Purification and characterization of two high-density-lipoprotein-binding proteins from rat and human liver.

Newly absorbed retinol is transported in association with chylomicrons and their remnants. In addition, after intake of high doses of retinol, significant amounts are also found in low-density lipoprotein (LDL). As both chylomicron remnants and LDL may be taken up by cells via the LDL receptor, and retinoids inhibit proliferation of some leukaemic cells, we have studied the uptake of retinol in leukaemic cells via the LDL-receptor pathway. HL-60 cells contain saturable binding sites for LDL. The binding of LDL to its receptor has a dissociation constant of about 3.2 x 10(-9) M, and the number of receptors per cell was calculated to be about 2700. Uptake of 125I-LDL by HL-60 cells was increased 2-fold by preincubating the cells with mevinolin. The presence of specific receptors for LDL on HL-60 cells was further confirmed by the finding that exogenous LDL cholesterol was able to up-regulate the ACAT (acyl-CoA: cholesterol acyltransferase) activity of HL-60 cells. We then tested the uptake of retinyl ester in leukaemic cells via the LDL-receptor pathway. HL-60 cells were incubated with LDL or chylomicron remnants labelled with [3H]retinyl palmitate. Uptake of retinyl ester associated with both LDL and chylomicron remnants was observed. Furthermore, the presence of excess LDL decreased the uptake by 75-100%, supporting the hypothesis that the uptake of retinyl ester occurred via the LDL receptor in HL-60 cells.     

Effect of AML serum on normal human myeloid differentiation and hexamethylene bisacetamide-induced granulocytic differentiation of the human HL-60 promyelocytic leukaemic cell line.

The effect of serum from 32 AML patients on the normal human myeloid differentiation and the hexamethylene-bisacetamide induced granulocytic differentiation of HL-60 promyelocytic leukaemic cell line was studied. Nonadherent normal mononuclear marrow cells were cultured in vitro at a concentration of 5 x 10(5) cells/ml for 6 days with each of the 32 AML sera. Ten normal human AB sera were used as control. The results showed an inhibitory activity on both morphological and functional differentiation of normal human myeloid immature marrow cells by 29 out of the 32 AML sera tested. These 29 AML sera were added to cultures of HL-60 (2.5 x 10(5)/ml) leukaemia cell line which incorporated 2 mM hexamethylene-bisacetamide for 6 days. The results showed no significant inhibition of hexamethylene-bisacetamide induced granulocytic differentiation by any of the 29 AML sera. The efficacy of hexamethylene-bisacetamide in inducing differentiation in the presence of inhibitory factors suggests a possible role in the treatment of AML patients.     

Susceptibility of the human promyelocytic cell line HL-60 to an endogenous antileukaemic factor in suspension cultures

HL-60 human leukaemic cell line a suitable homogeneous target population for the selective endogenous inhibitor of myelopoiesis, isolated in our laboratory, was submitted to multiparameter analysis of cell proliferation in suspension cultures. As detected by 3H-TdR incorporation, a single dose of the regulator elicited a 6 to 8 hours arrest of DNA synthesis. The inhibition could be prolonged by repeated applications. As affected by the factor, alteration of population kinetics is characterized, revealed by flow cytofluorometric analysis, in G1 arrest of a fraction of cells, and diminishing those in hyperdiploid and tetraploid stage. 51Cr-release detection of vitality proved, that the endogenous factor, chemically determined as nucleopeptide, affected non-toxically and reversibly HL-60 cell proliferation.     

Changes in surface antigens of HL-60 cells during differentiation in vitro

We have examined the pattern of binding of monoclonal antibodies OKM 1, FMC 10, FMC 12, FMC 13, FMC 17 and FMC 33 to human promyelocytic leukaemia (HL-60) cells. We found that the expression of antigens detectable with FMC 17 and FMC 33 (specific for monocytes and macrophages) was increased by exposure of HL-60 cells to 1,25-dihydroxyvitamin D3 but not by exposure of HL-60 cells to 12-tetradecanoyl phorbol-13-acetate (TPA). The antigen detected with the OKM 1 antibody was highly induced by TPA. The expression of granulocyte-specific antigens detected by FMC 10 and FMC 13 was increased during induction of granulocytic maturation; these antigens were retained during monocyte-macrophage differentiation of HL-60 cells. We conclude that in some cases the expression of particular antigens during maturation of malignant cells proceeds normally while in other cases antigenic differences between leukaemic and normal cells at equivalent levels of maturation can be detected.     

