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1.
J Nat Med ; 72(1): 260-266, 2018 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-29151157

RESUMO

Erypoegin K is an isoflavone isolated from the stem bark of Erythrina poeppigiana. It contains a furan group at the A-ring of the core isoflavone structure and can inhibit the activity of glyoxalase I, an enzyme that catalyzes the detoxification of methylglyoxal (MG), a by-product of glycolysis. In the present study, we found that erypoegin K has a potent cytotoxic effect on human leukemia HL-60 cells. Its cytotoxic effect was much stronger than that of a known glyoxalase I inhibitor S-p-bromobenzylglutathione cyclopentyl diester. Conversely, erypoegin K demonstrated weak cytotoxicity toward normal human peripheral lymphocytes. The treatment of HL-60 cells with erypoegin K significantly induced caspase-3 activity, whereas the pretreatment of the cells with caspase-3 inhibitor suppressed erypoegin K-induced cell death. Furthermore, nuclear condensation and apoptotic genome DNA fragmentation were observed in erypoegin K-treated HL-60 cells. These results indicated that the observed cell death was mediated by apoptosis. In addition, the toxic compound MG was highly accumulated in the culture medium of erypoegin K-treated HL-60 cells, suggesting that cell apoptosis was triggered by extracellular MG. The present study showed that erypoegin K has a potent apoptosis-inducing effect on cancerous cell lines, such as HL-60.


Assuntos
Benzofuranos/química , Erythrina/química , Células HL-60/química , Isoflavonas/química , Leucemia/tratamento farmacológico , Apoptose , Humanos , Leucemia/patologia
2.
J Org Chem ; 79(17): 7895-904, 2014 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-25089636

RESUMO

Seven new prenylated indole diketopiperazines, versicamides A-G (1-7) and a novel chemical derivative from 7, versicamide H (8), along with three known analogic diketopiperazines (9-11) were obtained from the marine-derived fungus Aspergillus versicolor HDN08-60. Their structures were determined by spectroscopic techniques, including 2D NMR, ECD calculations, and single-crystal X-ray diffraction analysis, together with the assistance of further chemical conversions. The cytotoxicities of 1-8 were tested against the HeLa, HCT-116, HL-60, and K562 cell lines, but only 8 exhibited moderate activity against HL-60 cells, with an IC50 value of 8.7 µM. Further investigation with target screening showed that 8 exhibited selective PTK inhibitory activities.


Assuntos
Aspergillus/química , Dicetopiperazinas/química , Dicetopiperazinas/farmacologia , Fungos/química , Células HL-60/efeitos dos fármacos , Cristalografia por Raios X , Células HL-60/química , Humanos , Concentração Inibidora 50 , Espectroscopia de Ressonância Magnética , Biologia Marinha , Estrutura Molecular , Prenilação
3.
Iran Biomed J ; 11(4): 209-14, 2007 10.
Artigo em Inglês | MEDLINE | ID: mdl-18392081

RESUMO

BACKGROUND: Microtubules (MT) are important components of cell cytoskeleton and play key roles in cell motility mitosis and meiosis. They are also the targets of several anticancer agents which indicating their importance in maintaining cell viability. Microtubular reorganization contributing to apoptotic morphology occurs in normal and neoplastic cells undergoing apoptosis induced by cytotoxic drugs. The aim of this study was to correlate the changes in the MT with behavior of the gamma-tubulin in apoptotic cell, and to see if apoptitic MT showed biochemical characteristics of stable MT. METHODS: Apoptosis was induced in the human leukemia cells (HL-60) by treatment with 1 microM of all-trans retinoic acid over a 5-day period. The time course of changes was assessed using flow cytometry, DNA fragmentation and immunocytochemistry in cells labeled for alpha-tubulins, acetylated alpha-tubulin and gamma-tubulin. RESULTS: The results indicated that gamma-tubulin content is increased after cells have gone through the apoptosis with a diffuse cytoplasmic pattern. Alpha-tubulin did not reveal any specific pattern of polymerization in apoptotic cells and acetylated alpha-tubulin content was also decreased in comparison with non-apoptotic cells. CONCLUSION: Our results support the idea that microtubule reorganization is an important factor of the mammalian cells response to apoptosis, and the altered properties of the MT did not reflect changes in function as apoptosis progresses.


