microRNA-181b targets MLK2 in HL-60 cells

microRNAs(miRNAs) play critical roles in many different cellular processes,including metabolism,apoptosis,differentiation and development.We showed miR-181b to be highly expressed in acute myeloid leukemia(AML).Furthermore,miR-181b contributed to proliferation of AML cells by targeting MLK2.Our results demonstrated that miR-181b plays an important role in the biology of AML and may be useful in developing therapies targeting miRNAs.     

Arsenic suppresses necrosis induced by selenite in human leukemia HL-60 cells

Selenium, an essential trace element for humans, has been shown to have anticancer effects. Arsenic, a possibly essential ultratrace element for humans, has been used in the treatment of leukemia. Anticancer effects of selenium and arsenic have been related to their ability to induce apoptosis. Because humans are exposed to diverse trace elements simultaneously, it is important to learn their interrelationship. In this study, we demonstrate that sodium selenite (Na₂SeO₃) causes apoptosis at 3 μM and necrosis at high concentrations (>3 μM) in HL-60 cells. Similarly, both sodium arsenite (NaAsO₂) at 50 μM and sodium arsenate (Na₂HAsO₄) induce apoptosis at 500 μM and necrosis at higher concentrations (>50 μM and >500 μM, respectively) in HL-60 cells. Arsenite/arsenate, but not selenite, enhances AP-1 DNA-binding activity. This finding indicates different mechanisms through which apoptosis is induced by these two elements. Interestingly, we observed that HL-60 cell necrosis induced by a high concentration (>3 μM) of selenite was essentially inhibited by arsenic (50 μM of NaAsO₂ or 500 μM of Na₂HAsO₄), which resulted in a net effect of apoptosis. Because AP-1 DNA-binding activity was not induced in the presence of a combination of necrotic amount of selenite and apoptotic amount of arsenite/arsenate, the observed apoptosis apparently was through the mechanism used by selenite. Our results suggest, for the first time, that the toxic necrotic effect of selenite can be neutralized by arsenite/arsenate at the cellular level. The U.S. Department of Agriculture, Agricultural Research Service, Northern Plains Area, is an equal opportunity/affirmative action employer and all agency services are available without discrimination. Mention of a trademark or proprietary product does not constitute a guarantee or warranty of the product by the U.S. Department of Agriculture and does not imply its approval to the exclusion of other products that may also be suitable.     

Apoptosis of human leukemic HL—60 cells induced to differentiate by treatment with RA or DMSO

In present study,we studied the effect of all-trans retinoic acid(ATRA)and dimethylsulfoxide(DMSO)on the induction of apoptosis in HL-60 cell line.Based on morphological changes by Hochest 33342 staining and identification of internuclesomal NDA celeavage by gel electrophoresis,we observed aberrant nuclear chromatin condensation and ladder-like pattern of DNA degradation. Using Flow Cytometric method.We found sub-G1 peak in RA-treated HL-60 cells starting 5 to 6d after the initiation of the treatment However,Such an obvious apoptotic peak was not identified in DMSO-differentiated cells.Combining the research accomplished before.our study approves further that apoptosis could be a common mode of death of terminally differentiated HL-60 cells.     

Effect of DNA methylation on protein-DNA interaction of HL-60 cells

HL-60 cells have been induced with differentiation index 16 % by S-adenosyl-L-rnethionine (SAM) as inducer in the presence of optimum conceptration of 10 μmol/L. The methylation level of genorne DNA determined by HPLC is increased during cell differentiation. When restriction endonuclease Hae Ⅲ, Sma I, Sal I, XhoI and Hind Ⅲ which are sensitive to 5-methylcytosine were used to cleave the genorne DNA, a resistance effect was found. The interaction between DNA and DNA binding proteins is changed by using gel retarding test.     