Thymosin beta4 and AcSDKP inhibit the proliferation of HL-60 cells and induce their differentiation and apoptosis

Our previous works have shown that bone marrow stromal cells secrete thymosin beta4 (Tbeta4) and AcSDKP. Tbeta4 and AcSDKP are existed in the conditioned medium of bone marrow endothelial cells. They exerted inhibitory effects on hematopoietic cells and then had protective effect on the early hematopoietic cells, which were cultured in the presence of hematopoietic stimulators. Thymosin beta4 consists of 43 peptides with a molecular weight of 4963. It contains at its N-terminal end the sequence of the acetylated tetrapeptide Ac-N-Ser-Asp-Lys-Pro (AcSDKP). This study was performed to evaluate the effect of Tbeta4 and AcSDKP on the growth of HL-60 cells. It was showed that Tbeta4 (10(-11)-10(-7)mol/L) and AcSDKP (10(-11)-10(-7)mol/L) had the dose-dependent inhibitory effect on the proliferation of HL-60 cells. Based on cell morphology and NBT reduction, Tbeta4 and AcSDKP induced differentiation of HL-60 cells. Morphologic and DNA fragment analysis proved that Tbeta4 and AcSDKP induced apoptosis of HL-60 cells. In order to analyze the mechanism of the effects of Tbeta4 and AcSDKP, intracellular free Ca(2+) concentration ([Ca(2+)](i)) of HL-60 leukemic cells was tested and Atlas cDNA Expression Array was performed. The results showed that Tbeta4 and AcSDKP could increased [Ca(2+)](i) by stimulating the release of Ca(2+) from intracellular Ca(2+) pool. Moreover, AcSDKP could also elicit a potent extracelluar calcium influx in HL-60 cells. Tbeta4 could also change apoptotic-related gene expression in leukemic cells, and resulted in the inhibition of proliferation and induction of differentiation and apoptosis of leukemic cells.     

Generation time of leukaemic blast progenitor cells

Previous studies have indicated that the generation time of human leukaemic cells is longer than that of normal haematopoietic cells. We have employed a modification of the thymidine (TdR)-suicide technique to measure directly the generation time of leukaemic progenitor cells capable of colony formation. The results obtained with two human leukaemic cell lines (KG-1 and HL-60) and with blast progenitor cells from two patients with acute myelogenous leukaemia indicate generation times ranging from 9 X 0-22 X 0 hr and S-phase durations ranging from 5 X 5-8 X 0 hr. Using the same technique, the generation time of normal bone marrow CFU-c was determined to be 9-11 hr. These findings suggest that the proliferation rate of human leukaemic blast progenitor cells is similar to that of normal haematopoietic stem cells.     

Dimethylsulfoxide exposure modulates HL-60 cell rolling interactions

Human leukaemic HL-60 cells are widely used for studying interactions involving adhesion molecules [e.g. P-selectin and PSGL-1 (P-selectin glycoprotein ligand-1)] since their rolling behaviour has been shown to mimic the dynamics of leucocyte rolling in vitro. HL-60 cells are neutrophilic promyelocytes that can undergo granulocytic differentiation upon exposure to compounds such as DMSO (dimethylsulfoxide). Using a parallel plate flow chamber functionalized with recombinant P-selectin-Fc chimaera, undifferentiated and DMSO-induced (48, 72 and 96?h) HL-60 cells were assayed for rolling behaviour. We found that depending on P-selectin incubation concentration, undifferentiated cells incurred up to a 6-fold increase in rolling velocity while subjected to an approximately 10-fold increase in biologically relevant shear stress. HL-60 cells exposed to DMSO for up to 72?h incurred up to a 3-fold increase in rolling velocity over the same shear stress range. Significantly, cells exposed for up to 96?h incurred up to a 9-fold decrease in rolling velocity, compared with undifferentiated HL-60 cells. Although cell surface and nuclear morphological changes were evident upon exposure to DMSO, flow cytometric analysis revealed that PSGL-1 expression was unchanged, irrespective of treatment duration. The results suggest that DMSO-treated HL-60 cells may be problematic as a substitute for neutrophils for trafficking studies during advanced stages of the LAC (leucocyte adhesion cascade). We suggest that remodelling of the cell surface during differentiation may affect rolling behaviour and that DMSO-treated HL-60 cells would behave differently from the normal leucocytes during inflammatory response in vivo.     

