Induction of differentiation of HL-60 cells by protein kinase C inhibitor, K252a

To clarify the role of protein kinase C and protein kinase A in cell proliferation and differentiation, the effects of K252a and its derivatives (K252b, KT5720), which have different inhibitory activity to these protein kinases, on the proliferation and differentiation of HL-60 cells were investigated. The proliferation and DNA synthesis of the HL-60 cells were inhibited by K252a in a dose dependent manner. However, K252b and KT5720 which are more specific inhibitors of protein kinase C or protein kinase A, respectively, had no observable effect on cell proliferation. K252a (40nM) enhanced the differentiation of HL-60 cells induced by 1,25(OH)2D3, retinoic acid and DMSO. K252b and KT5720 did not affect 1,25(OH)2D3-induced differentiation. K252a significantly inhibited the differentiation induced by PMA. These results demonstrate that K252a but not its derivatives can function as an antitumor drug and enhancer of the differentiation induced by various inducers.     

1,25-(OH)_2D_3诱导HL-60细胞分化后胞液Ca~(2+)浓度、磷酸化酶a和微粒体Ca~(2+)-ATP酶活性的改变

1,25-(OH)_2D_3对HL-60细胞具有促分化作用。本文报道了1,25-(OH)_2D_3在促进HL-60细胞分化前后胞液Ca~(2+)浓度、磷酸化酶a和微粒体Ca~(2+)-ATP酶活性的改变。结果表明,1,25-(OH)_2D_3加入HL-60细胞培养液后72小时,细胞NBT染色阳性率高于70％,形态向正常分化的细胞转化。同对,胞液Ca~(2+)浓度和微粒体Ca~(2+)-ATP酶活性明显降低,而磷酸化酶a活性显著升高。结果提示,在1,25-(OH)2_D_3作用下,HL-60细胞不仅杀菌功能增强,细胞内胞液Ca~(2+)浓度趋向正常,与钙恒稳有关的酶活性也同样发生改变。即1,25-(OH)_2D_3对HL-60细胞的诱导作用伴有钙恒稳的改变。这些变化与DMSO的作用相同。     

Failure of 1-oleoyl-2-acetylglycerol to mimic the cell-differentiating action of 12-O-tetradecanoylphorbol 13-acetate in HL-60 cells

Treatment of human promyelocytic leukemia cells (HL-60 cells) with 12-O-tetradecanoylphorbol 13-acetate (TPA) results in terminal differentiation of the cells to macrophage-like cells. Treatment of the cells with TPA induced marked enhancement of the phosphorylation of 28- and 67-kDa proteins and a decrease in that of a 75-kDa protein. When the cells were treated with diacylglycerol, i.e. 50 micrograms/ml 1-oleoyl-2-acetylglycerol (OAG), similar changes in the phosphorylation of 28-, 67-, and 75-kDa proteins were likewise observed, indicating that OAG actually stimulates protein kinase C in intact HL-60 cells. OAG (1-100 micrograms/ml), which we used, activated partially purified mouse brain protein kinase C in a concentration-dependent manner. Treatment of HL-60 cells with 10 nM TPA for 48 h caused an increase by about 8-fold in cellular acid phosphatase activity. Although a significant increase in acid phosphatase activity was induced by OAG, the effect was scant compared to that of TPA (less than 7% that of TPA). After 48-h exposure to 10 nM TPA, about 95% of the HL-60 cells adhered to culture dishes. On the contrary, treatment of the cells either with OAG (2-100 micrograms/ml) or phospholipase C failed to induce HL-60 cell adhesion. Ca2+ ionophore A23187 failed to act synergistically with OAG. In addition, hourly or bi-hourly cumulative addition of OAG for 24 h also proved ineffective to induce HL-60 cell adhesion. Our present results do not imply that protein kinase C activation is nonessential for TPA-induced HL-60 cell differentiation, but do demonstrate that protein kinase C activation is not the sole event sufficient to induce HL-60 cell differentiation by means of this agent.     

