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.     

C-FMS dependent HL-60 cell differentiation and regulation of RB gene expression

The dependence of induced myelomonocytic cell differentiation, and regulation of the RB tumor suppressor gene during this process, on the c-fms gene product, the CSF-1 lymphokine receptor, was determined in HL-60 promyelocytic leukemia cells. Adding a monoclonal antibody with specificity for the c-fms gene product to cells treated with various inducers of myelomonocytic or macrophage differentiation, including retinoic acid and 1,25-dihydroxy vitamin D3, inhibited the rate of differentiation. During the period of inducer treatment usually preceding onset of differentiation, longer periods of antibody exposure caused greater inhibition of differentiation. In a stable HL-60 transfectant overexpressing the CSF-1 receptor at the cell surface due to a constitutively driven c-fms trans gene, the rate of differentiation was enhanced compared to the wild type cell, consistent with a positive regulatory role for the CSF-1 receptor. The anti-fms antibody caused much less inhibition of differentiation in the transfectants than in wild type cells, consistent with a larger number of receptors causing reduced sensitivity. During the induced metabolic cascade leading to differentiation, the typical early down regulation of RB gene expression was inhibited by the antibody. The antibody itself caused an increase in RB expression per cell, which offset the decrease normally caused by differentiation inducers (1,25-dihydroxy vitamin D3 and retinoic acid). The changes in RB expression preceded changes in the RB protein to the hypophosphorylated state. Most of the RB protein in proliferating cells was phosphorylated and no significant accumulation of hypophosphorylated RB protein occurred until after onset of GO arrest. Thus the metabolic cascade leading to myelomonocytic differentiation of HL-60 cells appears to be driver by a function of the c-fms protein. Inhibiting that process by attacking this receptor impedes differentiation and also compromises the early down regulation of RB tumor suppressor gene expression which normally precedes differentiation. These findings provide additional support for a potential role for down regulating RB expression in promoting cell differentiation, and suggest the possibility that RB may be either a target or intermediate mediator of CSF-1 actions. © 1993 Wiley-Liss, Inc.     

Cell cycle specific induction of HL-60 cell differentiation and apoptosis by mycophenolic acid

HL-60 cells undergo terminal differentiation and apoptosis in response to different types of sub-toxic and toxic perturbations respectively. The mechanism by which cells sense different amounts of perturbation to activate pathways that lead to the engagement of a relevant biological response is not known. The response of HL-60 cells to treatment with the immunosuppressant mycophenolic acid (MPA), a specific inhibitor of dGTP/GTP-synthesis, allowed quantitation of a metabolic perturbation which triggered a cellular response. 1.5 microM MPA induced 38% terminal differentiation to CD14 positive, early monocyte-like cells and 22% cell death by apoptosis, whereas 3 microM MPA induced 70% apoptosis but no differentiation. Despite the difference in biological outcomes, 72 h exposure to both 1.5 microM and 3 microM MPA caused a similar ( approximately 75%) depletion of total GTP levels. Cells synchronized by centrifugal elutriation were treated with MPA. Elutriated cells were overall less sensitive to the effects of MPA but 3 microM MPA induced significantly less apoptosis and more differentiation in an elutriation-enriched G1-population than in a population normally distributed in the cell cycle, suggesting that the effects of MPA in S-phase may subsequently lead to cell death. However, analysis of apoptosis by using a terminal deoxynucleotidyltransferase assay and measurement of bromodeoxyuridine incorporation showed that apoptosis was engaged in G1. Analysis of the phosphorylation status of the retinoblastoma protein demonstrated that Rb was hypophosphorylated prior to apoptosis and that in apoptotic cells, separated by flow cytometry, Rb protein was absent, presumably due to proteolysis. The loss of Rb protein did not appear to permit transit to S-phase, and was not accompanied by an expression of c-Myc. Surprisingly, therefore, an antimetabolite inducing a loss of GTP brought about cell death by apoptosis in the G1 phase of the cell cycle.     

