Differentiation Of HL-60 Promyelocytic Cells Research Articles

Argonaute 2 Complexes Selectively Protect the Circulating MicroRNAs in Cell-Secreted Microvesicles

Cell-secreted miRNAs are highly stable and can serve as biomarkers for various diseases and signaling molecules in intercellular communication. The mechanism underlying the stability of circulating miRNAs, however, remains incompletely understood. Here we show that Argonaute 2 (Ago2) complexes and microvesicles (MVs) provide specific and non-specific protection for miRNA in cell-secreted MVs, respectively. First, the resistance of MV-encapsulated miRNAs to RNaseA was both depended on intact vesicular structure of MVs and protease-sensitive. Second, an immunoprecipitation assay using a probe complementary to human miR-16, a miRNA primarily located in the MVs and showed a strong, protease-sensitive resistance to RNaseA, identified Ago2 as a major miR-16-associated protein. Compared with protein-free miR-16, Ago2-associated miR-16 was resistant to RNaseA in a dose- and time-dependent fashion. Third, when the miR-16/Ago2 complex was disrupted by trypaflavine, the resistance of miR-16 to RNaseA was decreased. In contrast, when the association of miR-16 with the Ago2 complexes was increased during cell apoptosis, although the total amount of miR-16 and Ago2 remained unchanged, the resistance of miR-16 to RNaseA in the MVs was enhanced. A similar correlation between the increase of miR-223/Ago2 association and the resistance of miR-223 against RNaseA was observed during all trans retinoic acid (ATRA)-induced cell differentiation of promyelocytic HL60 cells. In conclusion, the association of miRNAs with Ago2 complexes, an event that is linked to cell functional status, plays a critical role in stabilizing the circulating miRNAs in cell-secreted MVs.

Inositol lipids and phosphates in the proliferation and differentiation of lymphocytes and myeloid cells.

It is established that receptor-stimulated hydrolysis of phosphatidylinositol 4,5-bisphosphate is an essential signalling reaction in the responses of many haemopoietic cells to stimuli: examples include platelet activation, antigen-driven initiation of cell proliferation in mature B and T lymphocytes and histamine release by mast cells, and chemotaxis and oxygen radical generation by neutrophils. However, the roles of inositol lipids and phosphates in the development of haemopoietic and immune cells are less well understood. This paper discusses three such situations: the sequential employment of phosphatidylinositol 4,5-bisphosphate hydrolysis and cyclic AMP accumulation as two signals essential to the action of the B lymphocyte-stimulatory cytokine interleukin 4; the involvement of antigen receptor-triggered inositol lipid hydrolysis in apoptotic elimination of immature anti-self T lymphocytes in the fetal mouse thymus; and the possible role of changes in the levels of abundant inositol polyphosphates in the differentiation of HL-60 promyelocytic cells and of normal human myeloid blast cells.

Increased responsiveness to TLR2 and TLR4 ligands during dimethylsulfoxide-induced neutrophil-like differentiation of HL-60 myeloid leukemia cells

Neutrophils express functional toll-like receptor2 (TLR2) and 4 (TLR4) that are crucial for the production of inflammatory cytokines. Here, we show that dimethylsulfoxide-induced neutrophil-like differentiation of promyelocytic HL-60 cells (dHL-60) results in cells that respond to TLR2 and TLR4 ligands similarly to primary neutrophils. Consistent with the increased responsiveness of the cells to TLR2 ligand, the TLR2 gene was strongly up-regulated in dHL-60 cells. On the other hand, increased surface expression of LPS receptor complex, TLR4/MD2/CD14, was observed without affecting TLR4 gene expression. Thus, the data demonstrate a higher responsiveness of dHL-60 cells to TLR2 and TLR4 ligands because of increased TLR2 and MD2/CD14 gene expression.

1,25-Dihydroxyvitamin D3 Suppresses Gene Expression of Eukaryotic Translation Initiation Factor 2 in Human Promyelocytic Leukemia HL-60 Cells

The physiologically active metabolite of vitamin D(3), 1alpha,25-dihydroxyvitamin D(3) (DVD), is a potent inducer of cell differentiation in human myeloid leukemia cells. In the present study, we examined changes in gene expression during DVD-induced cell differentiation of promyelocytic HL-60 cells employing a DNA microarray technique. The results identified 7 up-regulated and 9 down-regulated genes with a change greater than 1.5-fold after the DVD-treatment for both 2 and 6 days. Seven of these genes were further examined by reverse transcription-polymerase chain reaction (RT-PCR). The results showed that findings obtained from the DNA microarray analysis and RT-PCR are generally comparable with each other. Gene expression of the subunits of eukaryotic translation initiation factor 2 was then examined by methods including RT-PCR and real-time PCR. The results indicated the suppression of these genes, suggesting a linkage to differentiation-associated growth inhibition of these cells.