Ca2+ priming during vitamin D-induced monocytic differentiation of a human leukemia cell line

1,25-Dihydroxyvitamin D3 (1,25-(OH)2D3) induces monocytic differentiation of the human promyelocytic leukemia line, HL-60, and enhances Ca2+ transport in target cells of the mineral metabolism system. Hence, we determined whether the steroid's maturational effect on HL-60 involves alterations of intracellular calcium [( Ca2+]i). We found that, as detected by indo-1 fluorescence, [Ca2+]i increases in a slow tonic manner from 99 +/- 11 nM in virgin HL-60 to 182 +/- 19 nM (p less than 0.001) in those treated with 1,25-(OH)2D3 for 24 h. The first apparent rise in [Ca2+]i occurs at between 6 and 12 h and parallels expression of alpha-thrombin and N-formyl-methionyl-leucyl-phenylalanine (fMLP) receptors. This increase in [Ca2+]i is derived from extracellular calcium as its reduction abolishes the effect. The increase in [Ca2+]i is associated with an increase in inositol trisphosphate-stimulated Ca2+ flux from intracellular stores. Interestingly, 1,25-(OH)2D3-mediated HL-60 differentiation as manifest by expression of the macrophage-specific antigen, 63D3, is not blocked by low extracellular calcium. In contrast, the fMLP-induced superoxide ion generation is diminished if the increase in [Ca2+]i is prevented. Furthermore, fMLP-stimulated signal transduction is also reduced by limiting the stimulation of [Ca2+]i during 1,25-(OH)2D3 treatment. Thus, although differentiation of HL-60 to the monocytic phenotype by 1,25-(OH)2D3 is Ca2+-independent, expression of response to regulatory stimuli requires priming of cellular Ca2+ stores. The latter appears to be induced by 1,25-(OH)2D3 via stimulated Ca2+ entry through the plasma membrane.     

Ca2+-independent synergistic augmentation of O2- production by FMLP and PMA in HL-60 cells

N-Formyl-Met-Leu-Phe (FMLP) and phorbol 12-myristate 13-acetate (PMA) caused a synergistic augmentation of superoxide anion (O2-) production in neutrophil-like HL-60 cells differentiated with dibutyryl cAMP. The present study was undertaken to investigate the mechanism of the synergistic augmentation of O2- production. FMLP increased intracellular free Ca2+ concentration ([Ca2+]i), which was slightly suppressed by PMA and completely inhibited by an intracellular Ca2+ chelating agent, O,O'-bis(2-aminophenyl)ethyleneglycol-N,N,N',N'-tetraacetic acid tetraacetoxymethyl ester (BAPTA-AM). Although FMLP-induced O2- production was inhibited by BAPTA-AM, a major part of the synergistic augmentation of O2- production by FMLP and PMA remained after BAPTA-AM treatment, suggesting that a Ca2+-independent mechanism might be involved in the augmentation. FMLP and PMA caused an activation of phospholipase D (PLD) almost additively in a Ca2+-sensitive manner. The synergistic activation of mitogen-activated protein kinase (MAPK) was evoked by combined addition of PMA and FMLP in a BAPTA-AM resistant manner. However, PD98059, a MAPK kinase inhibitor, did not affect the synergistic augmentation of O2- production, although it potently inhibited the synergistic augmentation of tyrosine phosphorylation of MAPK. Wortmannin, a phosphatidylinositol 3-kinase inhibitor, inhibited FMLP-induced O2- production, but it did not inhibit the synergistic augmentation of O2- production by PMA and FMLP. In contrast, staurosporine and GF109203X, protein kinase C inhibitors, reduced the synergistic augmentation induced by PMA and FMLP. In addition, pertussis toxin (PT) abolished the synergistic augmentation of O2- production. It is concluded that the synergistic augmentation of O2- production induced by PMA and FMLP is mediated through a PT-sensitive G protein and a protein kinase C in a Ca2+-independent manner.     

fMLP-induced arachidonic acid release in db-cAMP-differentiated HL-60 cells is independent of phosphatidylinositol-4, 5-bisphosphate-specific phospholipase C activation and cytosolic phospholipase A(2) activation

