Expression of CD38 in Human Promyelocytic Leukemia HL-60 Cell Line during Differentiation by Niacin-related Compounds

It was found that three niacin-related compounds, isonicotinic acid, nicotinamide, and nicotinamide N-oxide, induced granulocytic differentiation in HL-60 cells. We investigated the expression of CD38, which catalyzes the synthesis of cyclic ADP-ribose, a Ca²⁺ mobilizer, during differentiation by niacin-related compounds. It was found that CD38 was induced by isonicotinic acid, whereas nicotinamide and nicotinamide N-oxide containing an amino group did not induce it. The difference in expression of CD38 may provide some useful information for the elucidation of the mechanisms of cell differentiation.     

ATRA-induced HL-60 myeloid leukemia cell differentiation depends on the CD38 cytosolic tail needed for membrane localization, but CD38 enzymatic activity is unnecessary

Leukocyte antigen CD38 expression is an early marker of all-trans retinoic acid (ATRA) stimulated differentiation in the leukemic cell line HL-60. It promotes induced myeloid maturation when overexpressed, whereas knocking it down is inhibitory. It is a type II membrane protein with an extracellular C-terminal enzymatic domain with NADase/NADPase and ADPR cyclase activity and a short cytoplasmic N-terminal tail. Here we determined whether CD38 enzymatic activity or the cytoplasmic tail is required for ATRA-induced differentiation. Neither a specific CD38 ectoenzyme inhibitor nor a point mutation that cripples enzymatic activity (CD38 E226Q) diminishes ATRA-induced differentiation or G1/0 arrest. In contrast a cytosolic deletion mutation (CD38 Δ11–20) prevents membrane expression and inhibits differentiation and G1/0 arrest. These results may be consistent with disrupting the function of critical molecules necessary for membrane-expressed CD38 signal transduction. One candidate molecule is the Src family kinase Fgr, which failed to undergo ATRA-induced upregulation in CD38 Δ11–20 expressing cells. Another is Vav1, which also showed only basal expression after ATRA treatment in CD38 Δ11–20 expressing cells. Therefore, the ability of CD38 to propel ATRA-induced myeloid differentiation and G1/0 arrest is unimpaired by loss of its ectoenzyme activity. However a cytosolic tail deletion mutation disrupted membrane localization and inhibited differentiation. ATRA-induced differentiation thus does not require the CD38 ectoenzyme function, but is dependent on a membrane receptor function.     

Induction of Differentiation in Human Promyelocytic Leukemia HL-60 Cell Line by Niacin-related Compounds

Water-soluble vitamin, niacin, and its related compounds were examined for their differentiation-inducing activity in human promyelocytic leukemia cells (HL-60). Among the compounds, which inhibited cell proliferation measured by MTT assay, isonicotinic acid, nicotinamide N-oxide, and nicotinamide induced NBT reducing activity. HL-60 cells were differentiated into granulocyte-like cells by these compounds, judging from morphological changes and loss of nonspecific esterase activity. The differentiation-inducing activity of water-soluble vitamin and its related compounds suggest that these compounds may be applicable for medical use.     

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.     

Molecular Mechanisms of Apoptosis in HL-60 Cells Induced by a Nitric Oxide-Releasing Compound

Nitric oxide (NO) generated from 1-hydroxy-2-0×0-3, 3-bis(2-aminoethyl)-l-triazene (NOC 18), an NO-releasing compound, induced monocytic differentiation of human promyelocytic leukemia HL-60 cells as assessed by expression of nonspecific esterases and morphologic maturation. Simultaneously, DNA fragmentation and morphological alterations typical of apoptosis were also induced. To investigate the mechanisms of apoptosis during differentiation of HL-60 cells induced by NO, the endogenous levels of Bcl-2 and Bax were assessed by immunoblotting. Treatment of cells with NOC 18 slightly reduced the level of Bcl-2 followed by Bax. These changes might be involved in the induction of apoptosis. The involvement of the activation of the interleukin-lβ converting enzyme (ICE) family of proteases (caspases), such as ICE and CPP32, in the pathways was also investigated. CPP32, but not ICE, was strongly activated in response to NOC 18 stimulation, thereby implicating CPP32-like activity in the induction of apoptosis. Moreover, the possible involvement of tyrosine phosphorylation in apoptosis was investigated. Pretreatment of cells with herbimycin A, an inhibitor of tyrosine kinases, suppressed DNA fragmentation and CPP32-like activity, whereas pretreatment with vanadate, an inhibitor of tyrosine phosphatases, enhanced both parameters, suggesting that tyrosine phosphorylation might be involved in the pathways of apoptosis in HL-60 cells induced by NO.     

