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.     

S-Adenosylmethionine metabolism in HL-60 cells: effect of cell cycle and differentiation

The effect of the cell cycle and differentiation on S-adenosylmethionine (SAM) metabolism in HL-60 cells has been investigated. Synthesis and pool sizes of SAM and S-adenosylhomocysteine (SAH) were cell-cycle-independent (SAM, 315, μM; SAH, 4.6 μM). The SAM-synthase (ATP: l-methionine S-adenosyltransferase) of HL-60 cells has a K_m for methionine of 12.8±2.0 μM and thus appears to be of the intermediate K_m type found in other malignant tissues. The enzyme does not show cell-cycle regulation. Treatment of cells with DMSO resulted in a rapid and marked decrease of SAM and SAH levels without affecting pool turnover or the SAM/SAH ratio. A decrease in SAM concentration could also be observed in a variant cell line resistant to differentiation with DMSO. DMSO inhibited SAM-synthase in cell-free extracts. This inhibition was noncompetitive with respect to l-methionine. Inhibition of SAM-synthase by cycloleucine lowered SAM levels in intact cells, but resulted in differentiation of only a minor percentage of cells. These data indicate that changes in SAM and SAH levels in HL-60 cells seem to be a consequence rather than a cause of differentiation.     

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.     

Control of HL-60 monocytic differentiation. Different pathways and uncoupled expression of differentiation markers

Control of expression of the terminally differentiated phenotype was studied using the human promyelocytic leukemia cell line HL-60. Three known inducers of HL-60 monocytic differentiation were compared: 1,25-dihydroxy vitamin D3, tetradecanoylphorbol acetate (TPA), and sodium butyrate. At concentrations where all three inducers resulted in similar courses of G1/0-specific growth arrest, the kinetics of appearance of certain differentiation markers typically characteristic of mature monocytic cells was determined. The markers were inducible oxidative metabolism, non-specific esterase activity, and the cell surface determinants Mo1, My4, and Mo2. The results indicate that: Regulation of the expression of these markers during induced monocytic differentiation is not controlled in common. The three monocytic inducers do not induce the same metabolic cascade leading to differentiation. Similar states of differentiation could thus be reached by different pathways apparently due to the fact that control of expression of different differentiation markers was not tightly coupled.     

Ceramide kinase expression is altered during macrophage-like cell differentiation of the leukemia cell line HL-60

Ceramide kinase (CerK) has important roles in leukocyte functions, including the role in degranulation of mast cells and the phagocytosis of polymorphonuclear leukocytes, so its expression levels should be strictly regulated. Here, we report that the mRNA expression and enzyme activity of CerK were decreased during macrophage-like cell differentiation of the leukemia cell line HL-60, yet neither was altered during granulocytic differentiation of the same cells. Our findings demonstrate that HL-60 cells are useful for studying CerK functions in leukocyte differentiation, and they also suggest that CerK might have an important role in such differentiation.     

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.     

Differentiation of HL-60 cells: cell volume and cell cycle changes

HL-60 promyelocytic leukemic cells can differentiate into more mature myeloid cells with the addition of dimethylsulfoxide, butyric acid or retinoic acid and can differentiate into macrophages with the addition of phorbol ester 12-0-tetradecanoylphorbol-13-acetate (TPA). After the addition of an inducer, the HL-60 cell volume shows a daily decrease while the cell number increases at a rate similar to the untreated control cells. Flow cytometry measurements show an increase in G1 cells and a decrease in S cells after day 1. Since the generation time is constant, the data suggest that the length of time spent in the different cell cycle stages has changed during differentiation. Within 3 hours after the addition of TPA to HL-60 cells, selective adhesion of G1 cells occurs. Smaller sized cells are recovered from the flask bottom and larger sized cells are recovered from the supernate. Flow cytometric analysis reveals a G1 and S block in cells obtained from both the supernatant and from the flask bottom. After 1 day of TPA incubation, there is preferential adhesion of G1 and G2 cells with the nonadherent cells being primarily in the S and G2 cell cycle stages and undergoing a cell cycle traverse.     

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.     

Interferon-inducible gene expression in HL-60 cells: effects of the state of differentiation.

The promyelocytic leukemia line HL-60 can be terminally differentiated in vitro to either monocyte/macrophages or granulocytes. We used this cell line to test whether the state of differentiation of a cell changes its response to interferon (IFN). The characteristics of expression of several IFN-alpha- and IFN-gamma-inducible genes in undifferentiated and differentiated HL-60 cells were examined. p67, an IFN-gamma-inducible protein, was induced similarly in three cell types, whereas another IFN-gamma-inducible protein, p56, was induced strongly only in undifferentiated cells. In contrast, two isozymes of 2,5(A)-synthetase were induced better in differentiated cells in response to either IFN. Several IFN-alpha-inducible mRNAs, e.g., 561, 6-16, 1-8, and 2A, were induced much more strongly in granulocytes than in macrophages or in undifferentiated cells. Electrophoretic mobility shift assays using the IFN-stimulated response element of gene 561 and nuclear extracts of IFN-alpha-treated cells revealed the appearance of one complex and the disappearance of another one, concomitant with differentiation of the cells to granulocytes. These observations suggest that expression of IFN-inducible genes in HL-60 cells is regulated by trans-acting factors whose activity changes with the state of differentiation of the cells. Our study may have implications in the optimal clinical use of IFNs. Inducing cellular differentiation may augment the efficacy of IFNs as antitumor agents.