Double-minute chromatin bodies in HL-60 leukemia cells sensitive and resistant to differentiation inducing agents

We studied the chromosome characteristics of HL-60 promyelocytic leukemia cells sensitive and resistant to differentiation inducing agents (DI). The karyotypic analysis of sensitive (HL-60 S) and resistant (HL-60 R) cells revealed the presence of identical chromosome abnormalities such as loss of chromosomes 5, 9, 10, 14, 16, 17 and X; and gain of chromosome 18. HL-60 S and HL-60 R cells also share five common markers. The difference between the two cell lines consisted essentially of the loss of an unidentifiable chromosome segment in the HL-60 R cell line. In addition, the two sublines showed marked differences in the content of double-minute chromatin bodies (DM), which were abundant in HL-60 S but rarely found in HL-60 R cells. Contrary to a previous report by others, there was no evidence of chromosome rearrangement of the DM as homogeneously stained regions (HSR) or abnormally banding regions (ABR) in the resistant HL-60 R cells. The presence of DM as an expression of gene amplification may be of relevance in the determination of susceptibility of HL-60 cells to DI.     

Myeloperoxidase is involved in H2O2-induced apoptosis of HL-60 human leukemia cells

We examined the mechanism of H(2)O(2)-induced cytotoxicity and its relationship to oxidation in human leukemia cells. The HL-60 promyelocytic leukemia cell line was sensitive to H(2)O(2), and at concentrations up to about 20-25 micrometer, the killing was mediated by apoptosis. There was limited evidence of lipid peroxidation, suggesting that the effects of H(2)O(2) do not involve hydroxyl radical. When HL-60 cells were exposed to H(2)O(2) in the presence of the spin trap alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone (POBN), we detected a 12-line electron paramagnetic resonance spectrum assigned to the POBN/POBN(.) N-centered spin adduct previously described in peroxidase-containing cell-free systems. Generation of this radical by HL-60 cells had the same H(2)O(2) concentration dependence as initiation of apoptosis. In contrast, studies with the K562 human erythroleukemia cell line, which is often used for comparison with the HL-60, and with high passaged HL-60 cells (spent HL-60) studied under the same conditions failed to generate POBN(.). Cellular levels of antioxidant enzymes superoxide dismutase, glutathione peroxidase, and catalase did not explain the differences between these cell lines. Interestingly, the K562 and spent HL-60 cells, which did not generate the radical, also failed to undergo H(2)O(2)-induced apoptosis. Based on this we reasoned that the difference in H(2)O(2)-induced apoptosis might be due to the enzyme myeloperoxidase. Only the apoptosis-manifesting HL-60 cells contained appreciable immunoreactive protein or enzymatic activity of this cellular enzyme. When HL-60 cells were incubated with methimazole or 4-aminobenzoic acid hydrazide, which are inhibitors of myeloperoxidase, they no longer underwent H(2)O(2)-induced apoptosis. Hypochlorous acid stimulated apoptosis in both HL-60 and spent HL-60 cells, indicating that another oxidant generated by myeloperoxidase induces apoptosis and that it may be the direct mediator of H(2)O(2)-induced apoptosis. Taken together these observations indicate that H(2)O(2)-induced apoptosis in the HL-60 human leukemia cell is mediated by myeloperoxidase and is linked to a non-Fenton oxidative event marked by POBN(.).     

Induction of differentiation in HL-60 promyelocytic cells: a comparative study in two sublines