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.     

Regulation of 25-hydroxyvitamin D3 metabolism in a human promyelocytic leukemia cell line (HL-60): 1,25-dihydroxyvitamin D3 stimulates the synthesis of 24,25-dihydroxyvitamin D3

The human promyelocytic leukemia cell line HL-60 undergoes macrophage-like differentiation after exposure to 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], the biologically active metabolite of vitamin D3. In the current study, we demonstrate that 1,25(OH)2D3 also regulates 25-hydroxyvitamin D3 [25(OH)D3] metabolism in HL-60 cells. The presence of 1,25(OH)2D3 in the culture medium of HL-60 cells stimulated the conversion of 7-10% of the substrate [25(OH)D3] to a more polar metabolite, which was identified as 24,25-dihydroxyvitamin D3 [24,25(OH)2D3] from the elution positions on sequential HPLC systems and the sensitivity to periodate treatment. The HL-60 subclone HL-60 blast, which is unresponsive to 1,25(OH)2D3 in terms of differentiation, also responded to 1,25(OH)2D3 treatment with the production of 24,25(OH)2D3. Maximal stimulation of 24,25(OH)2D3-synthesis (approximately 7 pmol/5 X 10(6) cells) in HL-60 cells was noted with a 12-h exposure to 10(-9) M 1,25(OH)2D3. The ability of vitamin D3 metabolites other than 1,25(OH)2D3 to induce the synthesis of 24,25(OH)2D3 in HL-60 cells was, with the exception of 1 alpha-hydroxyvitamin D3, in correlation with their reported affinities for the specific 1,25(OH)2D3 receptor which is present in HL-60 cells. Treatment of HL-60 cells with phorbol diesters abolished the 1,25(OH)2D3 responsiveness, while treatment with dimethylsulfoxide and interferon gamma did not markedly alter the 25(OH)D3 metabolism of HL-60 cells. Small amounts (approximately 1% of substrate) of two 25(OH)D3 metabolites, which comigrated with 5(E)- and 5(Z)-19-nor-10-keto-25-hydroxyvitamin D3 on two HPLC solvent systems, were synthesized by HL-60 cells, independently from 1,25(OH)2D3 treatment or stage of cell differentiation. Our results indicate that 1,25(OH)2D3 influences 25(OH)D3 metabolism of HL-60 cells independently from its effects on cell differentiation.     

Pivotal role of reactive oxygen species as intracellular mediators of hyperthermia-induced apoptosis

The effects of cellular antioxidant capacity on hyperthermia (HT)-induced apoptosis and production of antiapoptotic heat shock proteins (HSPs) were investigated in HL-60 cells and in HL-60AR cells that are characterized by an elevated endogenous catalase activity. Exposure of both cell lines to 43 degrees C for 1 h initiated apoptosis. Apoptosis peaked at 3-6 h after heat exposure in the HL-60 cells. Whereas HL-60AR cells were partially protected against HT-induced apoptosis at these early time points, maximal levels of apoptosis were detected later, i.e. 12-18 h after heat exposure. This differential induction of apoptosis was directly correlated to the induction of the antiapoptotic HSP27 and HSP70. In particular, in the HL-60 cells HSP27 was significantly induced at 12-18 h after exposure to 43 degrees C when apoptosis dropped. In contrast, coinciding with the late onset of apoptosis in HL-60AR cells at that time HL-60AR cells lacked a similar HSP response. In line with the higher antioxidant capacity HL-60AR cells accumulated reactive oxygen species to a lesser degree than HL-60 cells after heat treatment. Protection from HT-induced apoptosis as well as diminished heat-induced HSP27 expression was also observed after cotreatment of HL-60 cells with 43 degrees C and catalase but not with superoxide dismutase. These data emphasize the pivotal role of reactive oxygen species for HT induced pro- and antiapoptotic pathways.     