Nuclear CD38 in retinoic acid-induced HL-60 cells

The cell surface antigen, CD38, is a 45-kDa transmembrane protein which is predominantly expressed on hematopoietic cells during differentiation. As a bifunctional ectoenzyme, it catalyzes the synthesis of cyclic ADP-ribose (cADPR) from NAD(+) and hydrolysis of either NAD(+) or cADPR to ADP-ribose. All-trans-retinoic acid (RA) is a potent and specific inducer of CD38 in myeloid cells. In this report, we demonstrate that the nuclei of RA-treated human HL-60 myeloblastic cells reveal enzymatic activities inherent to CD38. Thus, GDP-ribosyl cyclase and NAD(+) glycohydrolase activities in the nuclear fraction increased very significantly in response to incubation with RA. With Western blotting, we detected in the nuclear protein fraction from RA-treated cells a approximately 43-kDa protein band which was reactive with the CD38-specific monoclonal antibody OKT10. The expression of CD38 in HL-60 nuclei was also shown with FACScan analysis. RA treatment gave rise to an increase in in vitro ADP ribosylation of the approximately 43-kDa nuclear protein. Moreover, nuclei isolated from RA-treated HL-60 cells revealed calcium release in response to cADPR, whereas a similar response was not observed in control nuclei. These results suggest that CD38 is expressed in HL-60 cell nuclei during RA-induced differentiation.     

3H]Ouabain binding and 86 rubidium uptake during the dimethyl sulfoxide-induced differentiation of human promyelocytic leukemia HL-60 cells

When HL-60 cells were induced to differentiate into granulocyte-like cells by culture in the presence of 1.3% dimethyl sulfoxide, an increase in the rate of ouabain-sensitive 86Rb transport was observed during the first 6 h followed by a constant decrease which became stable on day 4 at about 40% of control level. By contrast, the number of ouabain binding sites remained constant during the first 24 h then decreased with the same kinetics as that of the 86Rb transport rate. These results suggest that alterations in ionic fluxes, through activation of the sodium pump, are part of the early events resulting in HL-60 cell differentiation.     

Changes in the Susceptibility of 12-O-Tetradecanoylphorbol 13-Acetate (TPA)-Treated HL-60 Cells to Staphylococcal Leukocidin

Susceptibility of human promyelocytic leukemia HL-60 cells to staphylococcal leukocidin following treatment of cells with 12-O-tetradecanoylphorbol 13-acetate (TPA) was examined. TPA treatment for 6 hr rendered the cells very resistant transiently to leukocidin. There was no change in binding of leukocidin to the cells, but leukocidin-induced ⁴⁵CaCl₂ influx, phospholipase A₂ and C activities were inhibited. Further incubation with TPA rendered the cells sensitive again and then more sensitive than original HL-60 cells following increase of the binding, and leukocidin-induced activities described above appeared again. Those cells treated with TPA for more than 18 hr started to differentiate to macrophages morphologically and functionally. These data suggest that the differentiated cells were more sensitive than original HL-60 cells because of increased binding of leukocidin and that treatment of TPA for 6 hr may transiently impair the signal transduction system of leukocidin after binding of leukocidin to the specific receptor of the cell membrane. Using these TPA-treated cells, it was shown in this report that calcium influx, phospholipase A₂ and C activities were important to induce cytotoxic action of leukocidin after binding of leukocidin to specific receptors on the cells.     