PpGalNacT2 participating in vanadium-induced HL-60 cell differentiation

The current study demonstrates vanadium plays the role of antitumor, and its antitumor effect is dosage-dependent. N-acetyl-galactosamine-transferase 2 (polypeptide: N-acetyl-α-galactosaminyl-transferases 2, ppGalNAc-T2) is a member of ppGalNAcTs (polypeptide: N-acetyl-α-galactosaminyl-transferases) family, which proves to play a vital role in the tumor emergence and development process. In this study, we focused on ppGalNAc-T2 and vanadium and aimed to determine whether ppGalNAc-T2 is correlated with vanadium’s antitumor effect. We discovered that ppGalNAc-T2 changed with the variation of HL-60 cell growth induced by vanadium at mRNA level. Peanut agglutinin (PNA) is an exogenous lectin. PpGalNacT2 can be indirectly recognized by PNA. By means of flow cytometry and immunofluorescent staining, we found the deviation of PNA binding increased significantly at high concentration vanadium. Then we docked one of the possible compound substances of vanadium onto the body, VO₃ (molecular formula O₁₃V₄, partial vanadate tetramer) and ppGalNAcT2, and simulated them via molecular dynamics, which showed that VO₃ may inhibit the activity of the enzyme by stemming conformational changes of a key loop of ppGalNAcT2. To sum up, our results suggested that ppGalNacT2 participated in vanadium induced HL-60 cell differentiation, which might be able to provide a new mechanism of vanadium’s antitumor effect.     

Control of cell differentiation during proliferation. I. Monocytic differentiation of HL-60 promyelocytes

The cell proliferation relating an uncommitted precursor cell to a differentiated terminal cell has been quantitated. HL-60 promyelocytes, a bipotent precursor cell capable of differentiating along either the myeloid or monocytic pathway, were induced by a human lymphocyte-conditioned medium (CM) to differentiate into macrophage-like cells. The promyelocytes had a generation time of approx. 42 h. Most promyelocytes which differentiated became macrophage-like cells after only one cell division. Some, a minority, underwent more than one division. The time between induction of differentiation and expression of differentiated characteristics could thus be very short. Labelled S-phase promyelocytes could differentiate after traversing S. G2 and undergoing mitosis. Some, approx. 21%, required a subsequent complete cell cycle before differentiating. The data suggest a model in which cells must undergo a S-phase-specific differentiation control event in the presence of CM in order to differentiate in the subsequent G1 phase. This model proposes that a discrete time in S phase exists when cells are susceptible to exogenous regulation directing them to yield differentiated daughter cells.     

dsDNA-stimulated phosphorylation of a 72-kDa nucleoprotein accompanies PMA-induced HL-60 leukemic cell differentiation.

PMA treatment of human leukemic cells resulted in a significant increase in the phosphorylation of a 72-kDa protein, which was abrogated by treating the nuclear extracts with DNase I, but additionally stimulated by adding DNA. To be active, DNA must be double-stranded with an average size of 300 base pairs, but shows no apparent species- or sequence-specificity. NP-72 isolated from control or PMA-treated nuclei with 1 mM ATP lacked phosphorylating activity, suggesting it to be a substrate for a dsDNA-stimulated protein kinase(s). Simultaneous exposure of HL-60 cells to PMA and the protein kinase C inhibitor staurosporine diminished the phosphorylation of NP-72. These data suggest that leukemia cell differentiation is accompanied by the induction and/or activation of a dsDNA-stimulated protein kinase whose protein substrates include NP-72 and whose activity is directly or indirectly influenced by protein kinase C.     

Docosahexaenoic acid-containing phosphatidylethanolamine enhances HL-60 cell differentiation by regulation of c-jun and c-myc expression

18:1/docosahexaenoic acid (DHA)-containing phosphatidylethanolamine (PE) enhanced cell differentiation and growth inhibition of HL-60 induced by dibutyryl cAMP (dbcAMP) in a dose-dependent manner. The combined treatment of 200 μM dbcAMP and 50 μM 18:1/DHA-PE increased the NBT reducing activity, which is as an indicator of cell differentiation, to more than 75% from 40% of cells treated with 200 μM dbcAMP alone. In HL-60 cells treated with 50 μM 18:1/DHA-PE and 200 μM dbcAMP for 24 h, the expression level of c-jun mRNA and c-Jun protein were remarkably elevated compared to cells treated with dbcAMP alone. In contrast, there was no difference in the expression levels of c-fos mRNA and c-Fos protein between the combination of 18:1/DHA-PE + dbcAMP or dbcAMP alone. On the other hand, the combine treatment of 18:1/DHA-PE and dbcAMP markedly reduced the expression level of c-myc oncogene during 48 h incubation. The decreases of c-myc mRNA by 18:1/DHA-PE and/or dbcAMP was correlated with growth inhibition effect. Thus, 18:1/DHA-PE might enhance dbcAMP-induced HL-60 cell differentiation and growth inhibition by regulation of c-jun and c-myc mRNA and their products.     