Assuntos
Apoptose , Microtúbulos/química , Acetilação , Apoptose/efeitos dos fármacos , Células HL-60/química , Células HL-60/citologia , Humanos , Imuno-Histoquímica , Tretinoína/farmacologia , Tubulina (Proteína)/análise
4.
BMC Cancer ; 6: 20, 2006 Jan 24.
Artigo em Inglês | MEDLINE | ID: mdl-16433901

RESUMO

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 , Imagem 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 Genética
5.
Anal Biochem ; 346(2): 295-9, 2005 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-16213457

RESUMO

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.


Assuntos
Actinas/metabolismo , Quercetina/metabolismo , Fracionamento Celular , Cromatografia em Gel , Eletroforese em Gel de Poliacrilamida , Citometria de Fluxo , Células HL-60/química , Humanos , Ligantes , Proteínas Nucleares/isolamento & purificação , Ligação Proteica , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
6.
Z Naturforsch C ; 60(1-2): 143-51, 2005 Jan-Feb.
Artigo em Inglês | MEDLINE | ID: mdl-15787260

RESUMO

A human leukaemia cell line--HL-60--can be differentiated into neutrophils or macrophages and both differentiation processes are accompanied by changes of the lipid composition. Various methods were described for the extraction of lipids from cellular systems, but only two of them were applied to the HL-60 cell line so far. In this study we compared five selected extraction methods for the lipid extraction from HL-60 cells with regard to their qualitative analysis by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS): chloroform/methanol at volume ratios 2:1 and 1:2, isopropanol/ chloroform, isopropanol/hexane and butanol. In addition, the cholesterol and phospholipid concentrations in organic extracts were measured by colorimetric assays. Results can be summarized as follows: For the analysis of polar phospholipids obtained from HL-60 cells by MALDI-TOF MS, a chlorofom/methanol (1:2) or isopropanol/chloroform mixture or butanol can be applied as extraction systems On the other hand, if one would like to analyze changes in triacylglycerols, then chloroform/methanol (2:1) would be the method of choice.


Assuntos
Células HL-60/química , Lipídeos/química , Lipídeos/isolamento & purificação , Colesterol/análise , Humanos , Fosfolipases A , Fosfolipídeos/análise , Solventes , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
7.
Genes Chromosomes Cancer ; 42(2): 170-8, 2005 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-15540222

RESUMO

Mutation or dysregulation of related homeobox genes occurs in leukemia. Using RT-PCR, we screened members of the EHG family of homeobox genes, comprising EN1 (at 2q14), GBX2 (at 2q36), and EN2, GBX1, and HLXB9 (at 7q36), for dysregulation in acute myeloid leukemia (AML) cell lines indicated by chromosomal breakpoints at these sites. Only one EHG-family gene was expressed, HLXB9, in cell line GDM-1 (AML-M4). Karyotypic analysis of GDM-1 revealed a unique t(6;7)(q23;q35), also present in the patient. Fluorescence in situ hybridization analysis showed chromosomal breakpoints close to the region upstream of HLXB9, at 7q36, a region rearranged in certain AML patients, and at 6q23 upstream of MYB, a gene activated in leukemia. Detailed expression analysis suggested ectopic activation of HLXB9 occurred via juxtaposition with regions upstream of MYB, which was highly expressed in GDM-1. Our data identified a cell line model for a novel leukemic translocation involving MYB with HLXB9, further implicating HLXB9 in leukemogenesis.


Assuntos
Cromossomos Humanos Par 6/genética , Cromossomos Humanos Par 7/genética , Regulação Neoplásica da Expressão Gênica/genética , Genes myb/fisiologia , Proteínas de Homeodomínio/genética , Leucemia Mielomonocítica Aguda/genética , Fatores de Transcrição/genética , Translocação Genética/genética , Linhagem Celular Tumoral , Análise Citogenética/métodos , Células HL-60/química , Células HL-60/metabolismo , Células HeLa/química , Células HeLa/metabolismo , Proteínas de Homeodomínio/fisiologia , Humanos , Células Jurkat/química , Células Jurkat/metabolismo , Células K562/química , Células K562/metabolismo , Leucemia Mielomonocítica Aguda/patologia , Proteínas Proto-Oncogênicas c-myb/biossíntese , Fatores de Transcrição/fisiologia , Células U937/química , Células U937/metabolismo
8.
Arterioscler Thromb Vasc Biol ; 24(11): 2063-8, 2004 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-15388522