Two variants of HL-60 promyelocytic leukemia cells (HSC, OCI) that were indistinguishable by morphology, cell surface markers, DNA histograms, and by their inability to reduce nitroblue tetrazolium, were induced to differentiate by retinoic acid (RA), 12-O-tetradecanoyl-phorbol-13-acetate (TPA), and by phytohemagglutinin-leucocyte conditioned medium (PHA-LCM). Only OCI cells were induced to differentiate to mature granulocytes by TPA. Both cell lines expressed, however, the monocytic associated cell surface antigen detected by MO1 monoclonal antibody in response to TPA. MO1 expression was detected as early as 32 hours after initiation of differentiation by TPA, whereas partial morphologic changes were apparent only after 72 hours. Induction of differentiation by retinoic acid led to a significant inhibition of colony formation in HSC variant (from 1522 +/- 60 to 523 +/- 20/10(4) cells plated) and in the OCI variant (from 628 +/- 20 to 185 +/- 33 colonies/10(4) cells plated). The addition of PHA-LCM further inhibited colony growth of both RA-induced cell lines (155 +/- 7/10(4) cells plated in HSC, and 59 +/- 4 in OCI). PHA-LCM by itself reduced HL-60 colony numbers in a dose-related manner, and also increased the expression of MO1 on noninduced HSC and OCI cells. These observations suggest that differentiation of HL-60 cells is not necessarily accompanied by concomitant change in morphology, cell surface characteristics, and proliferation potentials, and may be dependent on different degrees of cellular commitment. They also suggest a role for growth factors in the induction to maturation of leukemic cells.     

Proteomic analysis on multi-drug resistant cells HL-60/DOX of acute myeloblastic leukemia

Multi-drug resistance (MDR) is an important factor that causes treatment failure in acute leukemia. However, the full development mechanisms of MDR still await [corrected] investigation. The purpose of this study is to investigate differentially expressed proteins in the multi-drug resistant acute myeloblastic leukemia (AML) cell line HL-60/DOX and the drug sensitive cell line HL-60, and to identify new potential multi-drug resistant related molecules with the proteomic approach. Two-dimensional gel electrophoresis (2-DE) maps of the proteins, extracted from two AML cell lines, HL-60/DOX and HL-60, were established respectively. The extracted proteins were digested by enzymes and identified with the matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS). The data of the peptide mass fingerprinting (PMF) was matched with databases of proteomics available on the Internet. Results showed that 16 proteins were identified to be differentially expressed between HL-60/DOX and HL-60 cells. They involved the protein disulfide isomerase precursor (PDI), the proteasomes alpha1 and other proteins which are related to drug resistance or cell metabolism, but their functional significances are required further investigation. Nevertheless, it is clear that this proteomic approach for studing the biology and development of MDR is a prerequisite in leukemia.     

Sodium Valproate Facilitates the Propagation of Granulocytic Ehrlichiae (Anaplasma phagocytophilum) in HL-60 Cells

As already shown, some inducers of the differentiation of promyelocytic cells along the granulocytic pathway, such as dimethylsulphoxide (DMSO) or all-trans retinoic acid, can enhance propagation of granulocytic ehrlichiae in HL-60 cell cultures. This study was conducted to prove whether sodium valproate, a salt of di-n-propylacetic acid (VPA) known to trigger cellular differentiation in several solid and hematopoietic malignancies is similarly efficient in ehrlichial cultures. Two cell lines derived from HL-60, that is, low-passage undifferentiated HL-60 (HL-60F) and high-passage HL-60 spontaneously differentiated towards monocytic phenotype (HL-60J) were grown in RPMI 1640 medium supplemented with 10% FBS. The respective HL-60F and HL-60J IC₅₀-values for NaVPA were estimated to be 0.8 and 2.2 mM under these culture conditions; to stimulate the differentiation, the respective doses of 0.3 and 1.2 mM were then applied. When the NaVPA-treated cells of both lines were challenged with an ehrlichial laboratory strain (HGE), maintained in splenectomized NMRI mice, the respective 1–2 and ≤0.1% primary infection rates in HL-60F and HL-60J cultures were observed 3 days post-inoculation. In comparison, only rare (≤0.1%) infected HL-60F and no infected HL-60J cells were recorded under the same experimental conditions in untreated control cultures. HGE continuously propagated in NaVPA-supplemented HL-60F cultures remained infectious to mice at least up to the 95th passage (12 months). NaVPA can thus facilitated continuous propagation of granulocytic ehrlichiae in cell cultures without a substantial loss of infectiveness. This revised version was published online in July 2006 with corrections to the Cover Date.     

