL induced disruption of the lipid raft structures and blunted PMA-induced redistribution of p47phox into lipid rafts. In contrast, treatment of endothelial cells with rHDL for up to 18 h had no effect on either basal or tumour necrosis factor-alpha-stimulated NADPH oxidase activity, but markedly suppressed the cytokine-induced expression of proinflammatory adhesion molecules. The results suggest that rHDL inhibits NADPH oxidase activation in leukocytes, probably by interrupting the assembly of NADPH oxidase sub-units at the lipid rafts. This effect may contribute to the vascular protective actions of rHDL against inflammation-mediated oxidative damage.
Assuntos
Células Endoteliais/efeitos dos fármacos , Leucócitos/efeitos dos fármacos , Lipoproteínas HDL/farmacologia , Microdomínios da Membrana/efeitos dos fármacos , NADPH Oxidases/antagonistas & inibidores , Apolipoproteína A-I/farmacologia , Sistema Livre de Células , Colesterol/metabolismo , Células Endoteliais/metabolismo , Células Endoteliais/ultraestrutura , Ativação Enzimática/efeitos dos fármacos , Células HL-60/efeitos dos fármacos , Células HL-60/metabolismo , Células HL-60/ultraestrutura , Humanos , Leucócitos/metabolismo , Leucócitos/ultraestrutura , Medições Luminescentes , Lipídeos de Membrana/metabolismo , Fagocitose/efeitos dos fármacos , Fosfatidilcolinas/farmacologia , Superóxidos/metabolismo , Acetato de Tetradecanoilforbol/farmacologia , Veias Umbilicais , Xantina/metabolismo , Xantina Oxidase/metabolismo , beta-Ciclodextrinas/farmacologiaRESUMO
We have synthesized novel heterocyclic organobismuth compounds that have potent antibacterial properties. In this study, we examined their anticancer activity and addressed the cellular mechanisms involved. Heterocyclic organobismuth compounds showed anticancer activities in various human cancer cell lines. These compounds have particularly potent anticancer activities against leukemia cell lines. One of them, bi-chlorodibenzo [c,f][1,5] thiabismocine (compound 3), inhibited the growth of the human promyelocytic leukemia cell line HL-60 at a concentration of 0.22 microM. Low concentrations of compound 3 (0.22-0.44 microM) induced apoptosis, whereas at a higher concentration (>1.1 microM) it causes acute necrosis. During the apoptosis, caspase-3, -8, and -9 were activated but caspase-12 was not. A broad caspase inhibitor (z-VAD-fmk), and caspase-3 (z-DEVD-fmk) and caspase-9 (z-LEHD-fmk) inhibitors suppressed the compound 3-induced apoptosis, but a caspase-8 inhibitor (z-IETD-fmk) was less effective, suggesting that the caspase-8 activity only partially participates in the apoptosis. In the apoptotic cells, cytochrome c was released from mitochondria to cytosol and a loss of mitochondrial transmembrane potential (DeltaPsi(m)) was detected. Compound 3-induced apoptosis was associated with enhanced generation of intracellular reactive oxygen species (ROS). Pretreatment of the cells with N-acetyl-L-cysteine or catalase suppressed the apoptosis. On the other hand, buthionine sulfoximine enhanced the compound 3-induced collapse of DeltaPsi(m) and apoptosis. Taken together, these results indicate that compound 3 is a potent inducer of apoptosis, triggering a caspase-3-mediated mechanism via the generation of ROS and release of cytochrome c from mitochondria, suggesting a potential mechanism for the anticancer activity of compound 3.
Assuntos
Apoptose/efeitos dos fármacos , Bismuto/farmacologia , Células HL-60/efeitos dos fármacos , Compostos Heterocíclicos/farmacologia , Compostos Organometálicos/farmacologia , Trifosfato de Adenosina/metabolismo , Anexina A5/metabolismo , Inibidores de Caspase , Caspases/efeitos dos fármacos , Caspases/metabolismo , Sobrevivência Celular/efeitos dos fármacos , DNA de Neoplasias/efeitos dos fármacos , Células HL-60/ultraestrutura , HumanosRESUMO
Three antitumor antibiotics, mitomycin C, bleomycin sulfate and peplomycin sulfate, were compared for their tumor-specific cytotoxicity, using human oral squamous cell lines (HSC-2, HSC-3, HSC-4, Ca9-22 and NA), human promyelocytic leukemic cell line HL-60 and human normal oral cell types (gingival fibroblast HGF, pulp cell HPC and periodontal ligament fibroblast HPLF). Among these three compounds, mitomycin C showed the highest tumor-specificity, due to its higher cytotoxic activity against human oral tumor cell lines than bleomycin and peplomycin. However, there was considerable variation of drug sensitivity among the six tumor cell lines. Mitomycin C induced internucleosomal DNA fragmentation and caspase-3, -8 and -9 activation in HL-60 cells only after 24 h. On the other hand, mitomycin C induced no clear-cut DNA fragmentation in HCS-2 cells, although it activated caspase-3, -8 and -9 to a slightly higher extent. Western blot analysis demonstrated that mitomycin C did not induce any apparent change in the intracellular concentration of anti-apoptotic protein (Bcl-2) and pro-apoptotic proteins (Bax, Bad). Electron microscopy of mitomycin C-treated HL-60 cells showed intact mitochondria (as regards to integrity and size) and cell surface microvilli, without production of an apoptotic body or autophagosome, at an early stage after treatment. The present study suggests the incomplete induction of apoptosis or the induction of another type of cell death by mitomycin C treatment.