Bisphenol A significantly enhances the neutrophilic differentiation of promyelocytic HL-60 cells

Bisphenol A (BPA) is a well-known endocrine disruptor. However, little information is available on its immunological effects. To investigate the effect of BPA on leukocyte differentiation, we investigated its action on the neutrophilic differentiation of HL-60 cells induced by dimethylsulfoxide and granulocyte colony-stimulating factor (G-CSF) for 6 days. At low concentrations (10 −10–10 −8 M), BPA significantly increased the superoxide production by differentiated HL-60 cells stimulated with opsonized zymosan (OZ) by about 20%, and expression of CD18, a component of the OZ-receptor, was increased to a similar extent by 10 −9 M BPA. To investigate the effect of BPA on the activity of PU.1, a transcription factor specific for granulocytic differentiation, we established a stable clone that expressed luciferase as a reporter of PU.1 activity. PU.1 activity increased during the neutrophilic differentiation of HL-60 cells, reaching a peak on day 3 and decreasing thereafter. Nanomolar BPA augmented the PU.1 activity on day 3 by about 60%. On the other hand, tamoxifen, a competitive inhibitor of estrogen receptors, did not suppress the effect of BPA on the differentiation of HL-60 cells. These results suggest that BPA exerts an enhancing effect on the neutrophilic maturation of leukocytes through an estrogen receptor-independent pathway. Long-term exposure to BPA might significantly affect the innate immunity of mammals, even at low doses.

Triphenyltin enhances the neutrophilic differentiation of promyelocytic HL-60 cells

Triphenyltin (TPT) is an environmental endocrine disruptor and toxic substance, but little information is available on its immunological effects. To assess the effect of TPT on leukocyte differentiation, we investigated its effect on the neutrophilic differentiation of HL-60 cells induced by dimethyl sulfoxide and granulocyte colony-stimulating factor (G-CSF) for 6 days. At a low concentration, 10 −7 M, TPT increased superoxide production by differentiated HL-60 cells stimulated with opsonized zymosan (OZ) by about 45% and increased expression of CD18, a component of the OZ-receptor, by about 90%. Real-time PCR analysis revealed that TPT augmented the expression not only of CD18 but also of components of superoxide-generating NADPH-oxidase, p47phox, 2.7-fold, and p67phox, 2.0-fold, and of granulocyte colony-stimulating factor receptor (G-CSFR), 3.0-fold, whereas various other endocrine disruptors, including parathion, vinclozolin, and bisphenol A, had no such enhancing effects. The results of a DNA macroarray analysis showed that TPT enhanced the expression of G-CSFR and certain other neutrophil functional proteins, including CD14 and myeloid leukemia cell differentiation protein (MCL-1), and that TPT induced a decrease in expression of LC-PTP, leukocyte protein–tyrosine phosphatase, to about half the control level. The TPT-dependent suppression of LC-PTP was confirmed by real-time PCR analysis, and the results of immunoblotting indicated that TPT enhances the expression of myeloid specific tyrosine kinase hck by about 30% at the protein level, and this together with the reduction of LC-PTP may enhance tyrosine phosphorylation, in turn resulting in enhancement of superoxide production. These findings suggest that TPT may have an enhancing effect on the neutrophilic maturation of leukocytes.

Novel esters of glaucarubolone as inducers of terminal differentiation of promyelocytic HL-60 cells and inhibitors of 7,12-dimethylbenz[a]anthracene-induced preneoplastic lesion formation in mouse mammary organ culture.

In an effort to discover new chemotherapeutic/chemopreventive agents from natural sources, brusatol (1) was found to induce HL-60 cellular differentiation, accompanied by strong antiproliferative and cytotoxic effects. A series of natural and semisynthetic quassinoids (1-48) was designed to effect both antiproliferative and differentiation-inducing properties. Compounds were assessed in vitro using the HL-60 promyelocytic cell model. Changes in activity due to structural modification of the core structure glaucarubolone (24) were consistent with activities reported in other cell systems. However, the following were novel SAR findings: (1) semisynthetic analogues with a hydroxylated ring at the beta-position of the ester side chain at C-15 were able to induce cellular differentiation at concentrations lower than those inducing cell growth arrest, and (2) quassinoids inhibiting DNA synthesis with greater efficacy than reducing cellular viability possessed alkyl substitutions at the alpha-position of the C-15 ester side chain. Analogues from this latter group and brusatol (1) and bruceantin (2) inhibited dimethylbenz(a)anthracene-induced preneoplastic lesion formation in a mouse mammary organ culture. The novel finding of 1 and glaucarubolone analogues as potent inducers of differentiation leads to potential novel applications in the field of cancer.

