Development of capping ability during differentiation of HL-60 human promyelocytic leukemia cells

Developmental changes in cell surface and cytoskeletal elements have been studied in human promyelocytic leukemia cells (line HL-60) which differentiate into functionally mature myeloid cells when grown in dimethyl sulfoxide (DMSO)-supplemented medium. Both differentiated and undifferentiated HL-60 cells bind fluorescent concanavalin A (F-Con A) in a diffuse pattern over the entire cell surface. As with normal neutrophils, pretreatment of the differentiated HL-60 cells with colchicine before incubation with Con A causes the formation of large cytoplasmic protrusions over which the lectin associates into a cap. On the other hand, similarly treated undifferentiated HL-60 cells do not form the cytoplasmic protuberances and are unable to cap the Con A. Transmission electron microscopy reveals that the number and distribution of microtubules and microfilaments change during differentiation. Thus, developing myeloid cells undergo important alterations in the structure and function of the cytoskeleton as they differentiate into mature phagocytes.     

Transmembrane potential responses during HL-60 promyelocyte differentiation

Myeloid cells, including granulocytes and monocyte/macrophages, are important in disease-associated inflammatory reactions. These cells come from a common progenitor, the promyelocyte. The human promyelocytic cell line, HL-60, can be induced to terminally differentiate into granulocytes or monocyte/macrophages in a controlled fashion providing a model to study various aspects of myelomonocytic differentiation. The expression of several ion channels is controlled in HL-60 cells in a differentiation specific pattern. The purpose of this study was to determine if lineage-specific ion channel expression during HL-60 differentiation resulted in differences in functional responses to external stimuli. This was investigated by examining transmembrane potential responses in HL-60 promyelocytes, HL-60-derived polymorphonuclear cells (PMNs), and monocytes to various stimuli using the transmembrane potential sensitive dye, diSBAC₂-(3). Exposure of HL-60 promyelocytes to ionomycin or ATP produced a membrane hyperpolarization. Studies using ion substitutions and ion channel blockers indicate that the hyperpolarization was mediated by K_Ca channels. During HL-60 promyelocyte differentiation to PMNs, the membrane potential response to ionomycin and ATP shifted from a hyperpolarization to a depolarization over 7 days. Conversely, HL-60-derived monocytes exhibited a membrane hyperpolarization in response to ionomycin and ATP. HL-60-derived monocytes also exhibit a Cl⁻ conductance specifically induced by ATP. Lineage-specific expression of ion channels during HL-60 cell differentiation is important in determining the transmembrane potential response of these cells. This may be translated into functional responses of various myelomonocytic cells during disease-associated inflammatory reactions. © 1996 Wiley-Liss, Inc.     

Retinoic acid induces expression of SLP-76: expression with c-FMS enhances ERK activation and retinoic acid-induced differentiation/G0 arrest of HL-60 cells

Retinoic acid (RA) is known to cause MAPK signaling which propels G0 arrest and myeloid differentiation of HL-60 human myeloblastic leukemia cells. The present studies show that RA up-regulated expression of SLP-76 (Src-homology 2 domain-containing leukocyte-specific phospho-protein of 76 kDa), which became a prominent tyrosine-phosphorylated protein in RA-treated cells. SLP-76 is a known adaptor molecule associated with T-cell receptor and MAPK signaling. To characterize functional effects of SLP-76 expression in RA-induced differentiation and G0 arrest, HL-60 cells were stably transfected with SLP-76. Expression of SLP-76 had no discernable effect on RA-induced ERK activation, subsequent functional differentiation, or the rate of RA-induced G0 arrest. To determine the effects of SLP-76 in the presence of a RA-regulated receptor, SLP-76 was stably transfected into HL-60 cells already overexpressing the colony stimulating factor-1 (CSF-1) receptor, c-FMS, from a previous stable transfection. SLP-76 now enhanced RA-induced ERK activation, compared to parental c-FMS transfectants. It also enhanced RA-induced differentiation, evidenced by enhanced paxillin expression, inducible oxidative metabolism and superoxide production. RA-induced RB tumor suppressor protein hypophosphorylation was also enhanced, as was RA-induced G0 cell cycle arrest. A triple Y to F mutant SLP-76 known to be a dominant negative in T-cell receptor signaling failed to enhance RA-induced paxillin expression, but enhanced RA-induced ERK activation, differentiation and G0 arrest essentially as well as wild-type SLP-76. Thus, SLP-76 overexpression in the presence of c-FMS, a RA-induced receptor, had the effect of enhancing RA-induced cell differentiation. This is the first indication to our knowledge that RA induces the expression of an adapter molecule to facilitate induced differentiation via co-operation between c-FMS and SLP-76.     