Assuntos
Antibióticos Antineoplásicos/farmacologia , Bleomicina/farmacologia , Carcinoma de Células Escamosas/patologia , Mitomicina/farmacologia , Neoplasias Bucais/patologia , Peplomicina/farmacologia , Apoptose/efeitos dos fármacos , Carcinoma de Células Escamosas/metabolismo , Caspases/metabolismo , Morte Celular/efeitos dos fármacos , Células Cultivadas , Ativação Enzimática/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Células HL-60/efeitos dos fármacos , Células HL-60/efeitos da radiação , Células HL-60/ultraestrutura , Humanos , Neoplasias Bucais/metabolismo , Raios UltravioletaRESUMO
BACKGROUND: Haematological cancer is characterised by chromosomal translocation (e.g. MLL translocation in acute leukaemia) and two models have been proposed to explain the origins of recurrent reciprocal translocation. The first, established from pairs of translocated genes (such as BCR and ABL), considers the spatial proximity of loci in interphase nuclei (static "contact first" model). The second model is based on the dynamics of double strand break ends during repair processes (dynamic "breakage first" model). Since the MLL gene involved in 11q23 translocation has more than 40 partners, the study of the relative positions of the MLL gene with both the most frequent partner gene (AF4) and a less frequent partner gene (ENL), should elucidate the MLL translocation mechanism. METHODS: Using triple labeling 3D FISH experiments, we have determined the relative positions of MLL, AF4 and ENL genes, in two lymphoblastic and two myeloid human cell lines. RESULTS: In all cell lines, the ENL gene is significantly closer to the MLL gene than the AF4 gene (with P value < 0.0001). According to the static "contact first" model of the translocation mechanism, a minimal distance between loci would indicate a greater probability of the occurrence of t(11;19)(q23;p13.3) compared to t(4;11)(q21;q23). However this is in contradiction to the epidemiology of 11q23 translocation. CONCLUSION: The simultaneous multi-probe hybridization in 3D-FISH is a new approach in addressing the correlation between spatial proximity and occurrence of translocation. Our observations are not consistent with the static "contact first" model of translocation. The recently proposed dynamic "breakage first" model offers an attractive alternative explanation.
Assuntos
Núcleo Celular/química , Proteínas de Ligação a DNA/genética , Genes , Imageamento Tridimensional , Hibridização in Situ Fluorescente/métodos , Proteína de Leucina Linfoide-Mieloide/genética , Proteínas de Neoplasias/genética , Proteínas Nucleares/genética , Fatores de Transcrição/genética , Adolescente , Adulto , Linhagem Celular Transformada/química , Linhagem Celular Transformada/ultraestrutura , Linhagem Celular Tumoral/química , Linhagem Celular Tumoral/ultraestrutura , Núcleo Celular/ultraestrutura , Cromossomos Humanos Par 11/genética , Cromossomos Humanos Par 11/ultraestrutura , Cromossomos Humanos Par 19/genética , Cromossomos Humanos Par 19/ultraestrutura , Cromossomos Humanos Par 4/genética , Cromossomos Humanos Par 4/ultraestrutura , Células HL-60/química , Células HL-60/ultraestrutura , Herpesvirus Humano 4 , Histona-Lisina N-Metiltransferase , Humanos , Interfase , Leucemia Monocítica Aguda/genética , Leucemia Monocítica Aguda/patologia , Masculino , Modelos Genéticos , Mieloma Múltiplo/patologia , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras B/patologia , Fatores de Elongação da Transcrição , Translocação GenéticaRESUMO
BACKGROUND: During granulopoiesis in the bone marrow, the nucleus differentiates from ovoid to lobulated shape. Addition of retinoic acid (RA) to leukemic HL-60 cells induces development of lobulated nuclei, furnishing a convenient model system for nuclear differentiation during granulopoiesis. Previous studies from our laboratory have implicated nuclear envelope composition as playing important roles in nuclear shape changes. Specifically noted were: 1) a paucity of lamins A/C and B1 in the undifferentiated and RA treated cell forms; 2) an elevation of lamin B receptor (LBR) during induced granulopoiesis. RESULTS: The present study demonstrates that perturbation of cytoskeletal elements influences nuclear differentiation of HL-60 cells. Because of cytotoxicity from prolonged exposure to cytoskeleton-modifying drugs, most studies were performed with a Bcl-2 overexpressing HL-60 subline. We have found that: 1) nocodazole prevents RA induction of lobulation; 2) taxol induces lobulation and micronuclear formation, even in the absence of RA; 3) cytochalasin D does not inhibit RA induced nuclear lobulation, and prolonged exposure induces nuclear shape changes in the absence of RA. CONCLUSIONS: The present results, in the context of earlier data and models, suggest a mechanism for granulocytic nuclear lobulation. Our current hypothesis is that the nuclear shape change involves factors that increase the flexibility of the nuclear envelope (reduced lamin content), augment connections to the underlying heterochromatin (increased levels of LBR) and promote distortions imposed by the cytoskeleton (microtubule motors creating tension in the nuclear envelope).