Identification of differentially expressed gene sequences along granulocytic differentiation of promyelocytic HL60 cells

Identification of differentially expressed gene sequences along granulocytic differentiation of promyelocytic HL60 cells

Downregulation of Wilms' tumor gene (wt1) during myelomonocytic differentiation in HL60 cells

The putative Wilms' tumor-suppressor gene (wt1) encodes a zinc finger DNA binding protein that functions as a transcription repressor. The wt1 gene expression corresponds to kidney development, suggesting a role for this gene in nephroblast differentiation. Here we show that wt1 mRNA expression was downregulated during terminal differentiation of promyelocytic HL60 cells. When HL60 cells were induced to differentiate to granulocytes by dimethyl sulfoxide (DMSO) or retinoic acid (RA), a marked downregulation in the levels of wt1 transcripts was found. The wt1 transcripts were also downregulated in HL60 cells during differentiation to monocytes by vitamin D3 or 12-o-tetradecanoyl- phorbol-13-acetate. Nuclear run-on transcription studies showed the transcriptional rate of wt1 gene was not significantly altered during DMSO-induced granulocytic differentiation, suggesting the downregulation was mostly caused by posttranscriptional modification. Importantly, wt1 transcripts were not significantly altered in K562 cells by treatments with DMSO or RA, which do not induce differentiation of K562 cells. These findings suggest that wt1 gene expression may be downregulated as a differentiation-linked event in HL60 cells.

Sphinganine Potentiation of Dimethyl Sulfoxide-Induced Granulocytic Differentiation, Increase of Alkaline Phosphatase Activity and Decrease of Protein-Kinase C Activity in a Human Leukemia Cell Line (HL-60)

The differentiation of HL-60 promyelocytic cells toward mature granulocytic cells induced by dimethyl sulfoxide (DMSO) was accompanied by a quantitative similar increase in alkaline phosphatase (ALP) activity and decrease in protein kinase C (PKC) activity. The combination of DMSO and sphinganine (SP), a potent inhibitor of PKC, increased in parallel the percentage of mature cells and the ALP activity. The enhancement of DMSO-induced differentiation and the potentiation of the decrease of PKC activity by SP also seemed to correlate with each other. Our results indicate that both ALP and PKC may play a role in the DMSO-induced granulocytic differentiation.

Differentiation of HL60 promyelocytic cells is promoted by a ‘differentiation enhancing factor’ produced by erythroleukemia cells

A differentiation enhancing factor isolated from murine erythroleukemia cells is also a potent enhancer of the differentiation of HL60 human promyelocytic leukemia cells, induced by retinoic acid and by phorbol ester. This stimulating effect is the result of a large increase in the sensitivity of HL60 cells for retinoic acid and for phorbol 12-myristate 13-acetate (20-fold and 40-fold, respectively). Accelerated differentiation induced by the protein factor, and monitored by the appearance of marker enzymes, is accompanied by a large increase in the fluctuation of the levels of protein kinase C (PKC) isozymes in HL60 cells. These results provide further support for the role of this new protein factor in cell differentiation and indicate that other cell types are susceptible to its biological effect.

Protein kinase C activity during sphinganine potentiation of retinoic acid-induced differentiation in a human leukemia cell line (HL-60)

The differentiation of HL-60 promyelocytic cells towards mature granulocytic cells induced by retinoic acid (RA) was accompanied by a decrease in protein kinase C (PKC) activity. The enhancement of RA-induced differentiation and the potentiation of the decrease of PKC activity by sphinganine (SP) seemed to correlate with each other. Kinetically, PKC activity during RA-induced differentiation without SP decreased to its lowest (75% of the control) after 48h; about 50% of the reduction was observed at 24h. In the presence of SP, PKC activity decreased more rapidly to its lowest (60% of the control) within 24h of incubation of RA. SP, added 24h before or concomitantly with the addition of RA, could potentiate the RA-induced differentiation and the reduction of PKC activity. Our results indicate that the effect of SP and the role of PKC during RA-induced differentiation may be critical at the early stages of induction of differentiation (within 24h of RA exposure).