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.     

Regulatory properties and cellular redistribution of zinc during macrophage differentiation of human leukemia cells

Many proteins require the binding of trace metals such as Ca, Fe, Cu, or Zn, which may modulate their structure, function, or activity. To determine if there were any overall changes in metalloprotein distribution or metal concentration during the process of macrophage differentiation we induced human myeloid HL-60 leukemia cells with phorbol 12-myristate 13-acetate (PMA) and quantitatively mapped their metal content using hard X-ray fluorescence micro-analysis. We found a transient increase in the zinc content of HL-60 cell nuclei during the early stages of differentiation induction. This finding was confirmed by spectrofluorometry in HL-60 cells and extended to U-937 leukemia cells. A role for protein kinase C-beta (PKC-beta) in this process was established by examining zinc content in an HL-60 variant, HL-525, which is PKC-beta deficient, and in HL-525 cells in which PKC-beta was restored by stable overexpression. Chemical chelation of both Cu and Zn served to inhibit macrophage differentiation in HL-60 cells, indicating a requirement for these metals during this process. Finally, we demonstrate that growth of HL-60 cells in a low-zinc environment removes their susceptibility to PMA-induced differentiation, and that this capacity can be partially restored by the addition of exogenous zinc.     

Macrophage glycolipid receptors for human migration inhibitory factor (MIF): differentiated HL-60 cells exhibit MIF responsiveness and express surface glycolipids which both bind MIF and convert nonresponsive cells to responsiveness

The human promyelocytic leukemia line HL-60 when treated with a phorbol diester (TPA) differentiates into cells (HL60-TPA) that respond to human migration inhibitory factor (MIF). Unresponsive HL-60 cells became responsive to MIF when preincubated with a glycolipid-enriched preparation extracted from HL60-TPA cells, human monocytes, human macrophage-like (U937) cell line, or with the purified glycolipid receptor for MIF from guinea pig peritoneal macrophages. Human blood monocytes exhibited an increased response to MIF when preincubated with glycolipids from HL60-TPA and U937 cells but not from HL-60 cells. Finally, glycolipids from HL60-TPA cells but not from HL-60 cells were able to reversibly bind MIF when covalently coupled to agarose. These studies suggest that TPA induces the differentiation of HL-60 cells into MIF-responsive cells through the expression of a glycolipid receptor for MIF.     

Alterations in insulin receptor kinase activity during differentiation of HL-60 cells

Differentiation of the human promyelocytic leukemia cell line HL-60 into monocytes or macrophages is associated with increased expression of cell surface insulin receptors, while differentiation of these cells into granulocytes is associated with receptor loss. Here we demonstrate that differentiation of HL-60 cells into monocytes or granulocytes induced by 1;25(OH)2vitD3 or Bt2cAMP, respectively, has no major effect on the specific activity of the insulin receptor kinase (IRK). By contrast, when HL-60 cells are incubated with a combination of 1;25(OH)2vitD3 and Bt2cAMP, their differentiation into adherent macrophages-like cells is accompanied by a 50% reduction in the specific activity of IRK. These findings suggest that acquisition or loss of insulin receptors during differentiation of HL-60 involves selective alterations in the functional aspects of these receptors. Our results also implicate the generation of specific regulatory signals that inhibit IRK activity when HL-60 cells are stimulated with a combination of 1;25(OH)2vitD3 and Bt2cAMP.     