The effect of endotoxin lipopolysaccharide from different bacterial species on the generation of intracellular inositol trisphosphate and superoxide in a human phagocytic cell line

The acute effects of endotoxins and lipid A on two intracellular responses, inositol phosphate generation and superoxide production were analysed in the DMSO differentiated premyelocytic leukaemic HL-60 cell line. Short-term incubation (1-10 min) with Escherichia coli-type LPS, Salmonella-type LPS and Lipid A caused significant increases in cellular InsP1 and InsP3, compared with control cells (P less than 0.5-P less than 0.001). The Escherichia coli-type LPS released approximately twice the quantity of InsP3 compared with Salmonella-type LPS (P less than 0.001). Lipid A-dependent stimulation of InsP3 production was also detected. The rate of superoxide production increased 1-10 min after addition of both Escherichia coli- and Salmonella-type LPS and Lipid A. Endotoxins and Lipid A caused a dose-dependent increase in intracellular oxidative activity. The superoxide response showed less species dependence and a higher response to particulate lipid A compared with the inositol phosphate response.     

In vitro proliferation of normal and leukaemic human leukocytes controlled by an inhibitory endopeptide

GI-3, an endogenous inhibitory fraction isolated from leukocytes, selectively inhibits the proliferation of granuloid precursor cells in a non-toxic manner. Its active principle was determined as an acidic chlor-tolidine positive decapeptide [ 3 ]. The in vitro effect on normal and acute leukaemic human bone marrow and blood cells was examined. A dose dependent inhibition by GI-3 of 3H-TdR incorporation into myeloid cells of normal bone marrow was found, the sensitivity of human cells being higher than that of rat cells. The proliferation of the target leukaemic bone marrow and blood cells (AML, AMMoL) was also decreased by the endogenous inhibitor in a dose dependent manner in untreated subjects as well as in patients in remission or relapse. The rate of inhibition of leukaemic of well-known cytostatics (adriamycin hydrochloride, dianhydrogalactitol) applied for comparison. Beyond its direct cytostatic effect, GI-3 could be used in the differential diagnosis of blastic leukaemias, complementing the routine cytochemical methods.     

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.     

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 (≥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.     

Partial purification and characterization of phorbol ester-regulated translational inhibitor(s) in human HL-60 leukemic cells

Addition of nanomolar concentration of the phorbol ester 12-O-tetradecanoyl phorbol 13-acetate (TPA) to the human promyelocytic HL-60 leukemia cells is associated with a cessation of cellular proliferation and a subsequent differentiation into macrophage-like cells. Because the growth rate of mammalian cells is tightly coupled to the functions of the protein synthetic machinery, we have examined whether TPA induces a change in HL-60 translational functions. Addition of control HL-60 cell extracts to rabbit reticulocyte lysates results in a pronounced inhibition of protein synthesis, while TPA-treated HL-60 cell extracts are significantly less inhibitory. The reduction in TPA-induced translational inhibitory activity can be observed after a 3-6-h treatment and reaches a maximum after 24 h. Fractionation of control cell extracts on DEAE-cellulose columns reveals two inhibitory activities, eluting at 100 and 350 mM KCl, respectively. The DEAE-100 inhibitor(s) is further resolved into two activities by heparin-agarose column chromatography (HEP-100 and HEP-250). TPA treatment of HL-60 cells for 48 h completely eliminates the HEP-250 inhibitory activity and reduces the HEP-100 and the DEAE-350 inhibitory activities by 50 and 25%. Inhibition of protein synthesis in rabbit reticulocyte lysates by DEAE-100 inhibitory activities can be partially reversed by the addition of globin mRNA while translational inhibition by DEAE-350 inhibitor(s) can be reversed by addition of eukaryotic initiation factor (eIF) 2 or fructose 6-phosphate. The DEAE-100 inhibitor(s) causes extensive degradation of radioactive polynucleotides while the DEAE-350 inhibitor(s) is capable of phosphorylating both the alpha- and the beta-subunits of the highly purified rabbit reticulocyte initiation factor eIF-2. These data show that the DEAE-100 inhibitor(s) contains a nuclease while the DEAE-350 inhibitor(s) is associated with eIF-2 alpha and eIF-2 beta protein kinases.     