Evidence of multifactorial mechanisms in an adriamycin-resistant HL-60 promyelocytic leukemia cell line

HL-60/AR leukemia cells, which were 60-fold resistant to the growth inhibitory activity of adriamycin, remained sensitive to the antiproliferative and differentiation-inducing activities of aclacinomycin A. The replication of HL-60/AR and of adriamycin sensitive parental HL-60 cells was inhibited by greater than 80% by 30 nM aclacinomycin A and the majority of cells (about 60 to 70%) of each line underwent granulocytic differentiation when treated with this agent, as assessed by the reduction of nitroblue tetrazolium. Measurement of the initial rates of uptake of daunorubicin and steady-state levels of adriamycin in sensitive and resistant lines indicated that transport differences do not fully account for the insensitivity of HL-60/AR cells to these anthracyclines. Furthermore, 30-fold greater levels of cell-associated adriamycin were required in HL-60/AR cells for toxic effects equivalent to those occurring in parental HL-60 cells. Analysis of DNA histograms of adriamycin treated HL-60 cells indicated that cell-cycle progression was blocked in G2-M, while this antibiotic blocked progression of resistant HL-60/AR cells in the S phase. These results suggest that, in addition to alterations in membrane permeability, differential sensitivity of multiple biochemical targets may be important in the toxicity and the development of resistance to anthracyclines. Furthermore, the finding that HL-60/AR cells do not exhibit cross-resistance to aclacinomycin A indicates that this oligosaccharide-containing anthracycline may have utility in the treatment of adriamycin resistant neoplasms.     

Ethanol induced apoptosis in human HL-60 cells

In this study flow cytometric and morphologic methods of apoptosis detection in human promyelocytic leukemia cell line HL-60 were compared. HL-60 cells were harvested at 4, 7, 16, 24 a 48 hours after induction of apoptosis by 3 % ethanol. Little changes were observed both by flow cytometry (decrease of forward scatter, increase of unprocessed cells staining with APO2.7 antibody) and viability determination by Trypan-blue staining until after 7 hours. However, after 4 hours morphologic changes were observed in the nuclear and cytoplasmic structures using Diff-Quik stained cytospin preparations and standard light microscopic techniques (50% apoptotic cells). The same results were obtained by flow cytometric measurement of sub-diploid DNA content (sub-G1 cells), and an increase of staining with APO2.7 antibody in cells permeabilised by digitonin prior to staining. After 7 hours almost all cells exhibited apoptotic morphology. After 16 hours the cell size (forward scatter) decreased significantly, and 54% of unprocessed cells were APO2.7 positive. After 24 hours only 6% of cells were alive (high forward scatter) and these cells were APO2.7 negative. The HL-60 cells did not proliferate during the cultivation in 3% ethanol, and after 48 hours all stained by Trypan blue. HL-60 leukemic cells were CD34-/AC133-, CD33+/CD15+, and only 2% of the cells were CD95+. Induction of apoptosis by ethanol did not enhance CD95 antigen expression.     

Dystroglycan depletion inhibits the functions of differentiated HL-60 cells

Dystroglycan has recently been characterized in blood tissue cells, as part of the dystrophin glycoprotein complex but to date nothing is known of its role in the differentiation process of neutrophils. We have investigated the role of dystroglycan in the human promyelocytic leukemic cell line HL-60 differentiated to neutrophils. Depletion of dystroglycan by RNAi resulted in altered morphology and reduced properties of differentiated HL-60 cells, including chemotaxis, respiratory burst, phagocytic activities and expression of markers of differentiation. These findings strongly implicate dystroglycan as a key membrane adhesion protein involved in the differentiation process in HL-60 cells.     

Cell cycle-dependence of HL-60 cell deformability.