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.     

Expression of src family genes during monocytic differentiation of HL-60 cells

It has been reported that src family protein-tyrosine kinases were expressed specifically in a certain lineage or differentiation stage of hematopoietic cells. To understand the molecular basis for differentiation and function of monocyte/macrophage, we investigated the expressions of src family genes by the HL-60 cells stimulated with differentiation-inducing agents. TPA and vitamin D3 (D3) were used as stimulants for monocytic development, since each agent has been known to induce phenotypically specific differentiation of HL-60 cells. The fyn, fgr, and lyn genes were characteristically expressed concomitantly with phenotypic changes and expressions of nuclear proto-oncogenes, whereas src, lck, hck, and yes genes were not. In TPA-induced differentiation of HL-60 cells, both fyn and lyn genes, but not fgr gene, were expressed. In contrast, both fgr and lyn genes, but not fyn gene, were expressed in D3-induced differentiation of the cells. The independent and characteristic expressions of these genes were observed in the further advanced differentiation of HL-60 cells induced by TPA plus D3 or D3 plus human transforming growth factor-beta 1. The granulocytic differentiation of the cells treated with retinoic acid was accompanied by intense expression of fgr, but weak or no expression of lyn and fyn gene. These data indicate that each protein-tyrosine kinase encoded by src family genes may play distinct roles in development and/or functions of monocyte/macrophage-lineage cells.     

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…     

Retinoic acid-induced blr1 expression promotes ERK2 activation and cell differentiation in HL-60 cells

Retinoids are known to induce the differentiation and cell cycle arrest of human myeloid leukemia cells in vitro. Differential display was used to identify putative early regulatory genes that are differentially expressed in HL-60 human promyelocytic leukemia cells treated with retinoic acid. One of the cDNAs cloned encodes sequences identifying Burkitt's lymphoma receptor 1 (BLR1), a recently described chemokine receptor. Northern blot analysis demonstrates that blr1 mRNA expression increases within 9 h of retinoic acid treatment, well before functional differentiation or G(1)/G(0) growth arrest at 48 h or onset of morphological changes, suggesting a possible regulatory function. The expression of blr1 mRNA is transient, peaking at 72 h when cells are differentiated. blr1 mRNA also is induced by other differentiation-inducing agents, 1alpha,25-dihydroxyvitamin D(3) and DMSO. Induction of blr1 mRNA by retinoic acid is not blocked by the protein synthesis inhibitor cycloheximide. In HL-60 cells stably transfected with blr1 cDNA, ectopic expression of blr1 causes an increase in ERK2 MAPK activation and promotes retinoic acid-induced G(1)/G(0) growth arrest and cell differentiation. The early expression of blr1 mRNA during differentiation, its ability to increase ERK2 activation, and its enhancement of retinoic acid-induced differentiation suggest that blr1 expression may be involved in retinoic acid-induced HL-60 differentiation.     

Inducible gene expression of activin A/erythroid differentiation factor in HL-60 cells.

Treatment of HL-60 cells with 12-O-tetradecanoyl-phorbol 13-acetate (TPA) for 48 h induced expression of mRNA of beta A chain of activin A/erythroid differentiation factor. Under the same condition, interferon-gamma caused a slight increase in beta A chain mRNA, whereas 1 alpha, 25-dihydroxyvitamin D3, dimethylsulfoxide and all-trans-retinoic acid failed to induce this mRNA in HL-60 cells. Furthermore, 4 h-treatment with TPA or lipopolysaccharide (LPS) induced a marked increase in beta A chain mRNA levels in interferon-gamma-pretreated HL-60 cells. In the cells pretreated with 1 alpha, 25-dihydroxyvitamin D3, TPA and LPS induced as little increase in beta A chain mRNA as in the control cells. Neither alpha nor beta B chain mRNA was detected in any sample. These results indicate that interferon-gamma has a priming effect on the activation of activin A/erythroid differentiation factor gene by TPA or LPS in HL-60 cells.     