In inflammatory cells, agonist-stimulated arachidonic acid (AA) release is thought to be induced by activation of group IV Ca(2+)-dependent cytosolic phospholipase A(2) (cPLA(2)) through mitogen-activated protein kinase (MAP kinase)- and/or protein kinase C (PKC)-mediated phosphorylation and Ca(2+)-dependent translocation of the enzyme to the membrane. Here we investigated the role of phospholipases in N-formylmethionyl-l-leucyl-l-phenylalanine (fMLP; 1 nM-10 microM)-induced AA release from neutrophil-like db-cAMP-differentiated HL-60 cells. U 73122 (1 microM), an inhibitor of phosphatidyl-inositol-4,5-biphosphate-specific phospholipase C, or the membrane-permeant Ca(2+)-chelator 1, 2-bis?2-aminophenoxy?thane-N,N,N',N'-tetraacetic acid (10 microM) abolished fMLP-mediated Ca(2+) signaling, but had no effect on fMLP-induced AA release. The protein kinase C-inhibitor Ro 318220 (5 microM) or the inhibitor of cPLA(2) arachidonyl trifluoromethyl ketone (AACOCF(3); 10-30 microM) did not inhibit fMLP-induced AA release. In contrast, AA release was stimulated by the Ca(2+) ionophore A23187 (10 microM) plus the PKC activator phorbol myristate acetate (PMA) (0.2 microM). This effect was inhibited by either Ro 318220 or AACOCF(3). Accordingly, a translocation of cPLA(2) from the cytosol to the membrane fraction was observed with A23187 + PMA, but not with fMLP. fMLP-mediated AA release therefore appeared to be independent of Ca(2+) signaling and PKC and MAP kinase activation. However, fMLP-mediated AA release was reduced by approximately 45% by Clostridium difficile toxin B (10 ng/ml) or by 1-butanol; both block phospholipase D (PLD) activity. The inhibitor of phosphatidylcholine-specific phospholipase C (PC-PLC), D609 (100 microM), decreased fMLP-mediated AA release by approximately 35%. The effect of D609 + 1-butanol on fMLP-induced AA release was additive and of a magnitude similar to that of propranolol (0.2 mM), an inhibitor of phosphatidic acid phosphohydrolase. This suggests that the bulk of AA generated by fMLP stimulation of db-cAMP-differentiated HL-60 cells is independent of the cPLA(2) pathway, but may originate from activation of PC-PLC and PLD.     

Inhibition of phospholipid/Ca2+-dependent protein kinase and phosphorylation of leukemic cell proteins by CP-46,665-1, a novel antineoplastic lipoidal amine

CP-46,665-1, an antineoplastic lipoidal amine, was found to inhibit phospholipid/Ca2+-dependent protein kinase (PL/Ca-PK, or protein kinase C), with an IC50 (concentration causing a 50% inhibition) of 10 microM. Its inhibition of the enzyme was reversed by phosphatidylserine, but not by Ca2+. The agent also inhibited the enzyme activity which was further augmented by 12-0-tetradecanoylphorbol-13-acetate (TPA), mezerein or diolein. Phosphorylation of endogenous proteins from HL-60 cells by the enzyme, with or without being further augmented by TPA, was inhibited by CP-46,665-1 as well as by alkyllysophospholipid (an antineoplastic agent). CP-46,665-1, while without effect on cyclic AMP-dependent protein kinase, also inhibited myosin light chain kinase (a calmodulin/Ca2+-dependent protein kinase). The present findings suggest that inhibition of the Ca2+-effector enzymes may be related in part to the antimetastatic activity of the lipoidal amine.     

Phorbol myristate acetate inhibits phosphoinositol lipid-specific phospholipase C activity via protein kinase C activation in conditions inducing differentiation in HL-60 cells.

We have studied, in streptolysin O-permeabilized HL-60 cells and in HL-60 membrane preparations, the effects of phorbol 12-myristate 13-acetate (PMA) on polyphosphoinositide-specific phospholipase C (PLC) activity and on terminal differentiation towards macrophagic-like cells. We showed that terminal differentiation was induced when differentiating concentrations of the drug were present for only 1-2 h in the culture medium. Conditions inducing differentiation also inhibited PLC activity for a long lasting period (at least 5 h). When terminal differentiation affected only part of the cell population, inhibition of phospholipase C activity was found to be less marked and reversible over the period studied. Moreover in experiments done in an HL-60 clone resistant to PMA, no inhibition of PLC activity was provoked by this tumour promotor. In order to study the involvement of protein kinase C in this process, we measured modifications of PLC activity by PMA in the presence of two different protein kinase C inhibitors, staurosporine and H-7. They both prevented the inhibition of PLC activity by PMA indicating that this inhibition is likely to be related to the effect of PMA on protein kinase C activity. This was also confirmed by the fact that active protein kinase C, by itself, was able to decrease PLC activity when added to membrane preparations or to streptolysin O-permeabilized control HL-60 cells. These results indicate that PMA acts in inhibiting phospholipase C activity through its effect on protein kinase C activation and/or on protein kinase C translocation to the plasma membrane and that terminal differentiation, might be related to changes in both protein kinase C and PLC activities.     