RESUMO

OBJECTIVE: Inflammation is one of the initial repair processes after vascular injury. E-selectin facilitates adherence of leukocytes to vascular endothelium at the site of inflammation. Because the role of E-selectin in this process is not fully understood, we studied the role of E-selectin in vascular injury with a flow chamber model and a rat model of carotid artery injury. METHODS AND RESULTS: We established a rat aortic endothelial cell (RAEC) culture system from the aortas of adult male rats. When rat myelomonocytes were suspended in a flow chamber, rolling and adhesion to lipopolysaccharide (LPS)-stimulated RAECs were observed. Cell rolling and adhesion were greatly reduced by addition of anti-E-selectin monoclonal antibody (mAb). We then induced balloon injury in the left carotid arteries of rats. E-selectin expression was enhanced in endothelial cells at adventitial small vessels 7 days after injury. Rats with balloon injury were injected intraperitoneally with anti-E-selectin mAb for 8 days. Inflammatory cell infiltration was reduced by anti-E-selectin mAb treatment at the adventitia at 7 days after injury. This reduction was associated with attenuation of intimal hyperplasia in the rats treated with the mAb. CONCLUSIONS: These data suggest that E-selectin regulates adventitial inflammation through leukocyte adhesion and contributes to the process of intimal hyperplasia after balloon injury.


Assuntos
Anticorpos Bloqueadores/farmacologia , Lesões das Artérias Carótidas/complicações , Selectina E/imunologia , Hiperplasia/prevenção & controle , Animais , Anti-Inflamatórios/farmacologia , Anticorpos Monoclonais/farmacologia , Aorta/química , Aorta/citologia , Aorta/metabolismo , Células CHO/química , Células CHO/metabolismo , Artérias Carótidas/química , Artérias Carótidas/citologia , Artérias Carótidas/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Tecido Conjuntivo/irrigação sanguínea , Tecido Conjuntivo/imunologia , Tecido Conjuntivo/patologia , Cricetinae , Cricetulus/genética , Modelos Animais de Doenças , Selectina E/biossíntese , Selectina E/fisiologia , Células Endoteliais/química , Células Endoteliais/metabolismo , Endotélio Vascular/citologia , Endotélio Vascular/crescimento & desenvolvimento , Endotélio Vascular/imunologia , Células HL-60/química , Células HL-60/metabolismo , Células HL-60/patologia , Humanos , Técnicas In Vitro , Inflamação/prevenção & controle , Leucócitos/imunologia , Leucócitos/metabolismo , Masculino , Neovascularização Patológica/prevenção & controle , Ratos , Ratos Sprague-Dawley , Túnica Íntima/crescimento & desenvolvimento , Túnica Íntima/imunologia , Túnica Íntima/patologia
9.
Zhongguo Shi Yan Xue Ye Xue Za Zhi ; 12(4): 436-40, 2004 Aug.
Artigo em Chinês | MEDLINE | ID: mdl-15363126

RESUMO

To study the importance of chemokine SDF-1 in surviving of acute myelocytic leukemia cells HL-60, the adhesion ability of HL-60 and expression of Bcl-2, Fas protein when SDF-1 activity was blocked by anti-CXCR4 monoclonal antibody (12G5) were compared with those detected before MAb incubation, in this experiment, HL-60 cell were cultured and co-cultured with normal marrow stromal cell. The adhesion rate was detected while the expression of Bcl-2 and Fas proteins were assayed by immunohistochemical technique when SDF-1 activity was inhibited. The results showed that cell adhesion rate of HL-60 decreased while the expression Bcl-2 decreased and Fas increased. It is concluded that inhibition of SDF-1 activity increases cell apoptosis and thus reduces life-span of leukemia cell at certain level.