Assuntos
Núcleo Celular/ultraestrutura , Citoesqueleto/fisiologia , Células HL-60/ultraestrutura , Diferenciação Celular/efeitos dos fármacos , Forma Celular/efeitos dos fármacos , Citocalasina D/farmacologia , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/ultraestrutura , Células HL-60/química , Células HL-60/efeitos dos fármacos , Hematopoese , Humanos , Lamina Tipo A/análise , Lamina Tipo B/análise , Microscopia Confocal , Microscopia de Fluorescência , Nocodazol/farmacologia , Paclitaxel/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/fisiologia , Receptores Citoplasmáticos e Nucleares/análise , Tretinoína/farmacologia , Receptor de Lamina BRESUMO
Production of effective vaccine formulations is dependent on the availability of assays for the measurement of protective immune responses. The development and standardization of in vitro human cell-based assays for functional opsonophagocytic antibodies require critical evaluation and optimization of the preparation of cells for the assay. We report evaluation of a number of protocols with two continuous cell lines (NB-4 and HL-60) for the provision of differentiated cells for use in functional assays. Flow cytometric analysis of CD11b antigen expression, as a marker of differentiation, indicated that all-trans-retinoic acid (ATRA) gave improved differentiation (>80% of cells differentiated at 96 h) when compared with dimethylformamide (DMF) (<60% of cells differentiated at 96 h). Morphological changes during differentiation toward a neutrophil-like phenotype were assessed by scanning electron microscopy. HL-60 and NB-4 cells treated with ATRA showed more spreading and flattening than cells treated with DMF, further evidence that they may have achieved a more differentiated phenotype. The number of cell divisions in culture appeared to be critical because cell lines maintained in exponential growth for >40 passages failed to express CD11b antigen or show morphological changes associated with differentiation after exposure to either differentiation-inducing reagent. Late-passage cells also demonstrated increased tolerance to DMF. Our results indicated that ATRA supplemented with vitamin D(3) and granulocyte colony-stimulating factor affords robust, rapid, and reproducible differentiation of both cell types.
Assuntos
Técnicas de Cultura de Células/métodos , Diferenciação Celular/efeitos dos fármacos , Proteínas Opsonizantes/imunologia , Fagocitose/imunologia , Tretinoína/farmacologia , Análise de Variância , Antígeno CD11b/metabolismo , Dimetilformamida/farmacologia , Citometria de Fluxo , Células HL-60/ultraestrutura , Humanos , Microscopia Eletrônica de VarreduraRESUMO
A great deal of experimental evidence has accumulated in the past several decades, suggesting that polysaccharides have wide bioactivities. Cladonia furcata polysaccharide, CFP-2, a water-soluble lichenin with a mean Mr 7.6 x 10(4), was first obtained by 0.25 M NaOH solution extraction, ethanol precipitation, DEAE-cellulose, and Sephadex G-200 column chromatography. Gas chromatography of acid hydrolyzate of CFP-2 suggested that it was composed of D-glucose, D-galactose, and D-mannose in the molar ratios of 8:1:1. Periodate oxidation, Smith degradation, IR, and NMR spectroscopy analysis revealed that CFP-2 had a backbone consisting of alpha-(1-->3) and alpha-(1-->4)-linked D-glucopyranosyl residues substituted at O-6 with beta-(1-->6)-linked D-galactopyranosyl residue and alpha-(1-->6)-linked D-mannopyranosyl residue. CFP-2 was able to reduce viability of cultured HL-60 and K562 cells. The antiproliferative properties of CFP-2 appeared to be attributable to its induction of apoptotic cell death as determined by ultrastructural change, internucleosomal DNA fragmentation, and increased proportion of the subdiploid cell population. To elucidate molecular events in the apoptosis, protein expressions of Bcl-2, Bax, Fas, and FasL were measured by Western blotting using specific antibodies in HL-60 cells. The level of Bcl-2 remained largely unchanged, but the Bax, Fas, and FasL expression showed up-regulation. Moreover, the telomerase activity analyzed by TRAP-ELISA assay in HL-60 cells treated with CFP-2 decreased as compared with the untreated control cells. These results suggest that CFP-2 could have a possible cancer therapeutic potential.