苏云金芽孢杆菌Bt9875晶体蛋白诱导HL-60细胞凋亡

[目的]研究苏云金芽孢杆菌(Bacillus thuringiensis,Bt)Bt9875菌株晶体蛋白对人急性髓细胞性白血病细胞HL-60的影响.[方法]采用MTT比色、荧光显微观察、DNA凝胶电泳、流式细胞术等方法来检测不同浓度的Bt9875晶体蛋白处理后HL-60细胞的凋亡特征.[结果]Bt9875晶体蛋白对HL-60细胞的生长具有明显的抑制作用,且随着蛋白质浓度的增加对HL-60细胞生长抑制愈加明显,而对正常人外周血单个核细胞(PBMC)无作用;荧光显微镜下观察发现经该蛋白作用后HL-60细胞核的形态呈现凋亡特征;流式细胞术分析表明,HL-60细胞经100 μg/mL晶体蛋白作用后,凋亡率达到52%;琼脂糖凝胶电泳显示细胞DNA呈梯状降解.[结论]初步证明了Bt9875晶体蛋白在体外能够明显抑制HL-60细胞的增长,并诱导其凋亡,这为苏云金芽抱杆菌晶体蛋白的应用开创了新的思路.     

Plasma membrane redox system during HL-60 induced differentiation

Summary HL-60 cells were induced to differentiate by exposure to TPA or 1,25-dihydroxyvitamin D₃ (calcitriol). Induction with TPA was in parallel with a modulation of transmembrane redox system. After addition of 2 ng/m1 TPA, transient increases in ferricyanide reductase activity, NAD(H) intracellular levels and short-term response of NAD(H) to 0.4 mM ferricyanide were observed. The role of ascorbate on the differentiation induced by calcitriol also was studied. When HL-60 cells were exposed to 10^–8 M calcitriol in the presence of 0.2 mM ascorbate, specific differentiation markers as NBT reduction or surface antigen CD11b increased significantly with respect to values obtained from treatments with calcitriol alone.     

Altered expression of nuclear matrix proteins in etoposide induced apoptosis in HL－60 cells

INTRODUCTIIONThe nuclear matrix is an essential component ofthe nucleus which is important for the nuclear structural integrity and specific genomic functions[1, 2].Several articles have reported that the nuclear matrix, as a higher order framework structures, mightbe disassembled du-ring the apoptotic process[3-5].Accordingly3 nuclear lamins A/C or B have beenfound to decrease in apoptotic thymocytes[6], Tcells[7], and carcinoma cell line[8, 9]. The nucleolar protein B23, an obscure ma…     

Glutamine promotes colony formation in bone marrow and HL-60 cells; Accelerates myeloid differentiation in induced HL-60 cells

Summary Several studies indicate that glutamine is a critical requirement for growth of cultured cells. The present studies describe the effect of deprivation of glucose or glutamine on mouse bone marrow cell or HL-60 cell colony formation in soft agar. The mouse bone marrow cells were induced to undergo granulocyte/macrophage type differentiation by colony-stimulating factor. Glutamine, but not glucose, was found to be an indispensable metabolite for the cloning of HL-60 cells or differentiated mouse bone marrow cells. In addition, the effect of glucose or glutamine on the rate of differentiation of dimethylsulfoxide (DMSO)-induced HL-60 cells in liquid culture was studied. Glutamine was found to be superior to glucose in its ability to support the proliferation and myeloid differentiation of HL-60 cells. When an optimal concentration of DMSO was used, the rate of differentiation of induced HL-60 cells was found to be a function of the concentration of glutamine. In addition to these studies glutamine utilization and product formation was studied in induced and uninduced HL-60 cells after 60 min incubation with 1 mM initial glutamine concentration. The fractional distribution of the glutamine carbon into its metabolic products remained unchanged in induced versus uninduced HL-60 cells. However, the rate of utilization of glutamine and product formation by terminally differentiated HL-60 cells was less than the rate of utilization of glutamine by undifferentiated HL-60 cells. The data do not explain the role of glutamine in the complex process of differentiation but establish the critical requirements for glutamine, but not glucose, in myelopoiesis. This work has been supported by USPHS Grants AM 31624 and CA 00859 and a Faculty Research Grant from Texas College of Osteopathic Medicine.     