Relationships between the cell cycle and the expression of c-myc and transferrin receptor genes during induced myeloid differentiation

We examined the relationship of cellular oncogene c-myc and transferrin receptor (TfR) gene expression to cell proliferation and cell cycle progression during myeloid differentiation in the HL-60 myeloid leukemia cell line. In order to determine levels of mRNA for these genes in HL-60 cells induced to differentiate along the myeloid pathway, RNA was isolated from HL-60 cells incubated with retinoic acid for 24 h and Northern blots were probed with labeled cDNAs for c-myc and TfR. c-myc mRNA decreased within 3 h of retinoic acid addition, and TfR mRNA decreased after 9 h; both mRNAs continued to decrease over 24 h. RNA was also isolated from HL-60 cells separated by centrifugal elutriation into cell cycle phases. TfR and c-myc cDNA probes hybridized equally to RNA from uninduced cells in all phases of the cell cycle. However, after 24 h incubation with the differentiation inducer retinoic acid, TfR mRNA was expressed substantially less in the G1 stage, whereas c-myc mRNA was still expressed equally in all cell cycle phases. These data indicate that, although TfR and c-myc expression are both associated with cell proliferation in the HL-60 line, TfR is down-regulated specifically in G1 upon induction of terminal differentiation whereas c-myc expression is disassociated from cell cycle control in these cells.     

Retinol bound to physiological carrier molecules regulates growth and differentiation of myeloid leukemic cells

We have tested effects of retinol bound to its physiological carrier molecules, i.e. low density lipoprotein chylomicron remnants, and retinol binding protein (RBP) on differentiation and proliferation of myeloid leukemic cells in concentrations that can be obtained in vivo. Data presented in this study show that physiological concentrations of retinyl ester in chylomicron remnants induce differentiation and inhibit proliferation of the cell line HL-60 and promyelocytic leukemic cells in primary culture. Retinyl ester in low density lipoprotein showed no effect either on cell differentiation or proliferation of any of the myeloid cells tested. Retinol bound to RBP induced differentiation of HL-60 cells only in concentrations above those that can be found in vivo. However, cell proliferation was reduced both in HL-60 cells and in primary culture of leukemic cells using physiological concentrations of holo-RBP. These results suggest that retinyl ester in chylomicron remnants is the most effective vehicle for transport of retinol into leukemic cells in vivo.     

Granulocytic differentiation of HL-60 cells is associated with increase of poly-N-acetyllactosamine in Asn-linked oligosaccharides attached to human lysosomal membrane glycoproteins

HL-60 cells were induced to differentiate into granulocytic cells by dimethyl sulfoxide, and structures of Asn-linked oligosaccharides attached to lysosomal membrane glycoproteins (lamp-1 and lamp-2) were elucidated before and after differentiation. Lamp-1 and lamp-2 were immunoprecipitated from the cells after labeling with radioactive sugars, and glycopeptides were prepared. The structures of glycopeptides obtained after serial lectin-affinity chromatography were elucidated by endo-beta-galactoside and methylation analysis. Glycopeptides bound to tomato lectin-Sepharose were found to be tetraantennary oligosaccharides that contain two or three poly-N-acetyllactosaminyl chains, of which one side chain contains three or more N-acetyllactosaminyl repeats, whereas those bound to Datura stramonium agglutinin-Sepharose were found to be tetraantennary oligosaccharides containing one or two short poly-N-acetyllactosaminyl side chains. Glycopeptides that were not bound to concanavalin A, tomato lectin, or D. stramonium agglutinin were found to be triantennary oligosaccharides with a negligible amount of poly-N-acetyllactosaminyl side chains. Comparison of Asn-linked oligosaccharides from undifferentiated and differentiated HL-60 cells reveals the following features. First, the number of Asn-linked oligosaccharides containing poly-N-acetyllactosaminyl side chains increases dramatically with a concomitant decrease in less complex Asn-linked oligosaccharides after differentiation. Second, the number of poly-N-acetyllactosaminyl side chains per Asn-linked oligosaccharides increases significantly. These increases in poly-N-acetyllactosamine were associated with increased activity of UDP-GlcNAc:beta-D-Gal-beta 1----3-N-acetylglucosaminyltransferase "extension enzyme," a key enzyme in the formation of poly-N-acetyllactosamines. Furthermore, the increased amount of poly-N-acetyllactosamine in lamp-1 and lamp-2 resulted in longer half-lives of lamp-1 and lamp-2 in differentiated HL-60 cells. These results suggest strongly that the differentiation of HL-60 cells into more phagocytic cells is associated with an increase in the complexity of Asn-linked oligosaccharides attached to lysosomal membrane glycoproteins, which in turn may play a role in stabilizing lysosomes.     