Assuntos
Anticorpos Monoclonais/farmacologia , Quimiocinas CXC/fisiologia , Proteínas Proto-Oncogênicas c-bcl-2/análise , Receptores CXCR4/fisiologia , Receptor fas/análise , Apoptose , Adesão Celular , Quimiocina CXCL12 , Células HL-60/química , Células HL-60/citologia , Humanos
10.
Free Radic Biol Med ; 37(8): 1182-90, 2004 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-15451058

RESUMO

Oxidative stress is known to be an important contributing factor in many chronic diseases. We tested the hypothesis that in healthy normal volunteers zinc acts as an effective anti-inflammatory and antioxidant agent. Ten normal volunteers were administered daily oral zinc supplementation (45 mg zinc as gluconate) and 10 volunteers received placebo for 8 weeks. Plasma zinc, MDA, HAE, and 8-OHdG levels; LPS-induced TNF-alpha and IL-1beta mRNA; and ex vivo TNF-alpha-induced NF-kappaB activity in mononuclear cells (MNC) were determined before and after supplementation. In subjects receiving zinc, plasma levels of lipid peroxidation products and DNA adducts were decreased, whereas no change was observed in the placebo group. LPS-stimulated MNC isolated from zinc-supplemented subjects showed reduced mRNA for TNF-alpha and IL-1beta compared to placebo. Ex vivo, zinc protected MNC from TNF-alpha-induced NF-kappaB activation. In parallel studies using HL-60, a promyelocytic cell line, we observed that zinc enhances the upregulation of mRNA and DNA-specific binding for A20, a transactivating factor which inhibits the activation of NF-kappaB. Our results suggest that zinc supplementation may lead to downregulation of the inflammatory cytokines through upregulation of the negative feedback loop A20 to inhibit induced NF-kappaB activation. Zinc administration to human subjects with conditions associated with increased oxidative stress should be explored.


Assuntos
Antioxidantes/farmacologia , Desoxiguanosina/análogos & derivados , Zinco/farmacologia , Adulto , Células Cultivadas/química , Células Cultivadas/efeitos dos fármacos , Adutos de DNA/sangue , Desoxiguanosina/sangue , Feminino , Células HL-60/química , Células HL-60/efeitos dos fármacos , Humanos , Interleucina-1/genética , Peroxidação de Lipídeos/efeitos dos fármacos , Masculino , Pessoa de Meia-Idade , Modelos Biológicos , Monócitos/química , Monócitos/efeitos dos fármacos , NF-kappa B/metabolismo , RNA Mensageiro/sangue , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Necrose Tumoral alfa/genética
11.
BMC Cell Biol ; 5: 30, 2004 Aug 19.
Artigo em Inglês | MEDLINE | ID: mdl-15317658

RESUMO

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
12.
Cell Cycle ; 3(8): 1050-6, 2004 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-15254417

RESUMO

Paclitaxel (PTX), a microtubule-active drug, causes mitotic arrest leading to apoptosis in certain tumor cell lines. Here we investigated the effects of PTX on human arterial smooth muscle cell (SMC) cells. In SMC, PTX caused both (a) primary arrest in G(1) and (b) post-mitotic arrest in G(1). Post-mitotic cells were multinucleated (MN) with either 2C (near-diploid) or 4C (tetraploid) DNA content. At PTX concentrations above 12 ng/ml, MN cells had 4C DNA content consistent with the lack of cytokinesis during abortive mitosis. Treatment with 6-12 ng/ml PTX yielded MN cells with 2C DNA content. Finally, 1-6 ng/ml of PTX, the lowest concentrations that affected cell proliferation, caused G(1) arrest without multinucleation. It is important that PTX did not cause apoptosis in SMC. The absence of apoptosis could be explained by mitotic exit and G(1) arrest as well as by low constitutive levels of caspase expression and by p53 and p21 induction. Thus, following transient mitotic arrest, SMC exit mitosis to form MN cells. These post-mitotic cells were subsequently arrested in G(1) but maintained normal elongated morphology and were viable for at least 21 days. We conclude that in SMC PTX causes post-mitotic cell cycle arrest rather than cell death.


