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.     

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.     

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.     

Development of the superoxide-generating system during differentiation of the HL-60 human promyelocytic leukemia cell line

Utilizing the induced differentiation of HL-60 promyelocytic leukemia cells as a model of myeloid maturation, we examined the development of the superoxide-generating system, focusing on NADPH oxidase activity, membrane depolarization, and cytochrome b content. NADPH oxidase activity, measured as NADPH-dependent superoxide production, increased with both spontaneous and N,N-dimethylformamide-induced differentiation. Activity in particulate fractions from induced HL-60 cells and human peripheral blood polymorphonuclear leukocytes was proportional to their relative rates of superoxide production, but activity from uninduced cells was surprisingly high: one-third that from induced cells, despite only 7% their rate of superoxide generation. NADPH oxidase activities in phagocytic vesicles from induced HL-60 cells and polymorphonuclear leukocytes were equal, indicating the equivalence of the enzyme system in active portions of their cell membranes. Separation by centrifugal elutriation of the HL-60 cell population into fractions of varying maturity confirmed the relationship of NADPH oxidase activity to advancing differentiation in both dimethylformamide-induced and spontaneously maturing cells. Membrane potential change, an early event related to activation of the oxidase, was followed by 3,3'-dipropylthiodicarbocyanine dye fluorescence. The depolarization response increased dramatically in both magnitude and initial rate of change during differentiation. The cells' cytochrome b content increased 3-fold with induction of differentiation, in proportion to the change in NADPH oxidase activity.     

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.     

3H]Ouabain binding and 86 rubidium uptake during the dimethyl sulfoxide-induced differentiation of human promyelocytic leukemia HL-60 cells

When HL-60 cells were induced to differentiate into granulocyte-like cells by culture in the presence of 1.3% dimethyl sulfoxide, an increase in the rate of ouabain-sensitive 86Rb transport was observed during the first 6 h followed by a constant decrease which became stable on day 4 at about 40% of control level. By contrast, the number of ouabain binding sites remained constant during the first 24 h then decreased with the same kinetics as that of the 86Rb transport rate. These results suggest that alterations in ionic fluxes, through activation of the sodium pump, are part of the early events resulting in HL-60 cell differentiation.     

Matrix metalloproteinases expression in HL-60 promyelocytic leukemia cells during apoptosis

Human promyelocytic leukemia HL-60 cells have been used as a model to study both the expression of matrix-metalloproteinases and the mechanisms of programmed cell death. In the present study we examined the expression of these proteases in HL-60 cells stimulated by different apoptotic triggers. As shown by zymography, HL-60 cells released three major isofroms of the matrix-degrading proteases; when the leukemic cells were grown in serum-free conditions, as well as after hyperthermia and methotrexate treatment, we found a significant loss of the constitutive production of the 92 kDa matrix-metalloprotease, with an unequivocable molecular and ultrastructural evidence of programmed cell death. These results suggest that in HL-60 cells the expression/release of matrix metalloproteases can be down-regulated in the presence of the apoptotic-induced alterations, and that the decreased matrix-degrading capacity of this leukemic cell line during apoptosis may reduce its invasive potential.     

Neolacto-series gangliosides induce granulocytic differentiation of human promyelocytic leukemia cell line HL-60

Neolacto-series gangliosides having linear poly-N-acetyl-lactosaminyl oligosaccharide structure have been demonstrated to be increased characteristically during granulocytic differentiation of human promyelocytic leukemia cell line HL-60 cells induced by dimethyl sulfoxide or retinoic acid (Nojiri, H., Takaku, F., Tetsuka, T., Motoyoshi, K., Miura, Y., and Saito, M. (1984) Blood 64, 534-541). When HL-60 cells were cultured in the presence of neolacto-series gangliosides prepared from mature granulocytes, the cells were found to be differentiated into mature granulocytes on the basis of the changes of morphology, surface membrane antigens, nonspecific esterase activity, and the activity of phagocytosis and respiratory burst. The differentiation of cells was dependent on the concentration of gangliosides and accompanied with inhibition of cell growth. These findings suggest that the particular ganglioside molecules play an important role in regulation of cell differentiation and that the appearance of neolacto-series gangliosides on cell surface membrane not only triggers the differentiation but also determines the direction of differentiation in HL-60 cells.     

Loss of transferrin receptors following induced differentiation of HL-60 promyelocytic leukemia cells

Human promyelocytic leukemia (HL-60) and lymphoblastoid (Daudi) cells were studied: for transferrin receptors before and after induced differentiation with dimethyl sulfoxide (DMSO), sodium butyrate or retinoic acid. None of these reagents affected the morphology or presentation of receptors in Daudi cells, but many HL-60 morphologically matured to banded neutrophils and demonstrated a concomitant loss of transferrin binding, suggesting an important role for transferrin receptors in cellular differentiation.     