HL-60-AR白血病细胞诱导分化性状的持续性表达

HGPRT~-人早幼粒白血病细胞突变株(HL-60-AR)与RA保温一定时间后,洗去药物继续培养,细胞分化性状(NBT还原能力、细胞膜C_3补体受体及形态变化)不但继续存在,而且能持续表达。撤去RA后连续传代培养,至少在传三代后细胞分化性状仍高度表达。然而,DMSO对HL-60-AR细胞的作用特点明显不同于RA。HL-60-AR细胞分化伴随增殖能力的降低。核酸分子杂交结果表明,细胞c-myc癌基因表达受抑先于细胞分化性状的获得和增殖能力的下降。     

Induction of the differentiation of HL-60 promyelocytic leukemia cells by L-ascorbic acid

The present study was undertaken to examine the effect of L-ascorbic acid (LAA) on the growth of HL-60 promyelocytic leukemia cells, besides induction of apoptosis. LAA (> or = 10(-4) M) was found to markedly inhibit the proliferation of HL-60 in liquid culture and clonogenicity in semisolid culture. Moreover, LAA-treated HL-60 showed activity to produce chemiluminescence and expressed CD 66b cell surface antigens, indicating that LAA induces the differentiation of HL-60 mainly into granulocytes. The results are supported by morphological changes of LAA-treated HL-60 into segmented neutrophils. Therefore, the inhibitory effect of LAA on the growth of HL-60 cells seems to arise from the induction of differentiation. To assess the potential role of LAA, cells were exposed to oxygen radical scavengers in the absence or presence of LAA. Catalase abolished and superoxide dismutase promoted LAA-induced differentiation of HL-60. Thus, H2O2 produced as a result of LAA treatment seems to play a major role in induction of HL-60 differentiation.     

5－氮－2＇－脱氧胞苷（5－aza－CdR）对HL－60细胞分化和DNA甲基化酶的影响

以５－氮－２＇－脱氧胞苷（５－ａｚａ－ＣｄＲ）为诱导物,在０．５μｍｏｌ／Ｌ的最佳浓度下,可诱导ＨＬ－６０细胞分化达１５％左右。同时,用［￣３Ｈ］－ｍｅｔｈｙｌ－ｓ－ａｄｅｎｏｓｙｌｍｅｔｈｉｏｎｉｎｅ（￣３Ｈ－ＳＡＭ）为底物,通过同位素参入法,测定了不同浓度诱导物对ＨＬ－６０细胞ＤＮＡ甲基化酶活力的影响,发现在最佳诱导物浓度下,可使ＨＬ－６０细胞ＤＮＡ甲基化酶活力明显下降,此外,也比较了不同分化水平的ＨＬ－６０细胞中具有不同甲基化水平的ＤＮＡ在体外接受甲基的能力,从而证明５－ａｚａ－ＣｄＲ诱导ＨＬ－６０细胞分化与其ＤＮＡ甲基化状态密切相关。     

C/EBPalpha inactivation in FAK-overexpressed HL-60 cells impairs cell differentiation

