Isoverbascoside对HL-60细胞的诱导分化和细胞毒作用

不同时间、不同浓度的isoverbascoside体外处理HL-60细胞,以形态改变(光镜和透射电镜观察)、功能分化(化学发光检测吞噬能力)、恶性度降低(裸鼠成瘤试验)等指标观察其诱导分化作用;以台盼蓝拒染作用和电镜下形态变化确定其细胞毒作用;用流式细胞术测定其对HL-60细胞周期的影响。20—25μmol/L Isov 1—3天诱导HL-60细胞向粒系方向分化,细胞吞噬能力提高,裸鼠成瘤性降低。30—35μmol/L Isov 2—3天对HL-60细胞有强烈的细胞毒作用。20μmol/L Isov处理12h可引起HL-60细胞的G_1期阻滞,在72h时引起HL-60细胞的G_2/M期的阻滞。     

Effect of 4-hydroxynonenal,a lipid peroxidation product,on exocytosis in HL-60 cells

Our work analysed the effect of 4-hydroxynonenal (HNE), a chemotactic aldehydic end-product of lipid peroxidation, on exocytosis in HL-60 cells. We measured the release of beta-glucuronidase, an enzyme of azurophil granules, from the cells incubated at 37 degrees C for 10 min in the presence of HNE concentrations ranging between 10(-8) and 10(-5) M. The release of lactate dehydrogenase was assayed to test cell viability. HNE (1 microM) was able to induce a significant and strong stimulation of beta-glucuronidase secretion without leading to cytotoxic effects. The finding that HNE could increase the exocytotic secretion from HL-60 cells together with its known chemotactic property supports the hypothesis that this lipid peroxidation product may play an important role as a chemical mediator of inflammation; moreover it is noteworthy that micromolar concentrations of HNE have actually been found in exudates from acute and chronic inflammations.     

Formation of nuclear matrix filaments by p27(BBP)/eIF6

p27(BBP)/eIF6 is an evolutionarily conserved protein necessary for ribosome biogenesis which was cloned in mammals for its ability to bind the cytodomain of beta 4 integrin. In cultured cells, a conspicuous fraction of p27(BBP)/eIF6 is associated with the intermediate filaments/nuclear matrix (IF/NM) cytoskeleton. The mechanism of this association is not known. Here we show that in epidermis p27(BBP)/eIF6 is naturally associated with IF/NM. To analyze the intrinsic capability of p27(BBP)/eIF6 to generate cytoskeletal networks, the properties of the pure, recombinant, untagged protein were studied. Recombinant p27(BBP)/eIF6 binds beta 4 integrin. Upon dialysis against IF buffer, p27(BBP)/eIF6 forms polymers which, strikingly, have a morphology identical to NM filaments. Cross-linking experiments suggested that polymerization is favored by the formation of disulphide bridges. These data suggest that p27(BBP)/eIF6 is associated with the cytoskeleton, and contributes to formation of NM filaments. These findings help to settle the controversy on nuclear matrix.     

Hydrogen peroxide-induced apoptosis of HL-60 human leukemia cells is mediated by the oxidants hypochlorous acid and chloramines

We set out to identify whether HOCl, which is generated from H(2)O(2) /MPO/Cl(-), is a proximal mediator of H(2)O(2) programmed cell death in the HL-60 human leukemia cell. We found that authentic HOCl induces apoptosis in the HL-60 cell. Both the addition of methionine, an HOCl scavenger, and the removal of Cl(-) from the medium to prevent the formation of HOCl inhibited H(2)O(2)-induced apoptosis. HL-60 cells underwent apoptosis when exposed to HOCl in full medium, which gives rise to chloramines by the reaction of HOCl with amine groups, but not by HOCl in the amine-free HBSS, in which HOCl but not chloramines can be detected. Authentic chloramines induced apoptosis in this cell line in a concentration-dependent manner and at concentrations lower than HOCl. Full medium exposed to HOCl for 24 h would support methionine noninhibitable apoptosis, but did not react with 2-nitro-5-thiobenzoic acid (TNB), raising the possibility that the final inducer is a nonoxidant formed from HOCl and chloramines. We conclude that the signal for apoptosis induced by H(2)O(2) in the MPO-containing HL-60 cell involves the reaction of the diffusible oxidant HOCl with amines producing chloramines and a subsequent non-TNB-reactive product.     

