Carboxy-terminal fragment of osteogenic growth peptide regulates myeloid differentiation through RhoA

The carboxy-terminal fragment of osteogenic growth peptide, OGP(10-14), is a pentapeptide with bone anabolic effects and hematopoietic activity. The latter activity appears to be largely enhanced by specific growth factors. To study the direct activity of OGP(10-14) on myeloid cells, we tested the pentapeptide proliferating/differentiating effects in HL60 cell line. In this cell line, OGP(10-14) significantly inhibited cell proliferation, and enhanced myeloperoxidase (MPO) activity and nitroblue tetrazolium reducing ability. Moreover, it induced cytoskeleton remodeling and small GTP-binding protein RhoA activation. RhoA, which is known to be involved in HL60 differentiation, mediated these effects as shown by using its specific inhibitor, C3. Treatment with GM-CSF had a comparable OGP(10-14) activity on proliferation, MPO expression, and RhoA activation. Further studies on cell proliferation and RhoA activation proved enhanced activity by association of the two factors. These results strongly suggest that OGP(10-14) acts directly on HL60 cells by activating RhoA signaling although other possibilities cannot be ruled out.     

Large-scale purification of myeloperoxidase from HL60 promyelocytic cells: characterization and comparison to human neutrophil myeloperoxidase

A large-scale purification procedure was developed for the isolation of myeloperoxidase from HL60 promyelocytic cells in culture. Initial studies showed the bulk of peroxidase-positive myeloperoxidase activity to be located in the cetyltrimethylammonium bromide solubilized particulate fraction of cell homogenates. The myeloperoxidase was then chromatographically purified using concanavalin A followed by gel filtration. SDS-PAGE analysis of the final preparation showed the presence of only two proteins with molecular masses of approximately 55 and 15 kDa, corresponding to the large and small subunits of myeloperoxidase. These data, along with Reinheit Zahl (RZ) values (A(430)/A(280)) of greater than or equal to 0.72, indicate that the myeloperoxidase prepared by this method is apparently homogeneous. Preparations routinely yielded 12-20 mg of pure myeloperoxidase per 10 ml of cell pellet. The HL60 myeloperoxidase was shown to be indistinguishable from purified human neutrophil myeloperoxidase by size exclusion chromatography, analytical ultracentrifugation, SDS-PAGE, Western blot, and NH(2)-terminal sequence analysis. The activities of the two myeloperoxidase samples, as measured using either the tetramethylbenzidine or the taurine chloramine assay, were indistinguishable. Finally, both enzymes responded identically to dapsone and aminobenzoic acid hydrazide, known inhibitors of myeloperoxidase. A protocol is presented here for the rapid, large-scale purification of myeloperoxidase from cultured HL60 cells, as well as evidence for the interchangeability of this myeloperoxidase and that purified from human neutrophils.     

Molecular cloning and characterization of cDNA for human myeloperoxidase

Partial amino acid sequence of human myeloperoxidase was determined, and a 41-base oligonucleotide containing deoxyinosines at four positions was chemically synthesized. By using the oligonucleotide as a probe, cDNA clones for human myeloperoxidase were isolated from a cDNA library constructed with mRNA from human promyelocytic leukemia HL-60 cells. One of the clones containing a 2.6-kilobase insert was subjected to nucleotide sequence analysis. The sequence was found to contain an open reading frame, 2,235 nucleotides coding for a protein of 745 amino acids with a calculated Mr of 83,868. The heavy chain of myeloperoxidase, consisting of 467 amino acids, was located on the COOH terminus half of the protein. The RNA specified by the cDNA was prepared using SP6 RNA polymerase and translated in rabbit reticulocyte lysates, and the product was identified as human myeloperoxidase by immunoprecipitation with rabbit anti-human myeloperoxidase antibody. By Northern hybridization analysis of RNA from leukemic cells, it was shown that myeloperoxidase mRNA is abundantly expressed in human promyelocytic HL-60 and mouse myeloid leukemia NFS-60 cells. Furthermore, the results of Southern hybridization analysis of human genomic DNA suggest that there are one or two genes for myeloperoxidase in the human haploid genome.     