In this study, the role of cytoskeleton in HL-60 deformability during the cell cycle was investigated. G1, S, and G2/M cell fractions were separated by centrifugal elutriation. Cell deformability was evaluated by pipette aspiration. Tested at the same aspiration pressures, S cells were found to be less deformable than G1 cells. Moreover, HL-60 cells exhibited power-law fluid behavior: mu = mu c(gamma m/ gamma c)-b, where mu is cytoplasmic viscosity, gamma m is mean shear rate, mu c is the characteristic viscosity at the characteristic shear rate gamma c, and b is a material constant. At a given shear rate, S cells (mu c = 276 +/- 14 Pa.s, b = 0.51 +/- 0.03) were more viscous than G1 cells (mu c = 197 +/- 25, b = 0.53 +/- 0.02). To evaluate the relative importance of different cytoskeletal components in these cell cycle-dependent properties, HL-60 cells were treated with 30 microM dihydrocytochalasin B (DHB) to disrupt F-actin or 100 microM colchicine to collapse microtubules. DHB dramatically softened both G1 and S cells, which reduced the material constants mu c by approximately 65% and b by 20-30%. Colchicine had a limited effect on G1 cells but significantly reduced mu c of S cells (approximately 25%). Thus, F-actin plays the predominate role in determining cell mechanical properties, but disruption of microtubules may also influence the behavior of proliferating cells in a cell cycle-dependent fashion.     

Induction of HL-60 monocytic cell differentiation promoted by a perturbation of DNA synthesis: hydroxyurea promotes action of TPA

Control of terminal cell differentiation was studied using the human promyelocytic leukemia cell line, HL-60. HL-60 cells are known to undergo terminal monocytic differentiation when continuously exposed to 1.6 nM tetradecanoylphorbol acetate (TPA). The dose-response relationship between TPA concentration and induced differentiation is relatively steep. TPA (1.1 nM) induces little G1/0 specific growth inhibition or phenotypic differentiation. In contrast, pretreating the cells with a pulse exposure to hydroxyurea promotes their capability to terminally differentiate in response to TPA. Initially exponentially proliferating cells exposed for 20 h, approximately one doubling time, to 0.3 mM hydroxyurea, a subcytotoxic dose, underwent rapid G1/0 specific growth arrest and cell differentiation in response to subsequent exposure to 1.1 nM TPA. The extent of terminal differentiation was comparable to that induced by 1.6 nM TPA. The results support the hypothesis that early events in induction of terminal HL-60 cell differentiation depend on an S phase-specific process which may involve gene amplification.     

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

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

Altered expression profiles of microRNAs during TPA-induced differentiation of HL-60 cells

MicroRNAs (miRNAs) are highly conserved small non-coding RNAs that regulate gene expression through translational repression by base-pairing with partially complementary mRNAs. The expression of a set of miRNAs is known to be regulated developmentally and spatially, and is involved in differentiation or cell proliferation in several organisms. However, the expression profiles of human miRNAs during cell differentiation remain largely unknown. In an effort to expand our knowledge of human miRNAs, we investigated miRNAs during 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced differentiation of human leukemia cells (HL-60) into monocyte/macrophage-like cells. Several hundred RNAs ranging from 18 to 26 nucleotides were isolated from HL-60 cells with or without TPA-induction, and subsequently characterized by sequencing, database searching, and expression profiling. By removing non-miRNA sequences, we found three novel and 38 known miRNAs expressed in HL-60 cells. These miRNAs could be further classified into subsets of miRNAs that responded differently following TPA induction, either being up-regulated or down-regulated, suggesting the importance of regulated gene expression via miRNAs in the differentiation of HL-60 cells.     