We previously demonstrated that focal adhesion kinase (FAK)-overexpressed (HL-60/FAK) cells have marked resistance against various apoptotic stimuli such as oxidative stress, ionizing radiation and TNF-receptor-induced ligand (TRAIL) compared with vector-transfected (HL-60/Vect) cells. Here, we show that HL-60/FAK cells are highly resistant to all-trans retinoic acid (ATRA)-induced differentiation, whereas original HL-60 or HL-60/Vect cells are sensitive. Treatment with ATRA at 1 muM for 5 days markedly inhibited the proliferation and increased the expression of differentiation markers (CD38, CD11b) in HL-60/Vect cells, but showed no such effect in HL-60/FAK cells. Electrophoretic mobility shift assay (EMSA) using an oligonucleotide for the c/EBP consensus binding sequence showed that c/EBPalpha was activated in ATRA-treated HL-60/Vect cells but not in HL-60/FAK cells, indicating that c/EBPalpha activation by ATRA was impaired in HL-60/FAK cells. In addition, the association of retinoblastoma protein (pRb) and c/EBPalpha after treatment with ATRA was seen in HL-60/Vect cells but not in HL-60/FAK cells. Further, hyperphosphorylation of pRb was observed in HL-60/FAK cells. Finally, the introduction of FAK siRNA into HL-60/FAK cells resulted in the recovery of sensitivity to ATRA-induced differentiation, confirming that the inhibition of HL-60/FAK differentiation resulted from both the induction of pRb hyperphosphorylation and the inhibition of association of pRb and c/EBPalpha.     

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.     

Commitment of neutrophilic differentiation and proliferation of HL-60 cells coincides with expression of transferrin receptor. Effect of granulocyte colony stimulating factor on differentiation and proliferation.

To examine the regulatory mechanisms of proliferation and maturation in neutrophilic lineage cells, we have tried to sort dimethyl sulfoxide (Me(2)SO)-treated HL-60 cells into transferrin receptor (Trf-R) positive (Trf-R(+)) and negative (Trf-R(-)) cells. Differentiated Trf-R(-) cells expressed more formyl-Met-Leu-Phe receptor (fMLP-receptor) and ability of O-(2) genaration, as markers of differentiation, than Trf-R(+) cells, and Trf-R(-) cell differentiation was markedly accelerated by the incubation with granulocyte colony stimulating factor (G-CSF). On the other hand, Trf-R(+) cells had a tendency to proliferate rather than differentiate, and proliferation was enhanced by G-CSF. These results indicate that Trf-R expression coincides with the commitment to proliferate or differentiate of HL-60 cells, and G-CSF accelerates these commitments. G-CSF-induced tyrosine phosphorylation of STAT 3 in Trf-R(-) cells much more than in Trf-R(+) cells. Protein 70 S6 kinase expression was higher in Trf-R(+) cells than in Trf-R(-) cells. Furthermore, p70 S6 kinase was hyperphosphorylated by G-CSF in Trf-R(+) cells, but not in Trf-R(-) cells. Rapamycin, an inhibitor of p70 S6 kinase activity, inhibited G-CSF-dependent proliferation of Trf-R(+) cells and increased fMLP-R expression on these cells. These results suggest that commitment to proliferation and differentiation in Me(2)SO-treated HL-60 cells is preprogrammed and correlated with Trf-R expression, and G-CSF potentiates the cellular commitment. STAT 3 may promote differentiation of Me(2)SO-treated HL-60 cells into neutrophils, while p70 S6 kinase may promote proliferation and negatively regulate neutrophilic differentiation.     

