Changes in asparagine-linked sugar chains of human promyelocytic leukemic cells (HL-60) during monocytoid differentiation and myeloid differentiation. Appearance of high mannose-type oligosaccharides in neutral fraction

Structural changes in the asparagine-linked sugar chains of plasma membrane glycoproteins during myeloid and monocytoid differentiation were investigated by the use of an in vitro differentiation system for human promyelocytic leukemic cells (HL-60), which can be induced to more mature myeloid cells by exposure to dimethyl sulfoxide and to macrophage-like cells by a phorbol ester. The asparagine-linked sugar chains released from their plasma membranes by hydrazinolysis were separated into a neutral fraction and an acidic fraction composed of over ten components. The content of neutral oligosaccharides, which accounted for 8% of the total asparagine-linked sugar chains in HL-60 cells, increased slightly to 13% in dimethyl sulfoxide-induced cells and markedly to 33% in phorbol ester-induced cells. Structural analyses revealed that the neutral oligosaccharides of HL-60 cells are all of the complex type with a variety of Gal beta 1----4GlcNAc beta 1----units in their outer chain moieties and the following core structure: (sequence; see text) After myeloid and monocytoid differentiation, the total amount of neutral complex-type sugar chains did not change significantly, but newly found high mannose-type sugar chains contributed up to 3% and 24% of the total sugar chains, respectively.     

Amiloride inhibits phorbol ester-stimulated Na+/H+ exchange and protein kinase C. An amiloride analog selectively inhibits Na+/H+ exchange

The human leukemic cell line, HL-60, differentiates in response to tumor-promoting phorbol esters. Recently, we have reported that one of the first events evoked by phorbol esters in HL-60 cells is the stimulation of Na+-dependent H+ efflux. In efforts to determine whether stimulation of Na+/H+ exchange by phorbol esters is coupled to induction of cellular differentiation, we found that 1) amiloride, a frequently used inhibitor of Na+/H+ exchange, rapidly inhibits phorbol ester-stimulated protein phosphorylation in vivo and protein kinase C-mediated phosphorylation in vitro, both with potency similar to that with which amiloride inhibits Na+/H+ exchange; 2) an amiloride analog, dimethylamiloride, is a far more potent inhibitor of Na+/H+ exchange than is amiloride, while being no more potent than amiloride in inhibiting phorbol ester/protein kinase C-mediated phosphorylation; and 3) at concentrations sufficient to completely inhibit Na+/H+ exchange, amiloride blocked phorbol ester-induced adhesion of HL-60 cells (adhesion being a property indicative of the differentiated state), but dimethylamiloride (as well as ethylisopropylamiloride, another very potent amiloride analog) did not. Thus, dimethylamiloride represents a potential tool for distinguishing protein kinase C-coupled from Na+/H+ exchange-coupled events in phorbol ester-stimulated cells.     

Expression of intermediate filament proteins in TPA-induced MPC-11 and HL-60 cells.

Under normal culture conditions, the tumor cell lines MPC-11 and HL-60 exhibit high rates of proliferation and show a peculiar expression of intermediate filament proteins as they appear to synthesize only lamin B. A 48-h exposure of murine plasmacytomas MPC-11 to the phorbol ester TPA reduces their growth and induces vimentin synthesis without affecting the composition of their nuclear lamina. When applied to human leukemic promyelocytes HL-60, such treatment promotes their maturation into macrophage-like cells: their proliferative ability is suppressed, a differentiated phenotype is developed, and their content in intermediate filament proteins now includes vimentin and a full complement of lamins A, B, and C. In the present study, a kinetic analysis of vimentin and lamin A/C expression in relation to proliferation and differentiation has been performed in these two cellular systems. Proliferation rates of MPC-11 and HL-60 populations were evaluated by monitoring cell growth and measuring thymidine incorporation. Maturation of HL-60 cells was assessed by Giemsa staining and percentage of adherent cells. Expression of vimentin and lamins A/C was analyzed using immunofluorescence and immunoblotting techniques. Our data show that there is a relationship between the level of vimentin expression and the extent of growth inhibition in both systems. They also suggest that the expression of lamins A/C during the TPA-induced maturation of HL-60 promyelocytes might be part of the processes which lock these cells into the macrophage pathway.     