Retinoic acid-induced differentiation-specific, C-kinase-dependent phosphorylation of cytosolic 44 and 32 kDa proteins in HL-60 cells

The effect of various differentiation-inducers on the activity of Ca2+, phospholipid-dependent protein kinase (C-kinase) activity and endogenous protein phosphorylation by the kinase were examined in the extracts of HL-60 cells. Although all of the inducers, retinoic acid, dibutyryl cAMP, nicotinamide, dimethylsulfoxide, and 3-aminobenzamide increased the cytosolic C-kinase activity accompanied with the differentiation into mature myelocytes, only retinoic acid markedly enhanced Ca2+, phospholipid-dependent phosphorylation of 44 and 32 kDa proteins in the cytosol. These results suggest that the differentiation pathway induced by retinoic acid is different from the pathways induced by other inducers.     

Cytosolic calcium regulates phorbol diester binding affinity in intact phagocytes

The mobilization of internally sequestered stores of Ca2+ and activation of protein kinase C appear to be involved in neutrophil activation. We have examined the inter-relationship of these two pathways by investigating the effects of modulating Ca2+ activity on the binding of [3H]phorbol 12,13-dibutyrate (PDBU) to protein kinase C in intact phagocytes. Differentiated HL-60 cells were equilibrated with [3H]PDBU prior to stimulation with various agents known to alter Ca2+ homeostasis in cells. Agents that elevated cytosolic Ca2+, such as f-Met-Leu-Phe and A23187, up-regulated radioligand binding by increasing the affinity of the PDBU/protein kinase C interaction. These effects were time- and agonist concentration-dependent and temperature-sensitive. The kinetics of the up-regulation of binding by f-Met-Leu-Phe coincided with the kinetics of Ca2+ mobilization (by quin2 fluorescence measurements). The putative intracellular Ca2+ antagonist 8-(N,N-diethylamino)-octyl 3,4,5-trimethoxybenzoate alone down-regulated [3H]PDBU binding and inhibited the up-regulation of ligand binding by f-Met-Leu-Phe and A23187. Low concentrations of La3+ (0.1-10 microM) also inhibited up-regulation of radioligand binding to f-Met-Leu-Phe and A23187, whereas higher concentrations (0.1-1 mM) alone increased [3H] PDBU binding and supported further up-regulation of ligand binding by the Ca2+-mobilizing agents. These data suggest a role for Ca2+ in the regulation of phorbol diester binding to protein kinase C in intact cells.     

Ouabain- and Ca2(+)-sensitive ATPase activity of chimeric Na- and Ca-pump molecules.

HL-60 cells are very sensitive to the cytotoxic action of ether lipids. Several hypotheses have been proposed to explain this cytotoxicity. We investigated the influence of the alkylphospholipid ET-18-OCH₃ on the activity of protein kinase C. HL-60 cells were incubated with ET-18-OCH₃ at a concentration of 20 μg/ml for 4 h. After the incubation the membrane fraction of the HL-60 cells was isolated and the activity of protein kinase C was determined while it was still associated with the membrane, using the synthetic peptide substrate [Ser²⁵]-protein kinase C (19–31) as a protein kinase C specific substrate. The activity of the membrane-bound protein kinase C was increased in HL-60 cells treated with ET-18-OCH₃ compared to untreated HL-60 cells. The increase in protein kinase C activity was not a consequence of translocation and appeared to be additive to the effect of the phorbol ester 12-myristate 13-acetate. In contrast, solubilized protein kinase C from HL-60 cells could be inhibited or stimulated in vitro by ET-18-OCH₃, dependent on the mode of addition of ET-18-OCH₃ and phospholipids.     

1-(5-Isoquinolinesulfonyl)-2-methylpiperazine(H-7), a potent inhibitor of protein kinases, inhibits the differentiation of HL-60 cells induced by phorbol diester

Treatment of the human promyelocytic leukemia cell line HL-60, with 12-o-tetradecanoylphorbol acetate (TPA) results in the differentiation into macrophage-like cell. A potent inhibitor of protein kinase C, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine(H-7), suppressed the proliferation of HL-60 cells and also inhibited TPA-induced cell differentiation of these cells. N-(2-guanidinoethyl)-5-isoquinolinesulfonamide(HA-1004), a weaker analog of H-7, failed to inhibit this TPA-induced cell differentiation. H-7 also inhibited TPA-induced protein phosphorylation in these cells. Thus, protein kinase C-mediated phosphorylation may be involved in the process of TPA-induced HL-60 cell differentiation.     