Analysis by high-resolution two-dimensional electrophoresis of differentiation-dependent alterations in cytosolic protein pattern of HL-60 leukemic cells.

HL-60 leukemic cells were differentiated along the neutrophilic pathway with retinoic acid (RA) or along the monocytic pathway with 1,25-dihydroxyvitamin D3 (1,25(OH)2D3). Using a high-resolution two-dimensional electrophoresis technique and subsequent silver staining, differentiation-dependent changes in cytosolic protein pattern of HL-60 cells were analysed and were compared with the cytosolic protein pattern of human neutrophils. The amount of 64 and 50 out of a total of 632 proteins studied was increased or decreased in RA- and 1,25(OH)2D3-differentiated HL-60 cells, respectively, in comparison to undifferentiated HL-60 cells. Thirty-three of these proteins were similarly altered in RA- and 1,25(OH)2D3-differentiated HL-60 cells. Twenty-two and 25 of the proteins altered in amount in RA- or 1,25(OH)2D3-differentiated HL-60 cells versus undifferentiated HL-60 cells were similarly altered in human neutrophils in comparison to undifferentiated HL-60 cells. Seven and 10 of the proteins altered in amount in RA- or 1,25(OH)2D3-differentiated HL-60 cells had specific equivalents in neutrophil cytosol. Our results show (i) that neutrophilic and monocytic differentiation is associated with decreases and increases in amount of cytosolic proteins; (ii) that both differentiation processes share a common set of alterations; and (iii) are associated with specific alterations in protein amount.     

A 41 kDa transferrin related molecule acts as an autocrine growth factor for HL-60 cells

HL-60 cells produce an autostimulatory growth factor. Since the stimulatory effect of HL-60 conditioned medium is only observed in the absence of exogenous transferrin we have assayed HL-60 cells for the production of transferrin and found that they produce polypeptides which react with transferrin antibodies. 35S-methionine labelling, immunoprecipitation and subsequent separation by SDS-gel electrophoresis reveals the presence of a major transferrin related 41 +/- 2 kDa species released by HL-60 cells. Physiological levels of iron salts completely abolish the requirement of exogenous transferrin which indicates that the endogenous transferrin related polypeptides in the presence of exogenous inorganic iron salts are sufficient for the proliferation of HL-60 cells provided insulin or related growth factors are present. The addition of transferrin receptor antibodies inhibits the stimulatory action of the endogenous transferrin related activity.     

19-Nor-1alpha,25-dihydroxyvitamin D2 (paricalcitol) exerts anticancer activity against HL-60 cells in vitro at clinically achievable concentrations