Differentiation-linked expression of the plasminogen activator inhibitor type-2 gene in the human HL-60 promyelocytic cell line

HL-60 is a human promyelocytic cell line which was found to be capable of differentiating toward a macrophage-like or granulocyte-like phenotype. Histochemical analysis demonstrated that incubation of cells in the presence of phorbol myristate acetate (PMA) or 1,25-dihydroxyvitamin D3 induced varying degrees of monocytic differentiation, while incubation in the presence of retinoic acid (RA) or dimethyl sulfoxide (DMSO) induced granulocytic differentiation. The differentiation induced by PMA, RA, and to a lesser extent DMSO, was accompanied by the induction of plasminogen activator inhibitor expression. mRNA analysis of control and PMA-induced cultures revealed the induction of a 2-kb message in treated cells which hybridized with a PAI-2-specific oligonucleotide probe. This is consistent with the literature concerning the expression of PAI by macrophages and granulocytes. No hybridization was detected with a PAI-1 specific probe. Expression of PAI by cells of hematopoietic origin appears to be associated with differentiation or stimulation of committed cells. Furthermore, PAI-2 expression by HL-60 cells is not restricted to one specific hematopoietic lineage. Since other cells of hematopoietic origin such as platelets express PAI-1, future studies using pluripotential cell lines could provide information on the initial events of lineage commitment and gene expression.     

Changes in membrane gangliosides: differentiation of human and murine monocytic cells

Membrane ganglioside changes in murine peritoneal macrophages and the human promyelocytic leukemia cell line HL-60 have been assessed by two-dimensional thin-layer chromatography. C3H/HeJ mice respond to protein-containing endotoxin but are hyporesponsive to protein-free endotoxin preparations. Compared to unstimulated resident cells, protein-containing endotoxin produced an alteration in the C3H/HeJ macrophage ganglioside pattern whereas protein-free endotoxin did not. In comparison, differentiation of HL-60 cells to a neutrophil-like cell by dimethylsulfoxide gave a ganglioside pattern similar to unstimulated HL-60 cells. However, differentiation of HL-60 cells by phorbol myristate acetate to macrophage-like cells results in a large increase in the monosialoganglioside GM3. The evidence presented indicates that discrete ganglioside changes occur in murine monocytes and HL-60 cells upon induction to cells with increased macrophage functions.     

Inhibition by islet-activating protein, pertussis toxin, of retinoic acid-induced differentiation of human leukemic (HL-60) cells

Human promyelocytic leukemic (HL-60) cells were induced to differentiate into neutrophil- or macrophage-like cells by incubation of the cells with retinoic acid, dibutyryl cyclic AMP (Bt2cAMP) or phorbol 12-myristate 13-acetate (PMA). Differentiation was determined by an increase in the percentage of morphologically mature cells. The retinoic acid-induced differentiation of HL-60 cells was, but the Bt2cAMP- or PMA-induced one was not, inhibited by prior exposure of the cells to islet-activating protein (IAP), pertussis toxin. The IAP-induced inhibition was correlated with the toxin-catalyzed ADP-ribosylation of a membrane GTP-binding protein with a molecular mass of 40 kDa. Thus, the IAP-substrate GTP-binding protein appears to be involved in the retinoic acid-induced differentiation of HL-60 cells.     

A protein complex expressed during terminal differentiation of monomyelocytic cells is an inhibitor of cell growth

A protein complex (PC) composed of the MRP8 and MRP14 proteins has previously been shown to be a specific inhibitor of casein kinase I and II. This PC is expressed during the late stages of terminal differentiation induced in human promyelocytic HL-60 leukemia cells by 1 alpha,25-dihydroxyvitamin D3 and in human monocytic THP-1 leukemia cells by phorbol 12-myristate 13-acetate. This expression is associated with terminal cell differentiation because incubation of HL-60 cells with an agent or condition that causes suppression of growth but not induction of differentiation does not result in expression of the PC. At concentrations of 5-15 nM, the purified PC inhibited the growth of HL-60 cells and THP-1 cells, as well as other cell types belonging to different cell lineages. This growth inhibition was preceded by a reduction in [32P]phosphate incorporation and, at the higher PC concentrations, was associated with a reduction in [3H]thymidine, [3H]uridine, and [32S]methionine incorporation. The specific expression pattern and growth-inhibitory character of the PC suggests that the complex may have a role in suppressing cell growth during monomyelocytic terminal differentiation induced by specific chemical stimuli and during physiological and pathological events associated with monomyelocytic cell functions.     