Assuntos
Fase G1/efeitos dos fármacos , Músculo Liso Vascular/citologia , Miócitos de Músculo Liso/efeitos dos fármacos , Paclitaxel/farmacologia , Aorta/citologia , Apoptose/efeitos dos fármacos , Contagem de Células , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Vasos Coronários/citologia , Células HL-60/química , Células HL-60/metabolismo , Células HL-60/patologia , Humanos , Células Jurkat/química , Células Jurkat/metabolismo , Células Jurkat/patologia , Mitose/efeitos dos fármacos , Músculo Liso Vascular/efeitos dos fármacos , Ploidias , Proteínas Proto-Oncogênicas p21(ras)/metabolismo
13.
Biochem J ; 381(Pt 3): 861-6, 2004 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-15128268

RESUMO

Deoxynojirimycin (DNJ) analogues are inhibitors of ceramide glucosyltransferase (CGT), which catalyses the first step in the glucosphingolipid (GSL) biosynthetic pathway. We have synthesized a series of DNJ analogues to study the contribution of N-alk(en)yl side chains (C4, C9 or C18) to the behaviour of these analogues in cultured HL60 cells. When cells were treated for 16 h at non-cytotoxic concentrations of inhibitor, a 40-50% decrease in GSL levels was measured by HPLC analysis of GSL-derived oligosaccharides following ceramide glycanase digestion of GSL and 2-aminobenzamide labelling of the released oligosaccharides. Using a novel technique for short-term [14C]galactose labelling of cellular GSL, we used compound inhibition of GSL biosynthesis as a marker for compound uptake into cells. Surprisingly, the uptake of all three of the DNJ analogues was extremely rapid and was not dependent upon the length of the N-alk(en)yl moiety. Compound uptake occurred in less than 1 min, as shown by the complete inhibition of GSL labelling in cells treated with all the DNJ analogues. Greatly increased cellular retention of N-cis-13-octadecenyl-DNJ was observed relative to the shorter-chain compounds, N-butyl-DNJ and N-nonyl-DNJ, as indicated by complete inhibition of CGT 24 h after removal of inhibitor from the culture medium. The present study further characterizes the properties of N-alk(en)ylated DNJs, and demonstrates that increasing the length of the side chain is a simple way of improving imino sugar retention and therefore inhibitory efficacy for CGT in cultured cells.


Assuntos
1-Desoxinojirimicina/análogos & derivados , 1-Desoxinojirimicina/farmacologia , Glicoesfingolipídeos/antagonistas & inibidores , Glicoesfingolipídeos/biossíntese , Células HL-60/efeitos dos fármacos , Células HL-60/metabolismo , 1-Desoxinojirimicina/metabolismo , Radioisótopos de Carbono/metabolismo , Linhagem Celular Tumoral , Cromatografia Líquida de Alta Pressão , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacologia , Glicoesfingolipídeos/metabolismo , Células HL-60/química , Humanos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Coloração e Rotulagem/métodos
14.
Biochem J ; 381(Pt 3): 867-75, 2004 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-15128289

RESUMO

In the accompanying paper [Mellor, Neville, Harvey, Platt, Dwek and Butters (2004) Biochem. J. 381, 861-866] we treated HL60 cells with N-alk(en)yl-deoxynojirimycin (DNJ) compounds to inhibit glucosphingolipid (GSL) biosynthesis and identified a number of non-GSL-derived, small, free oligosaccharides (FOS) most likely produced due to inhibition of the oligosaccharide-processing enzymes a-glucosidases I and II. When HL60 cells were treated with concentrations of N-alk(en)ylated DNJ analogues that inhibited GSL biosynthesis completely, N-butyl- and N-nonyl-DNJ inhibited endoplasmic reticulum (ER) glucosidases I and II, but octadecyl-DNJ did not, probably due to the lack of ER lumen access for this novel, long-chain derivative. Glucosidase inhibition resulted in the appearance of free Glc1-3Man structures, which is evidence of Golgi glycoprotein endomannosidase processing of oligosaccharides with retained glucose residues. Additional large FOS was also detected in cells following a 16 h treatment with N-butyl- and N-nonyl-DNJ. When these FOS structures (>30, including >20 species not present in control cells) were characterized by enzyme digests and MALDI-TOF (matrix-assisted laser-desorption ionization-time-of-flight) MS, all were found to be polymannose-type oligosaccharides, of which the majority were glucosylated and had only one reducing terminal GlcNAc (N-acetylglucosamine) residue (FOS-GlcNAc1), demonstrating a cytosolic location. These results support the proposal that the increase in glucosylated FOS results from enzyme-mediated cytosolic cleavage of oligosaccharides from glycoproteins exported from the ER because of misfolding or excessive retention. Importantly, the present study characterizes the cellular properties of DNJs further and demonstrates that side-chain modifications allow selective inhibition of protein and lipid glycosylation pathways. This represents the most detailed characterization of the FOS structures arising from ER a-glucosidase inhibition to date.