Activation of CMP-N-acetylneuraminic acid:lactosylceramide sialyltransferase during the differentiation of HL-60 cells induced by 12-O-tetradecanoylphorbol-13-acetate

We previously reported that the synthesis of NeuAc(alpha 2-3)Gal(beta 1-4)GlcCer (GM3) ganglioside was preferentially enhanced during the differentiation of HL-60 cells into a monocyte/macrophage lineage induced by 12-O-tetradecanoylphorbol-13-O-acetate (TPA). Since exogenously added GM3 ganglioside was shown to be able to induce the differentiation of HL-60 cells into the monocyte/macrophage lineage in a synthetic medium, the functional role of the GM3 ganglioside increase during the differentiation of HL-60 cells has become the subject of much interest. In the present study, we investigated the activity of CMP-NeuAc:lactosylceramide sialyltransferase, which catalyzes the synthesis of GM3 ganglioside from lactosylceramide, in cells undergoing differentiation induced by two different reagents, TPA and 1 alpha,25-dihydroxy-vitamin D3, which induce the differentiation of HL-60 cells into the monocyte/macrophage lineage through different modes of action. We showed that the activation of CMP-NeuAc:lactosylceramide sialyltransferase and the increase in GM3 ganglioside were not related to the differentiated lineage but to the specific action of TPA, i.e. activation of protein kinase C.     

Anti-proliferative effect of two novel palmitoyl-carnitine analogs, selective inhibitors of protein kinase C conventional isoenzymes.

Previous studies have shown that palmitoyl-carnitine is an anti-proliferative agent and a protein kinase C inhibitor. Two new palmitoyl-carnitine analogs were synthesized by replacing the ester bond with a metabolically more stable ether bond. An LD50 value in the nM range was found in anti-proliferative assays using HL-60 cells and was dependent on the alkyl-chain length. The inhibitory action of these water-soluble compounds on protein kinase C in vitro was greatly increased with respect to palmitoyl-carnitine and was dependent on the length of the alkyl chain. Its effect was mediated by an increase in the enzyme's requirement for phosphatidylserine. Inhibition of the in situ phosphorylation of a physiological platelet protein kinase C substrate and of phorbol ester-induced differentiation of HL-60 cells was also observed. Finally, to test for isoenzyme selectivity, several human recombinant protein kinase C isoforms were used. Only the Ca2+-dependent classic protein kinase Cs (alpha, betaIota, betaIotaIota and gamma) were inhibited by these compounds, yet the activities of casein kinase I, Ca2+/calmodulin-dependent kinase and cAMP-dependent protein kinase were unaffected. Thus, these novel inhibitors appear to be both protein kinase C and isozyme selective. They may be useful in assessing the individual roles of protein kinase C isoforms in cell proliferation and tumor development and may be rational candidates for anti-neoplasic drug design.     

Modulation of monocytic differentiation of HL-60 cells by inhibitors of protein tyrosine kinases.

We showed previously that the expressions of various src family protein tyrosine kinases (PTKs) were induced independently during the monocytic differentiation of HL-60 cells. The role of PTKs was further assessed in the present study by investigating the effects of PTK inhibitors on the differentiation. It was demonstrated that PTK inhibitors such as genistein and herbimycin A modulated monocytic differentiation of HL-60 cells; they inhibited the differentiation induced by TPA, while promoting that induced by vitamin D3 (D3). Immunoblotting analysis of protein molecules which had been phosphorylated on their tyrosine residues demonstrated that TPA induced phosphorylation of certain molecules different from those induced by D3 in HL-60 cells. PTK inhibitors blocked the phosphorylation and modulated differentiation driven by the inducers. These data suggest that PTKs are involved both promotively and suppressively in signaling events that induce monocytic differentiation of HL-60 cells.     