Alkaline phosphatase activity during differentiation of the human promyelocytic leukemia cell line, HL-60

The differentiation of HL-60 promyelocytic cells toward mature myelocytic cells induced by retinoic acid (RA) was accompanied by a quantitative similar increase in alkaline phosphatase (ALP) activity. The potentiation of RA-induced differentiation and the enhancement of ALP activity by sphinganine seemed to correlate with each other. The combination of RA and sphinganine increased in parallel the percentage of mature cells and the ALP activities. Short exposures (4–8h) of HL-60 cells to RA promoted differentiation and ALP activity to a fraction (about 50%) of their maximums which were achieved in cells after 24h or longer RA exposure. Our results indicate that the key step for the induction of ALP activity and the differentiation by RA probably takes place within 24h of incubation.

Changes in the levels of inositol lipids and phosphates during the differentiation of HL60 promyelocytic cells towards neutrophils or monocytes

HL60 cells were adapted to grow in a serum-free medium containing 1 mg l-1 inositol, in which they differentiated normally towards neutrophils (in 0.9% by volume dimethylsulphoxide) and towards monocytes (in 10 nM phorbol myristate acetate). Cells that had been equilibrium-labelled with [2-3H]myo-inositol contained a complex pattern of inositol metabolites, several of which were at relatively high concentrations. These included InsP5 and InsP6, which were present at concentrations of about 25 microM and 60 microM, respectively. Striking and different changes occurred in the levels of some of the inositol polyphosphates as the cells differentiated towards either neutrophils or monocytes. Most notable were a large but gradual accumulation of Ins(1,3,4,5,6)P5 as HL60 cells decreased in size and acquired neutrophil characteristics, and much more rapid and sequential declines in InsP4, InsP5 and InsP6 as the cells started to take on monocyte character. There was a marked accumulation of free inositol and of phosphatidylinositol in the cells during neutrophil differentiation, probably caused at least in part by an increased rate of inositol uptake providing an increased intracellular inositol supply. The same accumulation of Ins(1,3,4,5,6)P5 occurred during neutrophil differentiation, whether it was induced by dimethylsulphoxide or by a combination of retinoic acid and a T-lymphocyte cell line-derived differentiation factor. Ins(1,4,5)P3, a physiological intracellular mediator of Ca2+ release from membrane stores, did not change in concentration during these differentiation processes. These observations suggest that some of the more abundant cellular inositol polyphosphates play some important, but not yet understood, role either in the processes of haemopoietic differentiation or in the expression of differentiated cell character in myeloid cells.

Fluorescent tetradecanoylphorbol acetate: a novel probe of phorbol ester binding domains.

Protein kinase C (PKC) has a prominent role in signal transduction of many bioactive substances. We synthesized the fluorescent derivative, phorbol-13-acetate-12-N-methyl-N-4-(N,N'-di(2-hydroxyethyl)amino)-7-n itr obenz-2-oxa-1,3-diazole-aminododecanoate (N-C12-Ac(13)) of 12-O-tetradecanoylphorbol-13-acetate (TPA) to monitor the location of phorbol ester binding sites and evaluate its potential use as a probe of PKC in viable cells. The excitation maximum wavelength of N-C12-Ac(13) is close to 488 nm, facilitating its use in argon-ion laser flow and imaging cytometry. When incubated with 100 nM N-C12-Ac(13) at 25 degrees C, P3HR-1 Burkitt lymphoma cells accumulated the dye rapidly, reaching maximum fluorescence within 25 min, 20-fold above autofluorescence. Addition of unlabeled TPA significantly decreased the fluorescence of N-C12-Ac(13) stained cells in a dose-dependent manner indicating specific displacement of the bound fluoroprobe. Competitive displacement of [3H]-phorbol-12,13-dibutyrate ([3H]-PBu2) from rat brain cytosol with N-C12-Ac(13) gave an apparent dissociation constant (Kd) of 11 nM. N-C12-Ac(13) possessed biological activity similar to TPA. Like TPA (final concentration 65 nM) N-C12-Ac(13), at a lower concentration (51 nM), induced expression of Epstein-Barr viral glycoprotein in P3HR-1 cells, differentiation of promyelocytic HL60 cells, and caused predicted changes in the mitotic cycle of histiocytic DD cells. Microspectrofluorometric images of single cells labeled with N-C12-Ac(13) showed bright fluorescence localized intracellularly and dim fluorescence in the nuclear region, consistent with dye binding mainly to cytoplasmic structures and/or organelles and being mostly excluded from the nucleus. Because of the high level of non-specific binding of N-C12-Ac(13), this probe is not ideal for visualizing PKC in intact cells, but would be a valuable fluoroprobe to investigate the kinetic properties of purified PKC. Also, knowledge gained from these studies allows us to predict structures of fluorescent phorbols likely to have less non-specific binding and, consequently, be potentially useful for monitoring PKC in viable cells.