1-0-Hexadecyl-2-acetyl-sn-glycerol stimulates differentiation of HL-60 human promyelocytic leukemia cells to macrophage-like cells

The human promyelocytic leukemia cell line HL-60 can be differentiated to cells resembling either neutrophils or mononuclear phagocytes by a diverse group of stimuli. However, the underlying mechanisms remain unknown. We report that 1-0-hexadecyl-2-acetyl-sn-glycerol inhibits the growth of HL-60 cells and induces differentiation to cells resembling mononuclear phagocytes. HL-60 cultures incubated for 6 days with 1-0-hexadecyl-2-acetyl-sn-glycerol (5 micrograms/ml) demonstrated a ten-fold increase in nonspecific esterase activity, and produced cells with morphological features similar to those of monocytes and macrophages. Higher concentrations of 1-0-hexadecyl-2-acetyl-sn-glycerol significantly inhibited the growth of HL-60 cells and resulted in the virtual absence of cells resembling the original HL-60 line. 1-0-Oleoyl-2-acetyl-rac-glycerol added under the same conditions did not induce cell differentiation or inhibit cell growth.     

No effect of pulsed electromagnetic fields on PC12 and HL-60 cells

The effect of pulsed electromagnetic fields (PEMF) similar to those used in transcranial magnetic stimulation (TMS) on two tumour cell lines, the human promyelocytic leukaemia cell line (HL-60) and the rat pheochromocytoma cell line (PC12), was investigated. The two cell lines were exposed to non-homogeneous pulsed electromagnetic fields (about 0.25–4.5 T peak magnetic field strength; 1–8 exponential pulses, 0.25 Hz) at different positions on the coil (2×25 mm). After exposure with various intensities, various numbers of pulses and at different coil positions, cell viability and the intracellular cyclic AMP content were determined in the two cell lines. Additionally, in HL-60 cells the intracellular Hsp72 content and in PC12 cells the release of the neurotransmitters dopamine, noradrenaline and acetylcholine were measured after PEMF treatment. The results of these analyses do not hint at alterations in the cell viability or in the content of cAMP, Hsp72, dopamine, noradrenaline, and acetylcholine in the two tumour cell lines after PEMF exposure under various conditions.     

The HL-60 transforming sequence: a ras oncogene coexisting with altered myc genes in hematopoietic tumors

The oncogene of the HL-60 human promyelocytic leukemia cell line has been passed serially through NIH/3T3 mouse fibroblasts. Oncogene-specific probes prepared from the resulting tertiary transfectants by molecular cloning have been used to show that loss of the transfected oncogene from NIH/3T3 cells correlates with reversion to nontransformed morphology. Analysis of cells transfected by the oncogenes of other tumors and tumor cell lines indicates that the transforming gene of the HL-60 leukemia cell line is closely related to oncogenes of a Burkitt's lymphoma, an acute myelogenous leukemia, an adenocarcinoma of the colon, a neuroblastoma, and two sarcomas. This oncogene is distantly related to the viral oncogenes of Kirsten and Harvey sarcoma viruses. It has been termed N-ras. The active N-ras oncogene coexists with altered versions of the myc oncogene in the HL-60 and AW Ramos human tumors. This suggests a multistep mechanism involving both ras and myc genes in the creation of these tumors.     

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.     

Stimulation of sialidase activity during cell differentiation of human promyelocytic leukemia cell line HL-60

Sialidase activity of human promyelocytic leukemia cell line HL-60 was assayed by a modification of the fluorometric method using 4MU-NANA as a substrate. The pH optimum was 4.1 and the apparent Km value was 0.10 mM. When the cells were induced to differentiate into granulocytes by either retinoic acid or DMSO, sialidase activity increased markedly. After incubation of HL-60 cells with 1 μM retinoic acid for 6 days and with 1.3% DMSO for 8 days, 91% and 75% of total cells, respectively, differentiated into morphologically mature myeloid cells and the sialidase activity increased to 2.5–2.7 times as much as that of the corresponding controls. In other human myeloid leukemia cell lines, K562 and KG-1, the sialidase activity was found to be 1.5- and 3.8-fold that of HL-60, respectively.     