Assuntos
Ascomicetos/química , Glucanos/química , Glucanos/farmacologia , Polissacarídeos/química , Polissacarídeos/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2 , Apoptose , Sequência de Carboidratos , Linhagem Celular Tumoral/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Fragmentação do DNA , Proteína Ligante Fas , Glucanos/isolamento & purificação , Células HL-60/efeitos dos fármacos , Células HL-60/metabolismo , Células HL-60/ultraestrutura , Humanos , Espectroscopia de Ressonância Magnética , Glicoproteínas de Membrana/análise , Dados de Sequência Molecular , Estrutura Molecular , Polissacarídeos/isolamento & purificação , Proteínas Proto-Oncogênicas/biossíntese , Telomerase/antagonistas & inibidores , Proteína X Associada a bcl-2 , Receptor fas/análiseRESUMO
We studied the mechanism of the cytotoxic effects of 5-aminolevulinic acid-based photodynamic therapy (ALA-PDT; induction with 1 mM ALA for 4 h followed by a blue light dose of 18 J/cm(2)) on the human promyelocytic leukemia cell line HL60 using biochemical and electron microscopy methods. The disruption of mitochondrial membrane potential, deltapsi(m), was paralleled by a decrease in ATP level, unmasking of the mitochondrial antigen 7A6, release of cytochrome c into the cytoplasm, activation of caspases 9 and 3 and cleavage of poly(ADP-ribose) polymerase (PARP). This was followed by DNA fragmentation. These data suggest that ALA-PDT activates the mitochondrial apoptotic pathway. The level of endoplasmic reticulum Ca(2+)-binding chaperones ERp57 and ERp72 and of anti-apoptotic proteins Bcl-2 and Bcl-x(L) was decreased whereas that of Ca(2+)-binding protein calmodulin and the stress protein HSP60 was elevated following ALA-PDT. Inhibition of the initiator caspase 9, execution caspase 3 and Ca(2+)-dependent protease m-calpain, did not prevent DNA fragmentation. We conclude that, in our in vitro model, ALA-based photodynamic treatment initiates several signaling processes in HL60 cells that lead to rapidly progressing apoptosis, which is followed by slow necrosis. Two apoptotic processes proceed in parallel, one representing the mitochondrial pathway, the other involving disruption of calcium homeostasis and activation of the endoplasmic reticulum stress-mediated pathway.