Expression of catalase and myeloperoxidase genes in hydrogen peroxide-resistant HL-60 cells.

We studied the expression of catalase and myeloperoxidase genes in the hydrogen peroxide-resistant variants of human myeloid leukemia HL-60 cells HP50-2 and HP100-1. Southern blot hybridization with catalase and myeloperoxidase cDNA probes indicated that the copy number of the catalase gene in HP50-2 and HP100-1 cells was two and eight times, respectively, higher than that in HL-60 cells, whereas the copy number of the myeloperoxidase gene was the same. The amplified catalase and c-myc genes in HP100-1 cells were not decreased by treatment of the cells with inhibitors of poly(ADP-Ribose) polymerase, such as nicotinamide and benzamide. RNA blot hybridization with cDNA probes indicated that the content of catalase mRNA in HP50-2 and HP100-1 cells was four and 16 times higher, respectively, than that in HL-60 cells. By contrast, the content of myeloperoxidase mRNA in HP50-2 and HP100-1 cells was only a few percent of that in HL-60 cells. Furthermore, fluorescent in situ hybridization of a catalase cDNA probe to chromosomes indicated that the catalase gene in HP100-1 was amplified in the p13 region of a derivative chromosome 11. These results indicate that the increased synthesis of catalase in these resistant cells is mainly due to increased expression of the catalase gene, and that the lack of myeloperoxidase synthesis in these cells is due to the absence of its mRNA.     

Cyclic AMP-dependent and -independent protein kinases and protein phosphorylation in human promyelocytic leukemia (HL60) cells induced to differentiate by retinoic acid

The human leukemia cell line HL60 which resembles promyelocytes can be induced to differentiate to cells displaying features of the mature myeloid phenotype by a variety of agents including retinoic acid (RA) and agents that elevate intracellular adenosine 3:5 cyclic monophosphate (cyclic AMP) levels, e.g., 8-bromo-cyclic adenosine 3:5 monophosphate (8-Br-cyclic AMP), cholera toxin. Since most, if not all the effects of cyclic AMP, are mediated by adenosine 3:5 cyclic monophosphate-dependent protein kinase (cyclic AMP-dPK), we investigated the role of cyclic AMP-dPK and adenosine 3:5 cyclic monophosphate-independent protein kinase (cyclic AMP-iPK) in the induced differentiation of HL60 cells. Marked stimulation of cyclic AMP-dPK and cyclic AMP-iPK appears to be intimately involved with and specific for HL60 myeloid differentiation as evidenced by: (1) Stimulation of cyclic AMP-dPK and cyclic AMP-iPK early during HL60 myeloid differentiation and prior to phenotypic changes. (2) RA and dimethylformamide (DMF), agents that induce differentiation along the myeloid pathway, cause a marked increase in the type I cytosolic cyclic AMP-dPK and cyclic AMP-iPK (protamine kinase) while no such increases are noted in cells treated with 12-0-tetradecanoyl-phorbol-13-acetate (TPA) which induces differentiation along the monocyte/macrophage pathway. (3) Both native polyacrylamide gel electrophoresis as well as photoaffinity labeling with 8-azido-cyclic AMP demonstrate marked increases in type I cyclic AMP-dPK in the cytosols of cells exposed to agents that induce myeloid differentiation but no increase in TPA-differentiated cells. (4) The appearance and disappearance of specific cyclic AMP-dependent and -independent protein phosphorylations are associated with the induced myeloid differentiated state.     