佛波醇酯诱导HL-60细胞巨噬细胞样分化时蛋白激酶C活力及分布的改变

本文对佛波醇酶诱导人早幼粒白血病细胞系HL-60细胞分化为巨噬细胞样细胞对蛋白激酶c活力及其在亚细胞分布的变化进行了研究。蛋白激酶c活力在TPA处理1小时即明显降低,此低水平的酶活力持续整个实验时期。酶的亚细胞分布研究提示TPA处理细胞胞质组分酶活力剧烈降低,而颗粒组分存在一高盐浓度洗脱的酶活力峰。蛋白激酶c抑制剂三氟过(口了)嗪单独处理HL-60细胞导致胞质和颗粒组分酶活力升高,但并不诱导细胞分化;若与TPA合并处理细胞,酶活力又降低,此时细胞又分化为巨噬细胞样细胞。对上述结果的可能机理进行了讨论。     

Morphologic differentiation of HL-60 cells is associated with appearance of RPTPbeta and induction of Helicobacter pylori VacA sensitivity

Phorbol 12-myristate 13-acetate (PMA) induces differentiation of human leukemic HL-60 cells into cells with macrophage-like characteristics and enhances the susceptibility of HL-60 cells to the Helicobacter pylori VacA toxin (de Bernard, M., Moschioni., M., Papini, E., Telford, J. L., Rappuoli, R., and Montecucco, C. (1998) FEBS Lett. 436, 218-222). We examined the mechanism by which HL-60 cells acquire sensitivity to VacA, in particular, looking for expression of RPTPbeta, a VacA-binding protein postulated to be the VacA receptor (Yahiro, K., Niidome, T., Kimura, M., Hatakeyama, T., Aoyagi, H., Kurazono, H., Imagawa, K., Wada, A., Moss, J., and Hirayama, T. (1999) J. Biol. Chem. 274, 36693-36699). PMA induced expression of RPTPbeta mRNA and protein as determined by RNase protection assay and indirect immunofluorescence studies, respectively. Vitamin D(3) and interferon-gamma, which stimulate differentiation of HL-60 cells into monocyte-like cells, also induced VacA sensitivity and expression of RPTPbeta mRNA, whereas 1. 2% Me(2)SO and retinoic acid, which stimulated the maturation of HL-60 into granulocyte-like cells, did not. RPTPbeta antisense oligonucleotide inhibited induction of VacA sensitivity and expression of RPTPbeta. Double immunostaining studies also indicated that newly expressed RPTPbeta colocalized with VacA in PMA-treated HL-60 cells. In agreement with these data, BHK-21 cells, which are insensitive to VacA, when transfected with the RPTPbeta cDNA, acquired VacA sensitivity. All data are consistent with the conclusion that acquisition of VacA sensitivity by PMA-treated HL-60 cells results from induction of RPTPbeta, a protein that functions as the VacA receptor.     

Selective decrease in cell surface expression and mRNA level of the 55-kDa tumor necrosis factor receptor during differentiation of HL-60 cells into macrophage-like but not granulocyte-like cells.