Assuntos
Aminoácidos Neutros/farmacologia , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/efeitos da radiação , Células HL-60/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/efeitos da radiação , Fotoquimioterapia/métodos , Trifosfato de Adenosina/metabolismo , Apoptose/efeitos dos fármacos , Apoptose/efeitos da radiação , Divisão Celular/efeitos dos fármacos , Divisão Celular/efeitos da radiação , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Retículo Endoplasmático/ultraestrutura , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos da radiação , Células HL-60/fisiologia , Células HL-60/efeitos da radiação , Células HL-60/ultraestrutura , Humanos , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/efeitos da radiação , Mitocôndrias/fisiologia , Proteínas de Neoplasias/metabolismo , Fármacos Fotossensibilizantes/farmacologia , Estresse Fisiológico/induzido quimicamente , Estresse Fisiológico/metabolismo , Estresse Fisiológico/patologiaRESUMO
Mutations in the gene of the hepatic glucose-6-phosphate transporter cause glycogen storage disease type 1b. In this disease, the altered glucose homeostasis and liver functions are accompanied by an impairment of neutrophils/monocytes. However, neither the existence of a microsomal glucose-6-phosphate transport, nor the connection between its defect and cell dysfunction has been demonstrated in neutrophils/monocytes. In this study we have characterized the microsomal glucose-6-phosphate transport of human neutrophils and differentiated HL-60 cells. The transport of glucose-6-phosphate was sensitive to the chlorogenic acid derivative S3483, N-ethylmaleimide, and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid, known inhibitors of the hepatic microsomal glucose-6-phosphate transporter. A glucose-6-phosphate uptake was also present in microsomes from undifferentiated HL-60 and Jurkat cells, but it was insensitive to S3483. The treatment with S3484 of intact human neutrophils and differentiated HL-60 cells mimicked some leukocyte defects of glycogen storage disease type 1b patients (ie, the drug inhibited phorbol myristate acetate-induced superoxide anion production and reduced the size of endoplasmic reticulum Ca(2+) stores). Importantly, the treatment with S3484 also resulted in apoptosis of human neutrophils and differentiated HL-60 cells, while undifferentiated HL-60 and Jurkat cells were unaffected by the drug. The proapoptotic effect of S3483 was prevented by the inhibition of nicotinamide adenine dinucleotide phosphate oxidase or by antioxidant treatment. These results suggest that microsomal glucose-6-phosphate transport has a role in the antioxidant protection of neutrophils, and that the genetic defect of the transporter leads to the impairment of cellular functions and apoptosis.
Assuntos
Apoptose , Glucose-6-Fosfato/metabolismo , Doença de Depósito de Glicogênio Tipo I/patologia , Microssomos/metabolismo , Neutrófilos/fisiologia , Neutrófilos/ultraestrutura , Ácido 4,4'-Di-Isotiocianoestilbeno-2,2'-Dissulfônico/farmacologia , Antioxidantes/farmacologia , Apoptose/efeitos dos fármacos , Transporte Biológico/efeitos dos fármacos , Diferenciação Celular , Ácidos Cicloexanocarboxílicos/farmacologia , Inibidores Enzimáticos/farmacologia , Etilmaleimida/farmacologia , Células HL-60/ultraestrutura , Humanos , NADPH Oxidases/antagonistas & inibidoresRESUMO
Secretion in neutrophils is thought to be regulated in different ways for the different granule types. Specific granules are endowed with proteins which are related to docking and fusion events and are absent on azurophilic granules. Furthermore, even if secretion of content from all neutrophil granules is a Ca(2+)-dependent process, a higher concentration of cytosolic calcium is required for azurophilic than for specific granule secretion. In this paper we show that human neutrophils and promyelocitic cells express neuronal calcium sensor-1 (NCS-1), a calcium binding protein involved in exocytosis in various cell types. Both mRNA and protein were found in mature cells and precursors. NCS-1 is shown to be mainly associated with azurophilic granules and, therefore could play an instrumental role in the calcium-dependent secretion of azurophilic granules.
Assuntos
Proteínas de Ligação ao Cálcio/análise , Células HL-60/química , Neuropeptídeos/análise , Neutrófilos/química , Western Blotting , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Compartimento Celular , Fracionamento Celular , Células Cultivadas , Grânulos Citoplasmáticos/química , Células HL-60/ultraestrutura , Humanos , Microscopia , Proteínas Sensoras de Cálcio Neuronal , Neuropeptídeos/genética , Neuropeptídeos/metabolismo , Neutrófilos/ultraestrutura , RNA Mensageiro/análise , Transcrição GênicaRESUMO
Microaxial tomography provides a good means for microscopic image acquisition of cells or sub-cellular components like cell nuclei with an improved resolution, because shortcomings of spatial resolution anisotropy in optical microscopy can be overcome. Thus, spatial information of the object can be obtained without the necessity of confocal imaging. Since the very early developments of microaxial tomography, a considerable drawback of this method was a complicated image acquisition and processing procedure that requires much operator time. In order to solve this problem the Heidelberg 2pi-tilting device has been mounted on the Brno high-resolution cytometer as an attempt to bring together advanced microscopy and fast automated computer image acquisition and analysis. A special software module that drives all hardware components required for automated microaxial tomography and performs image acquisition and processing has been developed. First, a general image acquisition strategy is presented. Then the procedure for automation of axial tomography and the developed software module are described. The rotation precision has been experimentally proved followed by experiments with a specific biological example. For this application, also a method for the preparation of cell nuclei attached to glass fibres has been developed that allows for the first time imaging of three-dimensionally conserved, fluorescence in situ hybridisation-stained cell nuclei fixed to a glass fibre.