Time- and concentration-dependent effects of resveratrol in HL-60 and HepG2 cells

Resveratrol, a phytochemical present in grapes, has been demonstrated to inhibit tumourigenesis in animal models. However, the specific mechanism by which resveratrol exerts its anticarcinogenic effect has yet to be elucidated. In the present study, the inhibitory effects of resveratrol on cell proliferation and apoptosis were evaluated in the human leukaemia cell line HL-60 and the human hepatoma derived cell line HepG2. We found that after a 2 h incubation period, resveratrol inhibited DNA synthesis in a concentration-dependent manner. The IC50 value was 15 microm in both HL-60 and HepG2 cells. When the time of treatment was extended, an increase in IC50 value was observed; for example, at 24 h the IC50 value was 30 microm for HL-60 cells and 60 microm for HepG2 cells. Flow cytometry revealed that cells accumulated in different phases of the cell cycle depending on the resveratrol concentration. Furthermore, an increase in nuclear size and granularity was observed in the G1 and S phases of HL-60 treated and HepG2-treated cells. Apoptosis was also stimulated by resveratrol in a concentration-dependent manner in HL-60 and HepG2 cells. In conclusion, resveratrol inhibits cell proliferation in a concentration- and time-dependent manner by interfering with different stages of the cell cycle. Furthermore, resveratrol treatment causes stimulation of apoptosis as well as an increase in nuclear size and granularity.     

The endogenous differentiation factor of the HL-60 cells shows a nuclease activity

A structural homology between the endogenous differentiation factor of the HL-60 cell line of promyelocyte leukemia (HLDF) and several DNA/RNA-binding and DNA/RNA-hydrolyzing proteins was revealed, and expression of the hldf gene in prokaryotic systems was studied. On the basis of these experiments, the amino acid sequence of an 8-membered fragment of HLDF with potential nuclease activity was identified. The synthetic octapeptide RRWHRLKE was shown to be capable of the cleavage of RNA, linear DNA from phage lambda, and all forms of plasmid DNA. We established that treatment of the HL-60 cell culture with this peptide (10(-6) M) results in an increase in the number of apoptotic cells and suggested that HLDF is involved in processes of apoptosis.     

Expression of leukocyte adherence-related glycoproteins during differentiation of HL-60 promyelocytic leukemia cells

We used the HL-60 human promyelocytic leukemia cell line to analyze the surface expression of a family of adherence-related leukocyte surface antigens during myeloid differentiation. These antigens are composed of discrete alpha subunits, designated alpha L, alpha M, and alpha X, that are each noncovalently associated with a common beta subunit. Monoclonal antibodies directed against the individual subunits served as markers in both indirect immunofluorescence studies and immunoprecipitations from HL-60 cells differentiated preferentially towards mature granulocytes (DMSO, retinoic acid) or monocyte/macrophages (PMA, vitamin D3). In undifferentiated HL-60 cells, the alpha L and alpha X subunits were constitutively expressed, whereas the alpha M subunit was not. Differentiation of HL-60 cells along the granulocytic pathway with DMSO resulted in a marked increase in alpha M and minimal increases in alpha L and alpha X. The phenotypic expression of these antigens on DMSO-treated HL-60 cells closely resembled that on normal circulating PMN. Differentiation along the monocyte/macrophage pathway when using PMA or vitamin D3 resulted in major increases in alpha L and alpha X expression, as well as alpha M. These changes resulted in a surface phenotype characteristic of that present on human monocyte-derived macrophages. Triggering of undifferentiated HL-60 cells with PMA caused no increase in subunit expression, whereas stimulation of DMSO-differentiated HL-60 cells with PMA produced more than a 1.5-fold enhancement of both the alpha M and alpha X subunits, and stimulation of human PMN with PMA increased the surface expression of alpha M more than fourfold and alpha X subunit twofold. Stimulation with PMA produced no change in expression of the alpha L subunit in any of the three cell populations. These results indicate that the alpha subunits of this glycoprotein family can be selectively regulated during in vitro differentiation of a human promyelocytic leukemia cell line. Second, DMSO-differentiated HL-60 cells and human PMN possessed an intracellular pool of alpha M and alpha X, but not alpha L, that could be translocated to the surface. Thus, despite structural and functional relationships among the alpha subunits in this glycoprotein family, they undergo disparate surface expression and intracellular regulation during differentiation.