Changes in the gene expression of C-myc and CD38 in HL-60 cells during differentiation induced by nicotinic acid-related compounds

Changes in gene expression levels of c-myc and CD38 were examined during the differentiation of HL-60 cells to granulocytes due to three nicotinic acid-related compounds. CD38 expression was increased by isonicotinic acid and all-trans-retinoic acid (ATRA). Nicotinamide and nicotinamide N-oxide drastically decreased c-myc expression, but isonicotinic acid had no effect, suggesting that these compounds differentiate HL-60 to granulocytes through different pathways. These results should provide useful information as to the mechanisms of cell differentiation.     

STAT3反义核酸对HL-60细胞周期及c-myc mRNA表达的影响

实验分反义组、无义组、脂质体组、空白组,转染后,应用四唑盐（MTT）比色法分析细胞增殖率,流式细胞仪检测细胞周期,RT-PCR检测细胞中STAT3 mRNA和c-myc mRNA的表达。探讨STAT3反义寡核苷酸对白血病细胞HL-60细胞周期及c-myc的影响。STAT3 ASODN抑制HL-60细胞增殖呈时间和浓度依赖性;反义寡核苷酸作用后,G0/G1期细胞明显减少,S期细胞增多,细胞周期进程受到明显阻滞;反义寡核苷酸作用48h后细胞内STAT3mRNA及c-myc mRNA的表达水平下降,与各对照组比较有显著性差异（P〈0.05）。STAT3 ASODN能够明显抑制HL-60细胞增殖,并能阻滞HL-60细胞于G0/G1期、并下调c-myc mRNA的表达。     

An Artificial Protein with the Biological Activity of the Differentiation Factor for the HL-60 Cell Line of Human Promyelocyte Leukemia

The ABB-df artificial protein was prepared by inserting the TGENHR biologically active peptide corresponding to the 41–46 sequence of the differentiation factor for the HL-60 cell line of the human promyelocyte leukemia into the N-terminus of the polypeptide chain of albebetin, an artificial protein with the preset structure. The ABB-df protein was found to induce the differentiation of HL-60 cells and to inhibit their proliferation; its efficiency was almost the same as that of the starting peptide. According to CD spectroscopy, the inclusion of the peptide fragment into albebetin exerts virtually no effect on the regular secondary structure of albebetin.     

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.     

外泌体分泌在1,4-苯醌诱导的HL-60细胞凋亡中起保护作用

慢性苯暴露损害造血系统,可引起再生障碍性贫血,甚至白血病。外泌体(exosomes)是细胞分泌的纳米级膜泡,在许多生理和病理过程中发挥重要作用。然而,苯及其代谢产物对外泌体分泌的影响仍不清楚。本研究旨在观察苯的活性代谢产物1,4-苯醌(1,4-benzoquinone,1,4-BQ)能否引起人早幼粒白血病细胞HL-60外泌体分泌量的变化以及外泌体释放在1,4-BQ诱导的细胞凋亡中的作用。应用不同浓度1,4-BQ处理细胞24 h,超高速离心法提取细胞培养基中的外泌体,结果发现1,4-BQ能促进外泌体分泌,呈剂量反应关系。进一步应用外泌体抑制剂GW4869抑制外泌体分泌,流式细胞仪检测细胞凋亡率、蛋白质免疫印迹法检测抑凋亡蛋白质Bcl-2、凋亡通路关键蛋白质cleaved caspase-9和cleaved caspase-3的表达,探讨外泌体分泌对1,4-BQ所致细胞凋亡的影响。结果显示:与对照组相比,1,4-BQ单独处理组的凋亡率、Bcl-2、cleaved caspase-9和cleaved caspase-3的表达均显著增高(P<0.05),而1,4-BQ+GW4869组的凋亡率及凋亡相关蛋白质的表达均显著高于1,4-BQ单独处理组(P<0.05),表明抑制外泌体分泌可增加1,4-BQ诱导的细胞凋亡。综上表明,1,4-BQ能促进外泌体分泌,并在1,4-BQ诱导的细胞凋亡中起保护作用。本研究为了解苯的毒性效应和毒性机制提供了新的实验证据。     