Expression of the two known receptors for TNF was studied in the promyelocytic leukemia cell line HL-60 before and after differentiation of the cells along the granulocyte lineage (induced by incubation with retinoic acid), or along the macrophage lineage (induced by incubation with the phorbol diester, PMA). The extent of inhibition of TNF binding by receptor-specific antisera, as well as the size of the complexes formed after cross-linking TNF to its receptors on intact cells, indicated that both receptor species were expressed on the surface of the undifferentiated HL60 cells. Differentiation into granulocyte-like cells resulted in some increase in TNF binding. The increase was apparently due to enhanced expression of the 75-kDa TNF-R, whereas the amounts of the 55-kDa TNF-R did not change significantly. In contrast, in HL-60 cells induced to differentiate into macrophage-like cells, expression of the 55-kDa TNF-R species was completely abolished. The pattern of TNF-R expression in the differentiated HL-60 cells was similar to that observed in leukocytes isolated from peripheral blood: on granulocytes, there were about equal amounts of both receptor species, whereas on monocytes the 75-kDa receptor was predominant. The loss of 55-kDa receptors during differentiation of HL-60 cells into macrophage-like cells was accompanied by a pronounced decrease in the level of the mRNA for that receptor, suggesting that at least part of the change in TNF-R expression is due to mechanisms that control the amounts of receptor mRNA. Although little is yet known regarding the functional differences between the two receptor species, marked changes in the pattern of their expression, as observed during HL-60 cell differentiation, are likely to alter the kind of response of the cells to TNF and may therefore play an important role in the coordination of TNF effects in the organism.     

Enhanced Expression of αV Integrin Subunit and Osteopontin during Differentiation of HL-60 Cells along the Monocytic Pathway

HL-60 cells, a promyelocytic leukemic cell line, provide a good model for studying the role of adhesion molecules and associated receptors involved in cell differentiation. When exposed to factors such as phorbol esters, these cells grown in suspension differentiate into monocytes and adhere to tissue culture dishes. In this study we showed that HL-60 cells exposed to phorbol esters express osteopontin (OPN), a cell adhesion molecule linked with osteoclast function. Moreover, the timed expression of OPN, in phorbol ester treated cells, was linked to increased cell adhesion. Subsequent to the expression of OPN, an increase in mRNA levels for α_V integrin subunit was observed. The α_Vβ₃ integrin, a cell surface receptor found in high concentrations in osteoclasts, is considered to be a receptor for OPN. Furthermore, during differentiation we detected an increase in two cell surface markers specific for osteoclasts, 75B and 121F. This is the first report to demonstrate expression of OPN during differentiation of HL-60 cells, indicating that HL-60 cells can be used as a tool to enhance our understanding as to the role of OPN in cell differentiation.     

Differentiation of HL-60 cells by phorbol ester is correlated with up-regulation of protein kinase C-alpha.

To clarify the mechanism of 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced macrophage-like differentiation of HL-60 cells, we investigated the correlation between the effects of protein kinase C (PKC) inhibitors on the induction of markers of TPA-induced differentiation and those on suggested critical steps of the differentiation. H-7, sphingosine, and trifluoroperazine significantly suppressed TPA-induced cell adhesion but their effects on the induction of acid phosphatase and nonspecific esterase differed among the inhibitors. The three inhibitors failed to affect on TPA-induced annexin I expression. In contrast, staurosporine markedly suppressed the induction of all these markers. The effects of the inhibitors on some suggested critical steps of the differentiation, a rapid phosphorylation of specific proteins, a rapid membrane association of PKC, and down-regulation of PKC at 18 h after addition of TPA, were not correlated with those on the differentiation marker induction. Only the effect of the inhibitors on up-regulation of PKC-alpha was closely correlated with TPA-induced annexin I expression; staurosporine inhibited up-regulation of PKC-alpha but other inhibitors did not similarly affect the induction of annexin I expression. These results suggest that PKC-alpha is intimately related to macrophage-like differentiation of HL-60 cells by TPA.     

蛋白激酶C亚型在HL—60细胞诱导分化中的变化

用全反式维甲酸（ＡＴＲＡ）或佛波酯（ＰＭＡ）处理人早幼粒白血病细胞（ＨＬ－６０）３天,用形态学,ＮＢＴ还原实验,特异性和非特异性酯酶测定,证明细胞分别向粒细胞或单核／巨噬细胞分化。通过免疫组化法观察了蛋白激酶Ｃ（ＰＫＣ）α,βⅠ和βⅡ亚型在分化后的变化。结果显示,ＡＴＲＡ可引起ＨＬ－６０细胞ＰＫＣα,βⅠ和βⅡ的含量升高,分别为对照的５．０,２．８和４．２倍,并存在从胞膜向胞质转位。ＰＭＡ则使ＰＣ