Humic acid induces apoptosis in human premyelocytic leukemia HL-60 cells

It has been shown that humic acid (HA), a phenolic polymer, exhibits pro-oxidant and cytotoxic effects. In this study, HA induction of apoptosis was studied using cultured human premyelocytic leukemia HL-60 cells. Treatment at a range of HA concentrations (50-400 microg/ml) resulted in dose-and time-dependent sequences of events marked by apoptosis, as demonstrated through by apoptotic features such as loss of cell viability, chromatin condensation, and internucleosomal DNA fragmentation. This HA-induced apoptosis in the HL-60 cells was mainly associated with the release of cytochrome c from the mitochondria. Furthermore, apoptosis in the HL-60 cells was accompanied by the activation of caspase-3 and the specific proteolytic cleavage of poly (ADP-ribose) polymerase (PARP), a major component in the apoptotic cell death mechanism. Although the HA-induced apoptosis was associated with Bax protein levels, negligible Bcl-2 reduction was observed. Analysis of the data reported herein reveals that HA exerts antiproliferative action and growth inhibition on HL-60 cells through induction of apoptosis, which may have anticancer properties potentially useful for the development of new drug products.     

Alkyllysophospholipid ET-18-OCH3 acts as an activator of protein kinase C in HL-60 cells

HL-60 cells are very sensitive to the cytotoxic action of ether lipids. Several hypotheses have been proposed to explain this cytotoxicity. We investigated the influence of the alkylphospholipid ET-18-OCH₃ on the activity of protein kinase C. HL-60 cells were incubated with ET-18-OCH₃ at a concentration of 20 μg/ml for 4 h. After the incubation the membrane fraction of the HL-60 cells was isolated and the activity of protein kinase C was determined while it was still associated with the membrane, using the synthetic peptide substrate [Ser²⁵]-protein kinase C (19–31) as a protein kinase C specific substrate. The activity of the membrane-bound protein kinase C was increased in HL-60 cells treated with ET-18-OCH₃ compared to untreated HL-60 cells. The increase in protein kinase C activity was not a consequence of translocation and appeared to be additive to the effect of the phorbol ester 12-myristate 13-acetate. In contrast, solubilized protein kinase C from HL-60 cells could be inhibited or stimulated in vitro by ET-18-OCH₃, dependent on the mode of addition of ET-18-OCH₃ and phospholipids.     

A biologically active fragment of the differentiation factor of the HL-60 line cells: Identification and properties

Six-membered peptide fragment TGENHR (HLDF-6) was identified in the HL-60 cell culture of human promyelocyte leukemia treated with retinoic acid when studying the differentiation factor HLDF of this cell line. HLDF-6 retains the ability of the full-size factor to induce the differentiation and arrest the proliferation of the starting HL-60 cells. It was shown that the synthetic peptide HLDF-6 has no specific receptors on the surface of the HL-60 cells but can affect the binding of interleukin IL-1β, a cytokine involved in proliferation, to the cell surface. It was found on a model of transplantable NSO myeloma that HLDF-6 has an antitumor activity.     

Induction of apoptosis by pterocarpans from Platymiscium floribundum in HL-60 human leukemia cells

(+)-2,3,9-Trimethoxy-pterocarpan (1) (+)-3,9-dimethoxy-pterocarpan [(+)-homopterocarpin] (2), (+)-3-hydroxy-9-methoxy-pterocarpan [(+)-medicarpin] (3) and (+)-3,4-dihydroxy-9-methoxy-pterocarpan [(+)-vesticarpan] (4) are cytotoxic pterocarpans isolated from the native Brazilian plant Platymiscium floribundum. The purpose of the present study was to examine whether induction of apoptosis and/or inhibition of DNA synthesis is involved in the cytotoxicity of these pterocarpans in human leukemia cells. The effect on cell viability determined using the trypan exclusion assay revealed that all compounds tested reduced the number of viable cells, while only in the presence of 3 and 4, there was an increase of nonviable cells. The analysis of membrane integrity and morphological modifications by flow cytometry in the presence of these two compounds indicated that treated cells undergo necrosis, while 1 and 2 trigger apoptosis. DNA synthesis seemed to be affected since BrdU incorporation was inhibited in a dose-dependent manner in the presence of all tested compounds. Pterocarpan treatment also induced an increase in the amount of subdiploid DNA, indicating internucleosomal DNA breakdown, mitochondrial depolarization and caspase-3 activation, which indicate apoptosis induction.     