Expression of vimentin and nuclear lamins during the in vitro differentiation of human promyelocytic leukemia cells HL-60

We have reported previously that the human promyelocytic leukemia cell line HL-60, in its undifferentiated state, is devoid of cytoplasmic intermediate filament proteins and nuclear lamins A and C, but does express lamin B. Using immunofluorescence and immunoblotting techniques, we have further investigated the expression of vimentin and lamins A and C during differentiation of these tumor cells along the macrophage or granulocytic pathway in response to the inducing effects of the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) or dimethyl sulfoxide. Our results show that, while the expression of lamin B remains largely unchanged, the synthesis of vimentin and lamins A and C is dramatically enhanced during the maturation of HL-60 cells along both hemopoietic pathways. Northern blot analysis of cellular RNAs isolated from untreated and TPA-treated HL-60 cell populations as well as from control HeLa cells was performed using two oligonucleotides, one complementary to the 5' region common to human lamin A/C mRNAs and the other to the 5' region of hamster vimentin mRNA. Very low but still detectable amounts of vimentin and lamin A/C mRNAs were found in untreated HL-60 cell population, in accordance with the detection of small quantities of vimentin and lamins A and C in these populations. This is probably due to the presence of a small number of spontaneously differentiating cells. On the other hand, strong signals comparable to those obtained with RNA from control HeLa cells were detected for the three mRNA species from TPA-treated cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential gene expression in retinoic acid-induced differentiation of acute promyelocytic leukemia cells, NB4 and HL-60 cells

Acute promyelocytic leukemia (APL) is characterized by a specific chromosome translocation t(15;17), which results in the fusion of the promyelocytic leukemia gene (PML) and retinoic acid receptor alpha gene (RARalpha). APL can be effectively treated with the cell differentiation inducer all-trans retinoic acid (ATRA). NB4 cells, an acute promyelocytic leukemia cell line, have the t(15;17) translocation and differentiate in response to ATRA, whereas HL-60 cells lack this chromosomal translocation, even after differentiation by ATRA. To identify changes in the gene expression patterns of promyelocytic leukemia cells during differentiation, we compared the gene expression profiles in NB4 and HL-60 cells with and without ATRA treatment using a cDNA microarray containing 10,000 human genes. NB4 and HL-60 cells were treated with ATRA (10(-6)M) and total RNA was extracted at various time points (3, 8, 12, 24, and 48h). Cell differentiation was evaluated for cell morphology changes and CD11b expression. PML/RARalpha degradation was studied by indirect immunofluoresence with polyclonal PML antibodies. Typical morphologic and immunophenotypic changes after ATRA treatment were observed both in NB4 and HL-60 cells. The cDNA microarray identified 119 genes that were up-regulated and 17 genes that were down-regulated in NB4 cells, while 35 genes were up-regulated and 36 genes were down-regulated in HL60 cells. Interestingly, we did not find any common gene expression profiles regulated by ATRA in NB4 and HL-60 cells, even though the granulocytic differentiation induced by ATRA was observed in both cell lines. These findings suggest that the molecular mechanisms and genes involved in ATRA-induced differentiation of APL cells may be different and cell type specific. Further studies will be needed to define the important molecular pathways involved in granulocytic differentiation by ATRA in APL cells.     

Expression of CD38 in human promyelocytic leukemia HL-60 cell line during differentiation by niacin-related compounds

It was found that three niacin-related compounds, isonicotinic acid, nicotinamide, and nicotinamide N-oxide, induced granulocytic differentiation in HL-60 cells. We investigated the expression of CD38, which catalyzes the synthesis of cyclic ADP-ribose, a Ca2+ mobilizer, during differentiation by niacin-related compounds. It was found that CD38 was induced by isonicotinic acid, whereas nicotinamide and nicotinamide N-oxide containing an amino group did not induce it. The difference in expression of CD38 may provide some useful information for the elucidation of the mechanisms of cell differentiation.     

Triphenyltin enhances the neutrophilic differentiation of promyelocytic HL-60 cells

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

Induction of differentiation in human promyelocytic leukemia HL-60 cell line by niacin-related compounds

Water-soluble vitamin, niacin, and its related compounds were examined for their differentiation-inducing activity in human promyelocytic leukemia cells (HL-60). Among the compounds, which inhibited cell proliferation measured by MTT assay, isonicotinic acid, nicotinamide N-oxide, and nicotinamide induced NBT reducing activity. HL-60 cells were differentiated into granulocyte-like cells by these compounds, judging from morphological changes and loss of nonspecific esterase activity. The differentiation-inducing activity of water-soluble vitamin and its related compounds suggest that these compounds may be applicable for medical use.     