Assuntos
Núcleo Celular/ultraestrutura , Células HL-60/ultraestrutura , Hibridização in Situ Fluorescente , Tomografia , Núcleo Celular/química , Humanos , Citometria por Imagem , Processamento de Imagem Assistida por Computador , Interfase , Microscopia/instrumentação , Microscopia/métodos , Software , Tomografia/instrumentação , Tomografia/métodosRESUMO
Localization of actin was studied in HL-60 leukemia cells after treatment with the anticancer agent etoposide for 3 days in a range of concentrations (0.02-200 microM). Significant changes in morphology of the cells and F-actin distribution patterns labelled with TRITC-phalloidin occurred only after treatment with 100 and 200 microM etoposide. In comparison with control cells, the number of cells decreased, cells were larger and almost all treated cells had irregular surfaces with lamellipodia. F-actin was distributed in a punctate pattern throughout the cytoplasm after treatment. In some treated cells, fluorescence appeared as a bright haze, whereas in other cells it formed a network. Treated cells also showed bright fluorescence at their periphery. Immunogold labelling of actin was observed in cells whether or not treated with etoposide. Labelling was found in the nucleus and also in the cytoplasm. At the ultrastructural level, cells treated with 100 and 200 microM etoposide showed increased positivity for actin in relation with blebbing, margination of nuclear chromatin and bodies containing recognizable nuclear fragments. These findings indicate that alterations in expression of actin in HL-60 cells after treatment with etoposide is dose-dependent and related with apoptosis.
Assuntos
Actinas/metabolismo , Etoposídeo/farmacologia , Células HL-60/patologia , Células HL-60/efeitos dos fármacos , Células HL-60/ultraestrutura , Humanos , Microscopia ImunoeletrônicaRESUMO
Permeability transition, and a subsequent drop in mitochondrial membrane potential (DeltaPsi(m)), have been suggested to be mechanisms by which cytochrome c is released from the mitochondria into the cytosol during apoptosis. Furthermore, a drop in DeltaPsi(m) has been suggested to be an obligate early step in the apoptotic pathway. Didemnin B, a branched cyclic peptolide described to have immunosuppressive, anti-tumour, and anti-viral properties, induces rapid apoptosis in a range of mammalian cell lines. Induction of apoptosis by didemnin B in cultured human pro-myeloid HL-60 cells is the fastest and most complete ever described with all cells being apoptotic after 3 h of treatment. By utilizing the system of didemnin B-induced apoptosis in HL-60 cells, and the potent inhibitors of mitochondrial permeability transition, cyclosporin A and bongkrekic acid, we show that permeability transition as determined by changes in DeltaPsi(m) and mitochondrial Ca2+ fluxing, is not a requirement for apoptosis or cytochrome c release. In this system, changes in mitochondrial membrane potential and cytochrome c release are shown to be dependent on caspase activation, and to occur concurrently with the release of caspase-9 from mitochondria, genomic DNA fragmentation and apoptotic body formation.
Assuntos
Apoptose/efeitos dos fármacos , Caspases/fisiologia , Grupo dos Citocromos c/metabolismo , Depsipeptídeos , Membranas Intracelulares/fisiologia , Canais Iônicos , Mitocôndrias/enzimologia , Peptídeos Cíclicos/farmacologia , Apoptose/fisiologia , Ácido Bongcréquico/farmacologia , Linfoma de Burkitt/patologia , Sinalização do Cálcio , Caspase 9 , Caspases/metabolismo , Ciclosporina/farmacologia , Fragmentação do DNA , Ativação Enzimática , Células HL-60/efeitos dos fármacos , Células HL-60/ultraestrutura , Humanos , Membranas Intracelulares/efeitos dos fármacos , Melanoma/patologia , Potenciais da Membrana/efeitos dos fármacos , Proteínas de Membrana/metabolismo , Mitocôndrias/efeitos dos fármacos , Proteínas de Transporte da Membrana Mitocondrial , Poro de Transição de Permeabilidade Mitocondrial , Permeabilidade/efeitos dos fármacos , Tapsigargina/farmacologia , Células Tumorais CultivadasRESUMO
Degradation of several intracellular proteins involved in cell cycle control and tumour growth is regulated by the ubiquitin-dependent multicatalytic protease complex (proteasome). We report that proteasome inhibitor Z-Ile-Glu(OtBu)-Ala-Leucinal (PSI) was cytotoxic on most human myeloid leukaemia cell lines at IC50 doses ranging from 5 to 25 nmol/l. Additionally, PSI pre-treatment enhanced cytotoxicity by taxol and cisplatinum. PSI was more active on leukaemic than on normal CD34(+) bone marrow progenitors because the 50% growth inhibition of colony-forming unit granulocyte macrophage (CFU-GM) from cases of chronic myelogenous leukaemia (CML) and normal subjects was achieved by 15 nmol/l and 50 nmol/l PSI respectively. PSI killed cells by apoptosis as revealed by ultrastructural changes, nuclear DNA fragmentation, cleavage of poly (ADP-ribose) polymerase (PARP) and of beta-catenin, and was antagonized by ectopic expression of Bcl-2 but not by inactivating mutations of p53. This event was associated with a slight accumulation of Bcl-2, a decrease of Bax but no changes in Bcl-X(L) protein expression at any time point. In Ph(+) cell lines BCR-ABL protein was only down-regulated after 48 h of treatment with 10 nmol/l PSI. Altogether, these results indicate that PSI, alone or in association with other cytotoxic agents, has anti-tumour activity against myeloid malignancies and is more effective on leukaemic than on normal haematopoietic progenitor cells.