Histamine stimulated synaptosomal Ca2+ uptake through activation of calcium channels

Histamine stimulated Ca2+ uptake in synaptosomes was completely inhibited by the slow Ca2+ channel antagonists verapamil, cinnarizine and flunarizine, and slightly inhibited by nifedipine and diltiazem. Ca2+ uptake in synaptosomes depolarized or predepolarized with varying K+ concentrations was increased by histamine, in both conditions, until 30mM K+. At higher K+ concentrations histamine was not able to alter K+ effects in either conditions. 30mM K+ stimulated uptake of Ca2+ in the absence or presence of histamine was not inhibited by verapamil and diltiazem. However nifedipine slightly inhibited K+ and K+ +histamine effects. 3-Isobutyl-1-methyl-xanthine and dibutyryl cyclicAMP potentiated (10%) the uptake of Ca2+ in synaptosomes induced by histamine. Dibutyryl cyclicAMP alone however decreased the basal Ca2+ uptake in a concentration-dependent manner. Verapamil, but not diltiazem, antagonized the effects elicited by 3-isobutyl-1-methyl-xanthine and dibutyryl cyclicAMP in the presence of histamine. The data suggest that the increase in synaptosomal Ca2+ uptake induced by histamine is mediated by the activation of the voltage sensitive calcium channels, and possibly a cyclicAMP-dependent protein kinase phosphorylation can modulate the opening of Ca2+ channels.     

Sphingosine-induced c-jun expression: differences between sphingosine- and C2-ceramide-mediated signaling pathways

Sphingolipids such as ceramide and sphingosine are putative intracellular signal mediators in cell differentiation, growth inhibition and apoptosis. Previously, we reported that C2-ceramide induced c-jun expression in apoptosis of human leukemia HL-60 cells. Here we report that sphingosine also induced c-jun expression in apoptosis of HL-60 cells. Sphingosine-induced c-jun expression was stimulated by H-89, a protein kinase A inhibitor, whereas C2-ceramide-induced c-jun expression was inhibited by protein kinase C inhibitors. Furthermore, H-89 potentiated sphingosine-induced but not C2-ceramide-induced growth inhibition. These results suggest that sphingosine and C2-ceramide might induce c-jun expression and apoptosis in distinct signaling pathways.     

Development of receptors for leukotriene B4 on HL-60 cells induced to differentiate by 1 alpha,25-dihydroxyvitamin D3

The incubation of HL-60 human promyelocytic leukemia cells for 7 days with 100 nM 1 alpha,25-dihydroxyvitamin D3 [1,25(OH)2D3] induced differentiation into monocyte-like cells, as assessed by morphologic and biochemical characteristics. Stereospecific receptors for leukotriene B4 (LTB4) developed on the surface of the HL-60 cell-derived monocytes that had the capacity to transduce LTB4 stimulation of a transient increase in the cytosolic concentration of calcium ([Ca+2]in). HL-60 cell-derived monocytes, but not undifferentiated HL-60 cells, expressed a high affinity subset of 6400 +/- 3700 receptors per cell with a dissociation constant (Kd) of 2.3 +/- 1 nM (mean +/- SD, n = 3) and a low affinity subset of approximately 2.2 X 10(6) receptors per cell with an apparent Kd of 680 +/- 410 nM. Derivatives of LTB4 inhibited the binding of [3H]LTB4 to HL-60 cell-derived monocytes with a rank order of potency of LTB4 greater than 20-OH-LTB4 greater than 3-aminopropyl amide-LTB4, which is similar to the order for LTB4 receptors of human blood PMNL. In contrast, leukotrienes C4 and D4 and formyl-methionyl chemotactic peptides did not inhibit the binding of [3H] LTB4, which demonstrates the specificity of these receptors for isomers of 5,12-dihydroxy-eicosatetraenoic acid. LTB4 stimulated an increase in [Ca+2]in in HL-60 cell-derived monocytes which reached 50% of the maximal level at an LTB4 concentration of 0.5 nM (EC50). Preincubation of HL-60 cell-derived monocytes with 10 nM LTB4 resulted in a selective loss of high affinity receptors, as assessed by binding of [3H]LTB4, and a 200-fold increase in the EC50 for stimulation by LTB4 of increases in [Ca+2]in, without alterations in either the low affinity receptors for LTB4 or the responsiveness of [Ca+2]in to formyl-methionyl chemotactic peptides. HL-60 cells that are induced to differentiate into monocytes thus develop stereospecific receptors for LTB4 with binding and transductional characteristics similar to those of human blood PMNL.