Role of the p70 S6 kinase cascade in neutrophilic differentiation and proliferation of HL-60 cells-a study of transferrin receptor-positive and -negative cells obtained from dimethyl sulfoxide- or retinoic acid-treated HL-60 cells

Previously, we suggested that p70 S6 kinase (p70 S6K) plays an important role in the regulation of neutrophilic differentiation of HL-60 cells; this conclusion was based on our analysis of transferrin receptor (Trf-R) positive (Trf-R(+)) and negative (Trf-R(-)) cells that appeared after treatment with dimethyl sulfoxide (Me(2)SO). In this study, we analyzed the upstream of p70 S6K in relation to the differentiation and proliferation of both cell types. The granulocyte colony-stimulating factor (G-CSF)-induced enhancement of phosphatidylinositol 3-kinase (PI3K) activity in Trf-R(+) cells was markedly higher than that in Trf-R(-) cells. Wortmannin, a specific inhibitor of PI3K, partially inhibited G-CSF-induced p70 S6K activity and G-CSF-dependent proliferation, whereas rapamycin, an inhibitor of p70 S6K, completely inhibited these activities. The wortmannin-dependent enhancement of neutrophilic differentiation was similar to that induced by rapamycin. From these results, we conclude that the PI3K/p70 S6K cascade may play an important role in negative regulation of neutrophilic differentiation in HL-60 cells. For the G-CSF-dependent proliferation, however, p70 S6K appears to be a highly important pathway through not only a PI3K-dependent but also possibly an independent cascade.     

23-Hydroxybetulinic acid-mediated apoptosis is accompanied by decreases in bcl-2 expression and telomerase activity in HL-60 Cells

23-Hydroxybetulinic acid, a derivative of betulinic acid, was investigated for its apoptotic effect and the associated telomerase activity in human leukemia HL-60 cells. Apoptosis and bcl-2 were determined by flow cytometry analysis. A PCR-based telomeric repeat amplification protocol assay was used to detect telomerase activity. Results showed that 23-hydroxybetulinic acid induced growth arrest and apoptotic cell death in HL-60 cells. The apoptotic events were associated with concurrent down-regulation of bcl-2 and the telomerase activity. Our data suggest that 23-hydroxybetulinic acid may be a potential cytotoxic agent for treatment of cancer.     

Characterisation and properties of the inclusion complex of 24-epibrassinolide with β-cyclodextrin

This paper reports the first study of an inclusion complex of abrassinosteroid with 60n716714487/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">-cyclodextrin. The formation of inclusion complexesbetween 24-epibrassinolide and 60n716714487/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">-cyclodextrin was confirmed by theirphysicochemical properties and the compounds were analysed by differentialscanning calorimetry, powder X-ray diffraction, nuclear magnetic resonancespectrometry and scanning electron microscopy. Theoretical calculations usingthe MM+ HyperChem force field showed a preference for inclusion of thesidechain of the epibrassinolide molecule into the 60n716714487/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">-cyclodextrin cavity toform a 1:1 inclusion complex, although complexes involving inclusion ofthe steroidal nucleus also possess a favourable interaction energy. Rice laminainclination assay, employing IAC-103 and IAC-104 cultivars, showed an improvedactivity for the epibrassinolide-cyclodextrin complex compared to theepibrassinolide itself. The results suggest that brassinosteroid complexationwith cyclodextrins may enhance the biological activity of these plant growthregulators.     