Assuntos
Apoptose/efeitos dos fármacos , Células-Tronco Hematopoéticas/efeitos dos fármacos , Leucina/análogos & derivados , Leucina/uso terapêutico , Leucemia Mieloide/tratamento farmacológico , Inibidores de Proteases/uso terapêutico , Transativadores , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Western Blotting , Linhagem Celular Transformada , Cisplatino/administração & dosagem , Proteínas do Citoesqueleto/análise , Relação Dose-Resposta a Droga , Proteínas de Fusão bcr-abl , Expressão Gênica/efeitos dos fármacos , Genes bcl-2 , Genes p53 , Células HL-60/efeitos dos fármacos , Células HL-60/ultraestrutura , Células-Tronco Hematopoéticas/ultraestrutura , Humanos , Leucemia Mieloide/patologia , Glicoproteínas de Membrana/análise , Microscopia Eletrônica , Paclitaxel/administração & dosagem , Fatores de Tempo , beta CateninaRESUMO
PSGL-1, a specific ligand for P-, E- and L-selectin, was isolated from in vivo [3H]-glucosamine labeled HL-60 cells by a combination of wheat germ agglutinin-agarose and P- or E-selectin-agarose chromatography. N-linked oligosaccharides were released from the purified, denatured ligand molecule by peptide: N-glycosidase F treatment and, following separation by Sephacryl S-200 chromatography, partially characterized using lectin, ion-exchange and size-exclusion chromatography in combination with glycosidase digestions. The data obtained suggest that the N-glycans on PSGL-1 are predominantly core-fucosylated, multiantennary complex type structures with extended, poly-N-acetyllactosamine containing outer chains. A portion of the outer chains appears to be substituted with fucose indicating that the N-glycans, in addition to the O-glycans on PSGL-1, may be involved in selectin binding.
Assuntos
Membrana Celular/química , Leucócitos/química , Glicoproteínas de Membrana/química , Polissacarídeos/química , Polissacarídeos/isolamento & purificação , Membrana Celular/ultraestrutura , Enzimas/farmacocinética , Fucose/química , Células HL-60/química , Células HL-60/ultraestrutura , Humanos , Leucócitos/ultraestrutura , Glicoproteínas de Membrana/ultraestrutura , Oligossacarídeos/química , Oligossacarídeos/isolamento & purificaçãoRESUMO
HL-60 leukemic cells were treated with 6 different doses of etoposide for 72 hours. Changes in the distribution of vimentin were found to be dependent on the concentration of etoposide. As compared with control cells there were distinct changes in cells incubated with 100 and 200 microM/L of the drug. The size of cells treated with 100 microM/L and especially with 200 microM/L increased, but the number of cells decreased. In control cells and those treated with 0.02, 0.2 and 2 microM/L etoposide, vimentin was seen rather as a ring often with the increased concentration near one pole of the cells. Cells at 20 microM/L etoposide showed the same staining pattern but more brighter cells were found. The addition of 100 microM/L and 200 microM/L etoposide to cells resulted in diffusely distributed fluorescence staining, which often appeared as a quite dense network around the nucleus. Immunogold labelling was observed in cells treated with all doses of etoposide and control cells. Labelling was localized in the nucleus but also in the cytoplasm but rather in the area of the nucleus.