CD11b/DNA双参数流式细胞术检测HL-60细胞分化的细胞周期

目的采用双参数流式细胞术研究全反式维甲酸(alltransretinoidacid,ATRA)诱导人类急性早幼粒白血病细胞HL-60细胞分化的细胞周期。方法HL-60细胞经分化诱导剂ATRA(终浓度为1μmol/L)诱导不同时间点后,利用CD11b/DNA双参数流式细胞术同时检测分化细胞表面抗原CD11b的表达及分化细胞DNA含量。结果HL-60细胞经ATRA诱导后,细胞表面分化抗原CD11b表达明显升高,细胞阻滞于G0/G1期,且CD11b阳性细胞主要位于G0/G1期。结论CD11b/DNA双参数流式细胞术能简便,快速,直观地检测细胞分化的细胞周期。     

Identification of Novel Genes Differentially Expressed in PMA-induced HL-60 Cells Using cDNA Microarrays

Identification of normal growth and differentiation-inducing proteins and their interaction in normal development have made it possible to elucidate the molecular basis of normal development and the mechanisms uncoupling growth and differentiation during tumor development. The development of cancer and the experimental reversal of tumorigenicity are accompanied by complex changes in patterns of gene expression. cDNA microarrays provide a powerful tool for studying these phenomena. In the present study, a high-density microarray of human cDNA elements was used to search for differences in gene expression associated with differentiation of human promyelic leukemia HL-60 cells. Microarrays containing 3,063 human cDNAs were printed on glass slides with high-speed robotics. These DNA chips were used to quantitatively monitor differential expression of the cognate human genes using a highly sensitive two-color hybridization assay. The identification of known and novel phorbol ester-regulated genes in hematopoietic progenitor cells demonstrates the sensitivity of the assay.     

Induction of the differentiation of human HL-60 promyelocytic leukemia cell line by succinoyl trehalose lipids

Four analogs of succinoyl trehalose lipid-3 (STL-3)with saturated even-number or odd-number carbonchains, and unsaturated or halogenated fatty acidswere examined for their ability to inhibit the growthand induce the differentiation of HL-60 humanpromyelocytic leukemia cells. The optimalconcentration of STL-3 at which such activities wererecognized was closed to the critical micelleconcentration of STL-3. Analog of STL-3 witheven-number or odd-number carbon chain and unsaturatedfatty acids strongly inhibited growth and induced thedifferentiation of HL-60 cells, as evaluated in termsof nitroblue tetrazilium-reducing activity and theappearance of the CD36 antigen. An analog of STL-3with halogenated fatty acids significantly inhibitedproliferation but only induced the differentiation ofHL-60 cells. Our results indicate that the effects ofSTL-3 and its analogs on HL-60 cells depend on thestructure of the hydrophobic moiety of STL-3.These authors contributed equally to this work     

Luminol and diazoluminomelanin as indicators of HL-60 cell differentiation

Summary This paper describes use of a novel substituted melanin which is useful in detection of differentiating leukemia cells and their membranes. Comparisons of luminol-(5-amino-2,3-dihydro-1,4-phthalazinedione) and diazoluminomelanin (DALM)-mediated chemiluminescence (CL) were made with various types of differentiated and undifferentiated HL-60 whole cells, cell lysates, and membrane fractions. Luminol had a greater CL response than DALM with HL-60 promyelocytic stem cells and differentiated macrophage-like or neutrophil-like whole cell and cell lysate preparations. However, DALM showed markedly greater CL than luminol for membrane fractions derived from each cell type. The greatest luminol-dependent CL was observed for cell types high in myeloperoxidase (MPO). The greatest DALM-mediated CL was seen with cell types that are high in MPO or strong producers of superoxide (O_2-) anions. In some cases, significant differences in CL could also be distinguished on the basis of inducing agent used [i.e. dimethylsulfoxide, all-trans retinoic acid or 12-o-tetradecanoylphorbol-13-acetate]. Both luminol- and DALM-dependent CL were strongly inhibited by preincubation of cellular preparations with 3-amino-l-tyrosine (a component of DALM). Taken together, these data suggest that the reaction mechanism of luminol favors interaction with cytoplasmic MPO whereas that of DALM favors membrane interactions. Thus, both reagents may be of use in assays to detect differentiating leukocytes or their cellular components.     

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.