Assuntos
1-Desoxinojirimicina/análogos & derivados , 1-Desoxinojirimicina/farmacologia , Células HL-60/efeitos dos fármacos , Oligossacarídeos/antagonistas & inibidores , Oligossacarídeos/metabolismo , Acetilglucosamina/química , Amino Açúcares/farmacologia , Configuração de Carboidratos , Sequência de Carboidratos , Linhagem Celular Tumoral , Cromatografia Líquida de Alta Pressão/métodos , Glucose/química , Glicosilação/efeitos dos fármacos , Células HL-60/química , Humanos , Manose/química , Oligossacarídeos/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , alfa-Glucosidases/metabolismo , alfa-Manosidase/metabolismo
15.
J Nat Prod ; 67(4): 527-31, 2004 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-15104478

RESUMO

In this paper we report the isolation and structural elucidation of a new flavonoid (1) and three known compounds, 6,8-di-C-methylkaempferol 3-methyl ether (2), oleanolic acid, and 2 alpha,3 beta-dihydroxyurs-12-en-28-oic acid, from aerial parts of Eucalyptus occidentalis collected in Algeria. Flavonoids 1 and 2 were used to study their biological activities on the human promyelocytic leukemia cell line, HL-60. Our data show that these compounds induce morphological changes and internucleosomal DNA fragmentation characteristic of apoptotic cell death, which is mediated by caspase-8/caspase-3 activation and cytochrome c release.


Assuntos
Antineoplásicos/isolamento & purificação , Apoptose/efeitos dos fármacos , Flavonoides/isolamento & purificação , Células HL-60/química , Quempferóis/isolamento & purificação , Quempferóis/farmacologia , Argélia , Antineoplásicos/química , Antineoplásicos/farmacologia , Caspase 3 , Caspase 8 , Caspases/metabolismo , Grupo dos Citocromos c/metabolismo , Fragmentação do DNA , Ativação Enzimática , Eucalyptus , Flavonoides/química , Flavonoides/farmacologia , Humanos , Quempferóis/química , Leucemia Mieloide , Estrutura Molecular , Ácido Oleanólico/farmacologia , Folhas de Planta/química
16.
Biochem J ; 381(Pt 2): 495-501, 2004 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-15018615

RESUMO

We studied the expression and function of the IL (interleukin)-3 and IL-5 family of receptors in male germ cells. RT (reverse transcription)-PCR showed expression of mRNAs encoding the alpha and beta subunits of the IL-3 and IL-5 receptors in human testis, and the presence of IL-3 and IL-5 receptors alpha and beta proteins was confirmed by immunoblotting with anti-alpha and anti-beta antibodies. The immunolocalization studies showed expression of these receptors in the germ line in the human testis and in human and bovine ejaculated spermatozoa. Functional studies with bull spermatozoa indicated that IL-3 signalled for increased uptake of hexoses in these cells at picomolar concentrations compatible with expression of functional high-affinity IL-3 receptors in these cells. In contrast, IL-5 failed to induce increased hexose uptake in bull spermatozoa. Experiments using HL-60 eosinophils that express functional IL-3 and IL-5 receptors confirmed that IL-3, but not IL-5, signalled for increased hexose uptake. Our findings suggest that differential signalling for increased hexose uptake by heteromeric high-affinity IL-3 and IL-5 receptors in mammalian spermatozoa is a property that depends on the identity of the alpha-subunit forming part of the alphabeta-complex and is not a property specific to the germ cells.