1,25-Dihydroxyvitamin D3-induced differentiation in a human promyelocytic leukemia cell line (HL-60): receptor-mediated maturation to macrophage-like cells

The human-derived promyelocytic leukemia cell line, HL-60, is known to differentiate into mature myeloid cells in the presence of 1,25-dihydroxyvitamin D3 (1,25[OH]2D3). We investigated differentiation by monitoring 1,25(OH)2D3-exposed HL-60 cells for phagocytic activity, ability to reduce nitroblue tetrazolium, binding of the chemotaxin N-formyl-methionyl-leucyl-[3H]phenylalanine, development of nonspecific acid esterase activity, and morphological maturation of Wright-Giemsa-stained cells. 1,25(OH)2D3 concentrations as low as 10(-10) M caused significant development of phagocytosis, nitroblue tetrazolium reduction, and the emergence of differentiated myeloid cells that had morphological characteristics of both metamyelocytes and monocytes. These cells were conclusively identified as monocytes/macrophages based upon their adherence to the plastic flasks and their content of the macrophage-characteristic nonspecific acid esterase enzyme. The estimated ED50 for 1,25(OH)2D3-induced differentiation based upon nitroblue tetrazolium reduction and N-formyl-methionyl-leucyl-[3H]phenylalanine binding was 5.7 X 10(-9) M. HL-60 cells exhibited a complex growth response with various levels of 1,25(OH)2D3: less than or equal to 10(-10) M had no detectable effect, 10(-9) M stimulated growth, and greater than or equal to 10(-8) M sharply inhibited proliferation. We also detected and quantitated the specific receptor for 1,25(OH)2D3 in HL-60 and HL-60 Blast, a sub-clone resistant to the growth and differentiation effects of 1,25(OH)2D3. The receptor in both lines was characterized as a DNA-binding protein that migrated at 3.3S on high-salt sucrose gradients. Unequivocal identification was provided by selective dissociation of the 1,25(OH)2D3-receptor complex with the mercurial reagent, p-chloromercuribenzenesulfonic acid, and by a shift in its sedimentation position upon complexing with anti-receptor monoclonal antibody. On the basis of labeling of whole cells with 1,25(OH)2[3H]D3 in culture, we found that HL-60 contains approximately 4,000 1,25(OH)2D3 receptor molecules per cell, while the nonresponsive HL-60 Blast is endowed with approximately 8% of that number. The concentration of 1,25(OH)2D3 (5 X 10(-9) M) in complete culture medium, which facilitates the saturation of receptors in HL-60 cells, is virtually identical to the ED50 for the sterol's induction of differentiation. This correspondence, plus the resistance of the relatively receptor-poor HL-60 Blast, indicates that 1,25(OH)2D3-induced differentiation of HL-60 cells to monocytes/macrophages is occurring via receptor-mediated events.     

Development of specific functionally active receptors for platelet-activating factor in HL-60 cells following granulocytic differentiation

A human promyelocytic leukemia cell line (undifferentiated HL-60 cells) as well as a granulocyte form of HL-60 cells induced in vitro by exposure to dimethyl sulfoxide were examined for binding, metabolism, and biological responses to platelet-activating factor (PAF). Undifferentiated and differentiated HL-60 cells each exhibit a high capacity to incorporate and metabolize [3H]PAF at 37 degrees C; however, the amount of [3H]PAF that is assimilated by both cell populations is greatly reduced and its metabolism abolished at less than or equal to 4 degrees C. At 0 degrees C HL-60 granulocytes bind more [3H]PAF than their undifferentiated counterparts. Binding to differentiated cells reaches equilibrium within 80 min and is saturable, reversible and specific; PAF receptor antagonists WEB 2086, L-659,989, BN 52021, and kadsurenone abolish this specific [3H]PAF binding. In contrast, [3H]PAF uptake by undifferentiated HL-60 cells is neither saturable nor sensitive to specific receptor antagonists. Scatchard analyses reveal 5850 +/- 850 binding sites per differentiated HL-60 cell with a dissociation constant of 0.66 +/- 0.15 nM. In the presence of cytochalasin B, PAF (200 nM) induces degranulation only in differentiated cells and this response also is blocked by PAF receptor antagonists. Our results demonstrate that HL-60 cells develop specific and functionally active PAF receptors only after chemically induced differentiation into granulocytes.