Curcumin abolishes apoptosis resistance of calcitriol-differentiated HL-60 cells

Exposure of HL-60 cells to 1,25-dihydroxyvitamin D(3) (calcitriol) induces their differentiation into monocytes. This terminal differentiation is associated with acquired resistance to many proapoptotic stimuli. Here we show that differentiated HL-60 cells undergo apoptosis upon curcumin treatment although they retain resistance to apoptosis induced by a topoisomerase poison - etoposide. Curcumin induced changes of nuclear morphology, DNA fragmentation, release of cytochrome c as well as caspase activation in both differentiated and undifferentiated cells. Experiments performed in other laboratories suggested that curcumin initiates apoptosis by DNA damage that results from topoisomerase II poisoning. We measured gammaH2AX expression, a marker of DNA double strand breaks, in both undifferentiated and differentiated HL-60 cells treated with curcumin or etoposide. In etoposide-treated undifferentiated cells early gammaH2AX expression correlated with initiation phase of apoptosis. In contrast, in curcumin-treated cells gammaH2AX expression correlated with apoptotic DNA fragmentation, which is characteristic for the execution phase of apoptosis. Our experiments show that curcumin overcomes the resistance of calcitriol-differentiated HL-60 cells to DNA-damage-induced apoptosis by activating other cell signaling pathways leading to cell death of HL-60.     

Identification and expression of haponin, a new protein from HL-60 cells

We found a new protein haponin (an HLDF-like protein) in promyelocyte HL-60 cells that is immunoreactive to polyclonal antibodies against HLDFβ. Determination of the partial primary structure of the protein allowed us to reveal an immunogenic peptide of haponin and, on the basis of the amino acid sequence of this peptide, the degenerate primers were synthesized, which enabled us to clone the full-size cDNA of haponin. The stable heterologous expression of this cDNA in E. coli cells (Rosetta? strain) was obtained. Preparations of natural and recombinant proteins exhibited antigenic cross-reactivity to polyclonal antibodies against this peptide.     

Characteristics of U-937 and HL-60 leukemia cells differentiated by spinach extract

Some characteristics of U-937 and HL-60 leukemia cell lines treated with a fraction of non-dialyzable extract of spinach are reported. The absorbed fraction separated by a DEAE-Tyopearl 650 column chromatography of the non-dialyzable extract induced NBT reducing activity of U-937 and HL-60 cells. This fraction also induced substrate adhesion of U-937 cells, and the non-specific esterase activity of HL-60 cells. The expression of CD11b, CD11c and CD36 antigens on the U-937 cell surface was enhanced by the treatment with the fraction, whereas CD24 antigen was not. The treatment of HL-60 cells with the fraction also induced the expression of CD11b and CD11c antigens, but CD24 and CD36 were not expressed. These results indicated that the non-dialyzable extract of spinach induced immature differentiation of U-937 and HL-60 cells into monocyte/macrophages.Abbreviations NBT nitroblue tetrazolium - TPA 12-O-tetradecanoyl-phorbol-13-acerate - PBS phosphate buffered saline - FITC fluorescein isothiocyanate     

Altered expression profiles of microRNAs during TPA-induced differentiation of HL-60 cells