Assuntos
Hexoses/metabolismo , Interleucina-3/metabolismo , Interleucina-5/metabolismo , Transdução de Sinais/genética , Espermatozoides/metabolismo , Animais , Bovinos , Linhagem Celular Tumoral , Regulação da Expressão Gênica no Desenvolvimento/genética , Células Germinativas/química , Células Germinativas/metabolismo , Células HL-60/química , Células HL-60/metabolismo , Células-Tronco Hematopoéticas/química , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/metabolismo , Humanos , Interleucina-3/genética , Interleucina-3/farmacologia , Interleucina-3/fisiologia , Interleucina-5/genética , Interleucina-5/farmacologia , Interleucina-5/fisiologia , Masculino , Subunidades Proteicas/genética , RNA Mensageiro/genética , Receptores de Interleucina/genética , Receptores de Interleucina-3/genética , Receptores de Interleucina-5 , Sêmen/citologia , Espermatozoides/química , Espermatozoides/citologia , Espermatozoides/efeitos dos fármacos , Testículo/química , Testículo/metabolismo
17.
Genes Chromosomes Cancer ; 40(1): 72-7, 2004 May.
Artigo em Inglês | MEDLINE | ID: mdl-15034872

RESUMO

Comparative genomic hybridization (CGH) to metaphase chromosomes is a method for genome-wide detection of chromosomal aberrations in DNA samples. Recent advances in microarray technology have improved CGH by replacing metaphase chromosomes with a collection of mapped genomic clones placed on glass slides. However, it is quite expensive and labor-intensive to prepare DNA from the genomic clones for use in constructing genomic microarrays. Here we used strand-displacement rolling circle amplification (RCA) to manufacture whole-genome microarrays by using a collection of about 4,500 mapped RPCI-11 BAC clones that cover the human genome at approximately a 1-Mb resolution. These genomic microarrays detected all major chromosomal aberrations in cancer cells lines and in cell lines with aneuploidy. In this article, we discuss the advantages of using RCA for the manufacturing of large genomic microarrays.


Assuntos
Genoma Humano , Técnicas de Amplificação de Ácido Nucleico/métodos , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Linhagem Celular Tumoral , Cromossomos Artificiais Bacterianos/genética , Primers do DNA/biossíntese , Primers do DNA/genética , DNA Circular/genética , DNA de Neoplasias/biossíntese , DNA de Neoplasias/genética , Feminino , Células HL-60/química , Células HL-60/metabolismo , Células HL-60/patologia , Humanos , Masculino , Hibridização de Ácido Nucleico , Moldes Genéticos
18.
Biochem J ; 380(Pt 3): 715-22, 2004 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-14998372

RESUMO

Ceramides, which are produced from the hydrolysis of sphingomyelin or synthesized from serine and palmitate in a de novo pathway, are regarded as important cellular signals for inducing apoptosis. However, controversy over this proposed role of ceramides exists. Using stable isotope labelling coupled with GC (gas chromatography)-MS and mass isotopomer distribution analysis, we have studied the metabolism of exogenous long-chain ceramides in HL60 cells. Our results do not support the concept of enhanced ceramide transport into cells induced by solvent mixtures of ethanol and hydrocarbons. In addition, cell toxicity does not correlate with the amount of intact ceramide in the cells. Our results are more consistent with a disturbance of sphingomyelin metabolism induced by the solvent mixture. The characteristics of this disturbed sphingolipid disposition are the inhibition of dihydroceramide desaturation and an enhanced degradation of sphingomyelin. As a consequence, dihydroceramides accumulate and the cellular sphingomyelin content decreases. Inhibition of these pathways is most likely to be induced by the increased production of novel ceramide metabolites instead of by intact ceramides. Octadecane-1,2-diol is identified as a possible mediator. Treatments that divert ceramide degradation to the novel pathway are potential strategies in cancer therapy for inducing cell toxicity.


Assuntos
Ceramidas/metabolismo , Ceramidas/toxicidade , Esfingosina/análogos & derivados , Divisão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Cromatografia Gasosa-Espectrometria de Massas/métodos , Células HL-60/química , Células HL-60/metabolismo , Humanos , Solventes/farmacologia , Esfingolipídeos/metabolismo , Esfingomielinas/biossíntese , Esfingomielinas/metabolismo , Esfingosina/farmacologia
19.
Genes Chromosomes Cancer ; 39(4): 311-23, 2004 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-14978792