Assuntos
Antineoplásicos/farmacologia , Etoposídeo/farmacologia , Células HL-60/efeitos dos fármacos , Vimentina/biossíntese , Tamanho Celular/efeitos dos fármacos , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/ultraestrutura , Relação Dose-Resposta a Droga , Imunofluorescência , Células HL-60/metabolismo , Células HL-60/ultraestrutura , Humanos , Microscopia Imunoeletrônica , Vimentina/ultraestruturaRESUMO
PCNA antigen was localized at the light and electron microscopes level in two human leukemia cell lines HL-60 and K-562. PCNA expression was used to discriminate cycling from non-cycling cells. PCNA protein at the level of the light microscope was present in 70% of the cell in HL-60 cell line and in 65% of the cells in K-562 line. Streptavidin immunogold method was used for localization of PCNA expression at the ultrastructural level. Positive staining for this protein was seen as granular pattern in the nucleus and in the cytoplasm. In the nucleus the gold particles were seen to be associated with heterochromatin and euchromatin of the leukemia cells. In cytoplasm it was found on the endoplasmic reticulum and associated with ribosomes. Controls of the leukemia cells incubation with normal mouse serum showed no labelling at the light and electron microscope level.
Assuntos
Células HL-60/citologia , Células K562/citologia , Antígeno Nuclear de Célula em Proliferação/análise , Citoplasma/ultraestrutura , Células HL-60/ultraestrutura , Humanos , Imuno-Histoquímica , Células K562/ultraestrutura , Microscopia Eletrônica , Microscopia ImunoeletrônicaRESUMO
Formation of the Bcr-Abl chimeric protein is the molecular hallmark of Philadelphia-positive leukemia. Normal Bcr is a complex protein which has been found in the cytoplasm, has serine kinase activity, and has been implicated in cellular signal transduction. However, we have recently demonstrated that Bcr can also associate with condensed chromatin. Since two major Bcr proteins have been characterized (p160Bcr and p130Bcr), we sought to determine if different forms of Bcr localized to the nucleus vs the cytoplasm. Metabolic labeling and Western blotting experiments were performed using nuclear and cytoplasmic extracts of three human Philadelphia-negative leukemia/lymphoma cell lines (KG-1, HL-60, and Jurkat). Both methodologies showed that p160Bcr and p130Bcr localized to the cytoplasm, but the p130 form predominated in the nucleus. These results suggest that Bcr serves both nuclear and cytoplasmic functions, and that different forms of Bcr may be preferentially involved in these distinct activities.
Assuntos
Núcleo Celular/química , Citoplasma/química , Isoformas de Proteínas/análise , Proteínas Tirosina Quinases , Proteínas Proto-Oncogênicas/análise , Doença Aguda , Western Blotting , Células HL-60/química , Células HL-60/ultraestrutura , Humanos , Células Jurkat/química , Células Jurkat/ultraestrutura , Leucemia Mieloide/patologia , Microscopia Imunoeletrônica , Peso Molecular , Proteínas de Neoplasias/análise , Proteínas de Neoplasias/química , Isoformas de Proteínas/química , Proteínas Proto-Oncogênicas/química , Proteínas Proto-Oncogênicas/fisiologia , Proteínas Proto-Oncogênicas c-bcr , Células Tumorais Cultivadas/química , Células Tumorais Cultivadas/ultraestruturaRESUMO
We previously reported a novel monoclonal antibody (MAb), designated mNI-11, recognizing an adhesion-associated antigen distinct from any previously reported ones. In this article, this adhesion-associated antigen with a molecular weight of about 97 kDa was found to be strongly expressed on human umbilical vein endothelial cells (HUVECs) by fluorescence-activated cell sorter (FACS) analysis. Expression of this antigen on HUVECs was slightly increased in response to the exposure to tumor necrosis factor-alpha (TNF-alpha) or phorbol myristate acetate (PMA). As a biological function exerted by this antigen, it was of great interest that immobilized mNI-11 directly and rapidly enhanced the spread formation of HUVECs, whereas MAbs binding other adhesion-associated antigens such as mNI-58A (anti-CD11a), L130 (anti-CD18), L133.1 (anti-CD31), L178 (anti-CD44), L25.3 (anti-CD49d), or LB-2 (anti-CD54) did not carry such activity under the same conditions. The HUVECs spread formation enhanced by mNI-11 was completely blocked in the presence of a microfilament formation inhibitor, cytochalasin D (CyD), a Ca2+ calmodulin inhibitor, W-7, EDTA, and was partially blocked by a microtubule formation inhibitor, nocodazole, a protein kinase C (PKC) inhibitor, H-7, and a protein synthesis inhibitor, cycloheximide (CHX). However, a protein tyrosine kinase (PTK) inhibitor, genistein, did not affect the spread formation under the same conditions. Taken together, it was suggested that the spread formation of HUVECs enhanced by mNI-11 was mainly associated with the influx of Ca2+ and microfilament reorganization. In addition, the novel property associated with mNI-11 to enhance the spread formation of HUVECs was possibly mediated through its reaction against a unique epitope on HUVECs.