Sodium Valproate Facilitates the Propagation of Granulocytic Ehrlichiae (Anaplasma phagocytophilum) in HL-60 Cells

As already shown, some inducers of the differentiation of promyelocytic cells along the granulocytic pathway, such as dimethylsulphoxide (DMSO) or all-trans retinoic acid, can enhance propagation of granulocytic ehrlichiae in HL-60 cell cultures. This study was conducted to prove whether sodium valproate, a salt of di-n-propylacetic acid (VPA) known to trigger cellular differentiation in several solid and hematopoietic malignancies is similarly efficient in ehrlichial cultures. Two cell lines derived from HL-60, that is, low-passage undifferentiated HL-60 (HL-60F) and high-passage HL-60 spontaneously differentiated towards monocytic phenotype (HL-60J) were grown in RPMI 1640 medium supplemented with 10% FBS. The respective HL-60F and HL-60J IC₅₀-values for NaVPA were estimated to be 0.8 and 2.2 mM under these culture conditions; to stimulate the differentiation, the respective doses of 0.3 and 1.2 mM were then applied. When the NaVPA-treated cells of both lines were challenged with an ehrlichial laboratory strain (HGE), maintained in splenectomized NMRI mice, the respective 1–2 and ≤0.1% primary infection rates in HL-60F and HL-60J cultures were observed 3 days post-inoculation. In comparison, only rare (≤0.1%) infected HL-60F and no infected HL-60J cells were recorded under the same experimental conditions in untreated control cultures. HGE continuously propagated in NaVPA-supplemented HL-60F cultures remained infectious to mice at least up to the 95th passage (12 months). NaVPA can thus facilitated continuous propagation of granulocytic ehrlichiae in cell cultures without a substantial loss of infectiveness. This revised version was published online in July 2006 with corrections to the Cover Date.     

Study of high- and low-current-configuration homes from the 1988 Denver Childhood Cancer Study

An epidemiological study conducted by Savitz et al. reported that residential wire codes were more strongly associated with childhood cancer than were measured magnetic fields, a peculiar result because wire codes were originally developed to be a surrogate for residential magnetic fields. The primary purpose of the study reported here, known as the Back to Denver (BTD) study, was to obtain data to help in the interpretation of the original results of Savitz et al. The BTD study included 81 homes that had been occupied by case and control subjects of Savitz et al., stratified by wire code as follows: 18 high current configuration (HCC) case homes; 20 HCC control homes; 20 low current configuration (LCC) case homes; and 23 LCC control homes. Analysis of new data acquired in these homes led to the following previously unpublished conclusions. The home-averaged (i.e., mean of fields measured in subjects' bedrooms, family/living rooms, and rooms where meals normally eaten) spot 60 Hz, 180 Hz, and harmonic (i.e., 60-420 Hz) magnetic fields were associated with wire codes. The 180 Hz and harmonic components, but not the 60 Hz component, were associated with case/control status. Measured static magnetic fields were only weakly correlated (rapproximately 0.2) between rooms in homes. The BTD data provide little support for, but are too sparse to definitively test, the 1995 resonance hypothesis proposed by Bowman et al. Case and control homes had similar concentrations of copper in their tap water. Copper concentration was not associated with wire codes nor with the level of electric current carried by a home's water pipe. These results of the BTD study suggest that future case/control studies investigating power frequency magnetic fields might wish to include measurements of 180 Hz or harmonic magnetic fields in order to examine their associations (if any) with disease status.     

No effect of extremely low-frequency magnetic field observed on cell growth or initial response of cell proliferation in human cancer cell lines

An effect on the tumor promotion process, as represented by accelerated cell growth, has been indicated as one example of areas that demonstrate the possibility of biological effects of extremely-low frequency magnetic fields. We, therefore, exposed the five cell lines (HL-60, K-562, MCF-7, A-375, and H4) derived from human tumors to a magnetic field for 3 days to investigate the effects on cell growth. Prior to exposure or sham exposure, the cells were precultured for 2 days in low serum conditions. The number of growing cells was counted in a blind manner. To investigate the effect on the initial response of cell proliferation, two cell lines were synchronized in G1 phase by serum starvation and then exposed to a magnetic field for 18 h (H4 cells) or 24 h (MCF-7 cells), both with and without serum stimulation. The rate of DNA synthesis, taken as a measure of the cell proliferation, was determined by following the incorporation of [(3)H]-thymidine into the DNA. Three different magnetic field polarizations at both 50 and 60 Hz were used: linearly polarized (vertical); circularly polarized; and an elliptically polarized field. Magnetic field flux densities were set at 500, 100, 20 and 2 microT (rms) for the vertical field and at 500 microT (rms) for the rotating fields. No effect of magnetic field exposure was observed on either cell growth or the initial response of cell proliferation.