RESUMO

Leukemias are considered malignant clonal disorders arising from the accumulation of mutations in hematopoietic cells; the majority of these mutations are thought to be acquired somatically. Measurement of mutation frequency (Mf) at the hypoxanthine phosphoribosyltransferase (HPRT) locus has been developed as a method for estimating genomic instability. We investigated the Mf in 16 leukemic cell lines to determine whether these cell lines showed evidence of genomic instability. Although some leukemic cell lines had markedly elevated Mfs, the Mfs at the HPRT locus in leukemic cell lines were not always higher than those of B-lymphoblastoid cell lines and T lymphocytes from normal individuals. We were able to identify the HPRT mutation for 159 of 160 individual HPRT mutants. The HPRT mutations were characterized at a molecular level and classified as either gross chromosomal rearrangements (GCRs) or point mutations, such as single-nucleotide substitutions, insertions, or deletions. With rare exceptions, individual leukemic cell lines showed either point mutations or GCR, but not both. Of note, all the cell lines that primarily showed point mutations are known to be defective in mismatch repair machinery.


Assuntos
Hipoxantina Fosforribosiltransferase/genética , Leucemia/genética , Mutação/genética , Adolescente , Adulto , Idoso , Linfócitos B/química , Linfócitos B/metabolismo , Linfócitos B/patologia , Linhagem Celular Tumoral , Criança , Pré-Escolar , Análise Mutacional de DNA/métodos , Feminino , Marcadores Genéticos/genética , Instabilidade Genômica/genética , Células HL-60/química , Células HL-60/metabolismo , Humanos , Células Jurkat/química , Células Jurkat/metabolismo , Células K562/química , Células K562/metabolismo , Leucemia/patologia , Masculino , Mutagênese/genética , Projetos Piloto , Processamento de RNA/genética , RNA Mensageiro/metabolismo , RNA Neoplásico/genética , Recombinação Genética/genética , Células U937/química , Células U937/metabolismo
20.
Genes Chromosomes Cancer ; 39(4): 324-34, 2004 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-14978793

RESUMO

The t(9;11) has been described in patients with acute myeloid leukemia (AML), and two genes [AF9 (at 9p21) and FBP17 (at 9q34)] have been cloned as fusion partners of the MLL gene. From an AML-M5 with a t(9;11)(q34;q23), we identified a novel MLL fusion partner, AF9Q34. The AF9Q34 protein shows high homology with nGAP, a RAS GTPase-activating protein (RASGAP), and contains the highly conserved GRD and FLR motifs characteristic of RASGAPs. Recently, the rat homologue (DAB2IP) also was identified and reported to act as a RASGAP both in vivo and in vitro. RASGAPs negatively regulate the activity of RAS proteins that modulate diverse cellular processes by cycling between an inactive GDP-bound and an active GTP-bound state. In addition, the NH(2) terminus harbors an amino acid stretch with homology to the pleckstrin homology (PH) domain implicated in regulating the interaction between RAS and the catalytic domain of RASGAP. As a result of the breakpoint in the AF9Q34-MLL fusion protein, this PH domain is disrupted. This suggests that because of the translocation, the normal function of the AF9Q34 gene is aborted. Thus, AF9Q34 encodes a novel RASGAP gene that appears to be deregulated as a result of the translocation. The identification of this RASGAP protein in a novel MLL fusion implies that an indirect RAS-deregulating mechanism could be involved in leukemic transformation.


Assuntos
Proteínas de Ligação a DNA/genética , Leucemia Monocítica Aguda/genética , Proteínas de Fusão Oncogênica/genética , Proto-Oncogenes , Fatores de Transcrição , Proteínas Ativadoras de ras GTPase/genética , Sequência de Aminoácidos/genética , Sequência de Bases/genética , Proteínas de Transporte/genética , Linhagem Celular Tumoral , Quebra Cromossômica/genética , Cromossomos Humanos Par 11/genética , Cromossomos Humanos Par 9/genética , Perfilação da Expressão Gênica/métodos , Regulação Neoplásica da Expressão Gênica/genética , Células HL-60/química , Células HL-60/metabolismo , Células HeLa/química , Células HeLa/metabolismo , Histona-Lisina N-Metiltransferase , Humanos , Células Jurkat/química , Células Jurkat/metabolismo , Células K562/química , Células K562/metabolismo , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Proteína de Leucina Linfoide-Mieloide , Especificidade de Órgãos/genética , Translocação Genética/genética , Células U937/química , Células U937/metabolismo
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