Arsenic trioxide promotes histone H3 phosphoacetylation at the chromatin of CASPASE-10 in acute promyelocytic leukemia cells

Arsenic trioxide (As(2)O(3)) is highly effective for the treatment of acute promyelocytic leukemia, even in patients who are unresponsive to all-trans-retinoic acid therapy. As(2)O(3) is believed to function primarily by promoting apoptosis, but the underlying molecular mechanisms remain largely unknown. In this report, using cDNA arrays, we have examined the changes in gene expression profiles triggered by clinically achievable doses of As(2)O(3) in acute promyelocytic leukemia NB4 cells. CASPASE-10 expression was found to be potently induced by As(2)O(3). Accordingly, caspase-10 activity also substantially increased in response to As(2)O(3) treatment. A selective inhibitor of caspase-10, Z-AEVD-FMK, effectively blocked caspase-3 activation and significantly attenuated As(2)O(3)-triggered apoptosis. Interestingly, the treatment of NB4 cells with As(2)O(3) markedly increased histone H3 phosphorylation at serine 10, an event that is associated with acetylation of the lysine 14 residue. Chromatin immunoprecipitation assays revealed that As(2)O(3) potently enhances histone H3 phosphoacetylation at the CASPASE-10 locus. These results suggest that the effect of As(2)O(3) on histone H3 phosphoacetylation at the CASPASE-10 gene may play an important role in the induction of apoptosis and thus contribute to its therapeutic effects on acute promyelocytic leukemia.     

Phosphorylation and dephosphorylation of human promyelocytic leukemia cell (HL-60) proteins by tumor promoter

Human promyelocytic leukemia cell line (HL-60) has been shown to be induced to the terminal differentiation into macrophage-like cells by a tumor promoter, 12-O-tetradecanoyl-phorbol-13-acetate (TPA). The present studies describe the effects of TPA on the phosphorylation of HL-60 cell proteins. A rapid decrease in the phosphorylation of a 75 kD protein was observed within a few minutes after treatment with TPA. On the other hand, TPA treatment of HL-60 cells caused rapid increase in the phosphorylation of a 67 kD protein and other minor proteins. Phorbol and 4α-phorbol-12,13-dodecanoate, both of which are biologically inactive derivatives of TPA, failed to cause any changes in protein phosphorylation in HL-60 cells. These results suggest that changes in protein phosphorylation are involved in mechanisms of the differentiation in HL-60 cells induced by TPA. Cell fractionation experiments revealed that 67K protein was located in cytosol. Though 75K protein also seemed to be located in cytosol, the phosphate moiety of 75K protein was almost lost during cell fractionation, suggesting that the phosphorylation of 75K protein was specifically regulated in HL-60 cells. Dimethyl sulfoxide (DMSO), retinoic acid (RA) and 1,25-dihydroxy-vitamin D₃, all of which induce the differentiation in HL-60 cells, did not cause any changes in protein phosphorylation. These results suggest that the changes in protein phosphorylation are specific for TPA. The possible mechanisms of changes in protein phosphorylation by TPA were discussed.     

Lipids of human promyelocytic leukemia cell HL-60: increasing levels of ether-linked phospholipids during retinoic acid-induced differentiation

Cells of the human promyelocytic leukemia cell line HL-60 as well as HL-60 granulocytes induced in vitro by retinoic acid were examined for lipid composition. One of our original aims was to clarify how human granulocyte (differentiated HL-60 cells) synthesized enough precursors of lipid mediators, such as prostaglandins and/or platelet activating factor. Comparison studies yielded the following results. 1) After granulocyte differentiation, total phospholipid of HL-60 cells decreased to about 70% of that of untreated cells, while the content of triglyceride increased to about 200% of the original level. 2) The subclass composition of ethanolamine-containing glycerophospholipid was greatly altered during differentiation; 1-alkenyl-2-acyl glycerophosphoethanolamine (GPE) increased to 166% of that in the untreated cells, while 1,2-diacyl GPE decreased to 46% of the original value. The resultant profile became very similar to that of human peripheral polymorphonuclear leukocytes. 3) During differentiation, the amount of arachidonic acid stored in both phospholipid and triglyceride of retinoic acid-treated HL-60 cells significantly increased. Its distribution was also modified; arachidonic acid in 1,2-diacyl GPE decreased to 63%, while those of 1-alkenyl-2-acyl GPE, choline-containing glycerophospholipids, and phosphatidylinositol increased to 169, 154, and 153%, respectively. These results suggested that the regulatory mechanism of lipid turnover in HL-60 cells was modified during retinoic acid-induced granulocyte differentiation. The alterations were not enough to explain fully the capability of differentiated HL-60 cells to produce lipid mediators upon stimulation, but they were probably one of the factors that regulate these reactions.     

Induction of the differentiation of human HL-60 promyelocytic leukemia cell line by succinoyl trehalose lipids

Four analogs of succinoyl trehalose lipid-3 (STL-3)with saturated even-number or odd-number carbonchains, and unsaturated or halogenated fatty acidswere examined for their ability to inhibit the growthand induce the differentiation of HL-60 humanpromyelocytic leukemia cells. The optimalconcentration of STL-3 at which such activities wererecognized was closed to the critical micelleconcentration of STL-3. Analog of STL-3 witheven-number or odd-number carbon chain and unsaturatedfatty acids strongly inhibited growth and induced thedifferentiation of HL-60 cells, as evaluated in termsof nitroblue tetrazilium-reducing activity and theappearance of the CD36 antigen. An analog of STL-3with halogenated fatty acids significantly inhibitedproliferation but only induced the differentiation ofHL-60 cells. Our results indicate that the effects ofSTL-3 and its analogs on HL-60 cells depend on thestructure of the hydrophobic moiety of STL-3.These authors contributed equally to this work