MicroRNAs (miRNAs) are highly conserved small non-coding RNAs that regulate gene expression through translational repression by base-pairing with partially complementary mRNAs. The expression of a set of miRNAs is known to be regulated developmentally and spatially, and is involved in differentiation or cell proliferation in several organisms. However, the expression profiles of human miRNAs during cell differentiation remain largely unknown. In an effort to expand our knowledge of human miRNAs, we investigated miRNAs during 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced differentiation of human leukemia cells (HL-60) into monocyte/macrophage-like cells. Several hundred RNAs ranging from 18 to 26 nucleotides were isolated from HL-60 cells with or without TPA-induction, and subsequently characterized by sequencing, database searching, and expression profiling. By removing non-miRNA sequences, we found three novel and 38 known miRNAs expressed in HL-60 cells. These miRNAs could be further classified into subsets of miRNAs that responded differently following TPA induction, either being up-regulated or down-regulated, suggesting the importance of regulated gene expression via miRNAs in the differentiation of HL-60 cells.     

灰毡毛忍冬次皂苷乙抑制白血病细胞HL-60的增殖及其机制研究

研究了灰毡毛忍冬次皂苷乙(MB)在体外对白血病细胞HL-60和结肠癌细胞LOVO增殖的抑制作用,并初步探讨其分子机制。采用MTT法检测MB的增殖抑制作用;利用RT2ProfilerTMPCR Array芯片实时定量PCR扩增肿瘤发生中84个关键基因。结果表明MB对两种肿瘤细胞生长均有抑制作用,且对HL-60效果更好。以HL-60作为细胞模型,总共发现差异基因20个,其中上调基因14个,下调基因6个,主要作用是阻滞细胞周期和降低细胞侵袭转移。     

Induction of Growth Inhibition and Differentiation of Human Leukemia HL-60 Cells by a Tunisian Gerboui Olive Leaf Extract

Cancer protection associated with the consumption of olive products is well established, but not for leukemia. The protective effects of olive (Olea europaea L.) leaves were investigated by incubating human promyelocytic leukemia HL-60 cells with olive leaf extracts (OLEs) from seven principal Tunisian olive varieties, namely, Chemchali, Chemlali, Chétoui, Gerboui, Sayali, Zalmati and Zarrazi. The results showed significant growth inhibition of HL-60 cells incubated for 48 h with a 100-fold dilution of each OLE which had been obtained by incubating 10 g of dried leaves in 100 ml of 70% ethanol for one week with subsequent ultrafiltration. DNA fragmentation was observed in the cells incubated for 19 h with a 100-fold dilution of the Chemchali, Chemlali and Zalmati extracts. The results of a nitroblue tetrazolium (NBT) assay revealed NBT reduction, a differentiation marker, by the OLE-treated cells after an overnight incubation. The Gerboui extract showed the highest NBT reduction ability at more than 90%. An HPLC analysis revealed the presence of apigenin 7-glucoside in the extract, which was found in subsequent experiments to be responsible for the Gerboui extract-mediated cell differentiation.     

Magnesium restriction induces granulocytic differentiation and expression of P27Kip1 in human leukemic HL-60 cells

When cultured in Mg restricted medium, human leukemic HL-60 cells develop morphological and functional granulocytic differentiation. In 0.03 mM Mg, cells display the distinctive features of differentiation, without appreciable inhibition of proliferation. In 0.01 mM Mg, cells show terminal differentiation, accompanied by clear inhibition of proliferation. Such cells accumulate in the G0/G1 phase and subsequently die via apoptosis, similar to HL-60 cells that have been induced to differentiate by DMSO. These phenotypic changes are associated with a marked increase in the expression level of the cyclin dependent kinase inhibitor p27^Kip1. Cyclin E expression is also slightly increased in Mg restricted cells, whereas no changes are observed in the expression level of cyclin D1. We also show that during differentiation cell total Mg decreases, whereas [Mg²⁺]_i increases in both Mg-depleted and DMSO-treated cells. These data suggest that the maturation process is paralleled by a redistribution of intracellular Mg, leading to a shift from the bound to the free form. These changes could modulate the kinetics of Mg-dependent enzyme(s) that are involved in the control of the differentiation pathway. We propose that this model may represent an useful tool for the study of the mechanisms of cell differentiation and related events, such as aging and death. J. Cell. Biochem. 70:313–322, 1998. © 1998 Wiley-Liss, Inc.