Sphingomyelinase action inhibits phorbol ester-induced differentiation of human promyelocytic leukemic (HL-60) cells

Prior studies showed that sphingomyelinase action and the free sphingoid bases inhibited protein kinase C (Kolesnick, R. N., and Clegg, S. (1988) J. Biol. Chem. 263, 6534-6537). The present studies investigated whether sphingomyelinase action also inhibited a biologic process mediated via protein kinase C, phorbol ester-induced differentiation of HL-60 promyelocytic cells into macrophages. The potent phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) stimulated time- and concentration-dependent conversion of HL-60 cells into macrophages, ED50 congruent to 5 x 10(-10) M. Differentiation involved growth inhibition, adherence of the suspended cells to tissue culture plastic, morphologic changes, and development of specific enzymatic markers. Sphingomyelinase, which increased the level of sphingoid bases and inactivated protein kinase C, prevented this event. In control incubations, cell number increased 2.10-fold over 24 h, and 2 +/- 1% of the cells were adherent. In incubations with TPA (0.5 nM), cell number increased only 1.75-fold, and 30% were adherent. Sphingomyelinase (3.8 x 10(-5) unit/ml) restored growth to incubations containing TPA to 2.02-fold and reduced adherence to 15%. Sphingomyelinase (3.8 x 10(-2) unit/ml) also restored growth partially and reduced adherence to a maximal concentration of TPA (3 nM). Similar results were obtained with the sphingoid base sphingosine (3-4.5 microM). Sphingomyelinase antagonized the morphologic changes associated with conversion to the macrophage phenotype. Untreated HL-60 cells presented typical promyelocytic morphology with large nuclei, little cytoplasm, and uniformity of nuclear and cell shape. TPA induced a larger cell population with abundant cytoplasm and unusual shape. Sphingomyelinase prevented these changes. Sphingomyelinase blocked TPA-induced increases in the macrophage marker enzymes, acid phosphatase and alpha-naphthyl acetate esterase. These studies indicate that the action of a sphingomyelinase, like the sphingoid bases, blocks phorbol ester-induced differentiation of HL-60 cells into macrophages and provides further support for the concept that sphingomyelinase action may be sufficient to comprise a physiologically relevant inhibitory pathway for protein kinase C.

Molecular cloning of a gene selectively induced by gamma interferon from human macrophage cell line U937.

A cDNA clone encoding a gamma interferon (IFN-gamma)-inducible mRNA in human cells of the macrophage lineage was isolated and characterized. The corresponding gene, gamma.1, was selectively induced by IFN-gamma, responding a hundredfold better to IFN-gamma than to IFN-alpha. The induction was rapid and transient, with maximal mRNA accumulation at about 3 h and decline to the basal level after 48 h. Transcriptional activation could be detected as early as 5 min after IFN-gamma stimulation and accounted entirely for the mRNA accumulation. The induction of gamma.1 by IFN-gamma was cell-type restricted, being seen only in macrophages and endothelial cells. In addition, phorbol ester-induced differentiation of promyelocytic HL-60 cells and promonocytic THP-1 cells rendered the gamma.1 gene inducible by IFN-gamma. The 1.0-kilobase gamma.1 cDNA sequence encoded a small predicted polypeptide of 38 amino acids and had a conserved sequence associated with rapidly turning over mRNAs. In vitro translation of the gamma.1 transcript yielded a 4,000-dalton polypeptide.

Molecular Cloning of a Gene Selectively Induced by Gamma Interferon from Human Macrophage Cell Line U937

A cDNA clone encoding a gamma interferon (IFN-gamma)-inducible mRNA in human cells of the macrophage lineage was isolated and characterized. The corresponding gene, gamma.1, was selectively induced by IFN-gamma, responding a hundredfold better to IFN-gamma than to IFN-alpha. The induction was rapid and transient, with maximal mRNA accumulation at about 3 h and decline to the basal level after 48 h. Transcriptional activation could be detected as early as 5 min after IFN-gamma stimulation and accounted entirely for the mRNA accumulation. The induction of gamma.1 by IFN-gamma was cell-type restricted, being seen only in macrophages and endothelial cells. In addition, phorbol ester-induced differentiation of promyelocytic HL-60 cells and promonocytic THP-1 cells rendered the gamma.1 gene inducible by IFN-gamma. The 1.0-kilobase gamma.1 cDNA sequence encoded a small predicted polypeptide of 38 amino acids and had a conserved sequence associated with rapidly turning over mRNAs. In vitro translation of the gamma.1 transcript yielded a 4,000-dalton polypeptide.