19-Nor-1alpha,25-dihydroxyvitamin D2 (paricalcitol) is an analogue of 1,25(OH)2D3 with reduced calcemic effects that is approved in the United States for the suppression of parathyroid hormone in chronic renal failure. Paricalcitol has anticancer activity in prostate cancer cells. We tested the effects of paricalcitol on the HL-60 leukemia cells, studying cellular differentiation, cell cycle changes, apoptosis and cellular proliferation. Paricalcitol at 10(-8)M concentration induced the maturation of HL-60 cells in a time-dependent manner, as shown by increased expression of CD11b differentiation surface antigen. The ability of HL-60 cells to reduce nitroblue tetrazolium (NBT) was markedly increased after exposure to paricalcitol at 10(-8)M for 72 h. Paricalcitol inhibited colony formation of HL-60 cells in a soft agar semisolid media after 10-day incubation (estimated IC50 of 5 x 10(-9) M. Exposure to 10(-8)M paricalcitol for 72 h increased the number of cells in G0/G1 phase, and decreased the number of cells in S phase, and significantly increased the number of HL-60 cells undergoing apoptosis. The concentration required to achieve inhibition of growth of HL-60 cells is comparable to clinically achievable levels. These findings support the clinical evaluation of paricalcitol as an antileukemia agent.     

Sphingomyelinase action inhibits phorbol ester-induced differentiation of human promyelocytic leukemic (HL-60) cells

Prior studies showed that sphingomyelinase action and the free sphingoid bases inhibited protein kinase C (Kolesnick, R. N., and Clegg, S. (1988) J. Biol. Chem. 263, 6534-6537). The present studies investigated whether sphingomyelinase action also inhibited a biologic process mediated via protein kinase C, phorbol ester-induced differentiation of HL-60 promyelocytic cells into macrophages. The potent phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) stimulated time- and concentration-dependent conversion of HL-60 cells into macrophages, ED50 congruent to 5 x 10(-10) M. Differentiation involved growth inhibition, adherence of the suspended cells to tissue culture plastic, morphologic changes, and development of specific enzymatic markers. Sphingomyelinase, which increased the level of sphingoid bases and inactivated protein kinase C, prevented this event. In control incubations, cell number increased 2.10-fold over 24 h, and 2 +/- 1% of the cells were adherent. In incubations with TPA (0.5 nM), cell number increased only 1.75-fold, and 30% were adherent. Sphingomyelinase (3.8 x 10(-5) unit/ml) restored growth to incubations containing TPA to 2.02-fold and reduced adherence to 15%. Sphingomyelinase (3.8 x 10(-2) unit/ml) also restored growth partially and reduced adherence to a maximal concentration of TPA (3 nM). Similar results were obtained with the sphingoid base sphingosine (3-4.5 microM). Sphingomyelinase antagonized the morphologic changes associated with conversion to the macrophage phenotype. Untreated HL-60 cells presented typical promyelocytic morphology with large nuclei, little cytoplasm, and uniformity of nuclear and cell shape. TPA induced a larger cell population with abundant cytoplasm and unusual shape. Sphingomyelinase prevented these changes. Sphingomyelinase blocked TPA-induced increases in the macrophage marker enzymes, acid phosphatase and alpha-naphthyl acetate esterase. These studies indicate that the action of a sphingomyelinase, like the sphingoid bases, blocks phorbol ester-induced differentiation of HL-60 cells into macrophages and provides further support for the concept that sphingomyelinase action may be sufficient to comprise a physiologically relevant inhibitory pathway for protein kinase C.     

The anti-angiogenic 8-epipuupehedione behaves as a potential anti-leukaemic compound against HL-60 cells

We have previously reported that 8-epipuupehedione, a synthetic derivative of sesquiterpenes found in several kinds of sponges, is a potent inhibitor of angiogenesis. Here, we show that 8-epipuupehedione is also a potent anti- leukaemic compound, targeting three hallmarks of malignancy: proliferation, survival and extra-cellular matrix re-modelling. To fulfil this goal, we use the HL-60 promyeolocytic cells as our model system and the following experimental procedures: cell growth assay, Hoetsch staining, cell cycle analysis and DNA fragmentation, caspase 3 activity and zymographic assays. Our results show that this compound inhibits proliferation and has potent and specific pro-apoptotic effects on HL-60 promyelocytic cells, inducing their nuclei and DNA fragmentation, as well as caspase 3 activity activation. Furthermore, 8-epipuupehedione strongly inhibits matrix metalloproteinase-2 and urokinase production by HL-60 cells. These results suggest that 8-epipuupehedione could be an attractive drug for further evaluation in the treatment of leukemia.