Influence of cell cycle synchronization on digital image analysis of HL-60 granulopoiesis.

Retinoic acid (RA)-treated HL-60 cells subjected to density arrest (DA) and double thymidine block (TB) synchronization demonstrated image feature changes associated with cellular proliferation and differentiation. RA-treated TB cells demonstrated an increased level of morphologic differentiation (assessed by differential counts and quantitation of nuclear shape) and more rapid functional differentiation (assessed by superoxide production and expression of complement receptors) than RA-treated DA cells. By comparison to DA cells, TB cells had less variation in virtually all image features values. A Kruskal-Wallis test of image features ranked total optical density (TOD) of Feulgen-stained cells, nuclear area and shape factor as the top three features regardless of synchronization method. Statistically significant changes in image feature values of RA-treated cells were first noted on day 1. The computer-assisted ability to discriminate RA-treated cells on a given day after induction from paired controls by means of an unsupervised learning algorithm increased over a seven-day period for both DA and TB cells. However, in the dichotomous (RA-treated versus untreated) classification scheme employed, which did not account for continuous levels of morphologic differentiation, there was no advantage in the use of the TB over DA procedure.     

Effects of protein kinase A and calcium/phospholipid-dependent kinase modulators in the process of HL-60 cell differentiation: their opposite effects between HL-60 cell and K-562 cell differentiation.

We have previously shown that HL-60 cells treated with 1 alpha, 25-(OH)2D3 in magnesium-deficient medium are committed to differentiate but do not express differentiation-related phenotypes. In the present study, we demonstrated that Mg2+ deprivation blocked the process of differentiation before the induction of lysozyme mRNA and that the process of HL-60 cell differentiation could be divided into two steps, i.e., a commitment step and a phenotypic expression step. We studied the effects of protein kinase A (PKA) and calcium/phospholipid-dependent protein kinase (PKC) modulators at each step. The results indicated that agonists of PKA enhanced both steps but that N-(2-[methylamino]ethyl-5-isoquinolinesulfonamide inhibited them. On the other hand, 1-oleyl-2-acetylglycerol and 12-O-tetradecanoylphorbol-13-acetate enhanced the commitment step but inhibited that of phenotypic expression. Staurosporine and 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine inhibited the commitment step and enhanced that of phenotypic expression. These results indicate that PKA acts as a positive regulatory signal and that PKC has a dual role in the process of HL-60 cell differentiation, i.e., as a positive regulatory signal in the commitment step and as a negative one in the phenotypic expression step. Recently, we have also shown that in K-562 cell differentiation into erythroid lineage, PKA may serve as a negative regulatory signal in both steps; however, PKC may act dually, namely as a negative regulatory signal in the commitment step and as a positive one in the phenotypic expression step.(ABSTRACT TRUNCATED AT 250 WORDS)     

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。     

Functional changes in human leukemic cell line HL-60. A model for myeloid differentiation

Polar solvents induce terminal differentiation in the human promyelocytic leukemia cell line HL-60. The present studies describe the functional changes that accompany the morphologic progression from promyelocytes to bands and poly-morphonuclear leukocytes (PMN) over 9 d of culture in 1.3 percent dimethylsulfoxide (DMSO). As the HL-60 cells mature, the rate of O(2-) production increase 18-fold, with a progressive shortening of the lag time required for activation. Hexosemonophosphate shunt activity rises concomitantly. Ingestin of paraffin oil droplets opsonized with complement or Ig increases 10-fold over 9 d in DMSO. Latex ingestion per cell by each morphologic type does not change significantly, but total latex ingestion by groups of cells increases with the rise in the proportion of mature cells with greater ingestion capacities. Degranulation, as measured by release of β-glucuronidase, lysozyme, and peroxidase, reaches maximum after 3-6 d in DMSO, then declines. HL-60 cells contain no detectable lactoferrin, suggesting that their secondary granules are absent or defective. However, they kill staphylococci by day 6 in DMSO. Morphologically immature cells (days 1-3 in DMSO) are capable of O(2-) generation, hexosemonophosphate shunt activity, ingestion, degranulation, and bacterial killing. Maximal performance of each function by cells incubated in DMSO for longer periods of time is 50-100 percent that of normal PMN. DMSO- induced differentiation of HL-60 cells is a promising model for myeloid development.