1alpha,25-dihydroxyvitamin D3 stimulates steroid sulphatase activity in HL60 and NB4 acute myeloid leukaemia cell lines by different receptor-mediated mechanisms

Steroid sulphatase is a key enzyme in the biosynthesis of bioactive estrogens and androgens from highly abundant inactive circulating sulphated steroid precursors. Little is known about how the expression/activity of this enzyme is regulated. In this article, we show that of 1alpha,25(OH)2D3 stimulates an increase steroid sulphatase activity in the HL60 myeloid leukaemic cell line that is inhibited by a specific nuclear VDR (VDRnuc) antagonist and unaffected by plasma membrane-associated vitamin D receptor (VDRmem) agonists and antagonists. 1alpha,25(OH)2D3-mediated up-regulation of steroid sulphatase activity in HL60 cells was augmented by RXR agonists, blocked by RXR-specific antagonists, and RAR specific agonists and antagonists had no effect. In contrast, the 1alpha,25(OH)2D3-mediated up-regulation of steroid sulphatase activity in the NB4 myeloid leukaemic cell line was unaffected by the specific VDRnuc and RXR antagonists, but was blocked by a VDRmem-specific antagonist and was increased by VDRmem-specific agonists. The findings reveal that VDRnuc-RXR-heterodimers play a key role in the 1alpha,25(OH)2D3-mediated up-regulation of steroid sulphatase activity in HL60 cells. However, in NB4 cells, VDRnuc-derived signals do not play an obligatory role, and non-genomic VDRmem-derived signals are important.     

Role of c-fes protooncogene in myeloid differentiation

The main purpose of this report is to provide a review of the present knowledge on the structure, function, and possible regulatory role of c-fes in the genetic programs underlying the proliferation and differentiation of hematopoietic myeloid cells. Fes encodes a non-receptor tyrosine kinase that is highly expressed in immature and differentiated cells of the granulocytic and mono-macrophagic lineages. It is therefore possible that c-fes is involved in the signal transduction of myeloid cell differentiation, even if the specific substrates phosphorylated by this protooncogene are only poorly characterised. Several experimental models have been established to evaluate the role of c-fes in myeloid differentiation, in particular: the differentiation capacity of HL60 cells lacking the p92(c-fes) protein, the transfection of c-fes gene into K562 cells and transgenic animals overexpressing c-fes. The results obtained point to the importance of c-fes in myeloid cells, since it appears to be involved in granulocytic maturation as an antiapoptotic gene, and in macrophagic maturation as a regulatory gene.     

Transferrin receptor regulation is coupled to intracellular ferritin in proliferating and differentiating HL60 leukemia cells

Cultured myeloid leukemia cells display transferrin receptors but decrease receptor display after differentiation induction or accumulation of intracellular iron. To determine whether regulation of transferrin receptors and ferritin were linked under these disparate conditions, serum-free and fetal bovine serum (FBS) cultures of HL60 promyelocytic leukemia cells were used to investigate relationships between transferrin receptor display and intracellular ferritin. Using 125I-transferrin binding and immunofluorescence staining for transferrin receptors, HL60 cells cultured in serum-free, transferrin-free medium expressed fewer transferrin receptors and contained increased ferritin when compared to cells cultured with FBS or transferrin supplemented, serum-free medium. When placed in medium containing transferrin, cells previously grown in transferrin-free medium rapidly re-expressed transferrin receptors and decreased their ferritin content. HL60 cells induced to differentiate into granulocytes or macrophages also decreased transferrin receptor display and increased their ferritin content. Transferrin receptor display and ferritin content in both proliferating and differentiating myeloid leukemia cells are inversely related and their regulation is closely linked. Regulation of transferrin receptor display and ferritin synthesis may be important events regulating myeloid cell growth and differentiation.     

Cytochrome P-450- and Peroxidase-Dependent Activation of Procarbazine and Iproniazid in Mammalian Cells

Metabolism of hydrazine derivatives, procarbazine and iproniazid, to reactive free radical intermediates has been studied using spin-trapping techniques in intact human promyelocytic leukemia (HL60) and mouse hepatic cell lines. While HL60 cells have been shown to contain both myeloperoxidase and cytochrome P-450 enzymes, the hepatic cell line shows only cytochrome P-450 activity. Both peroxidases and cytochrome P-450 have been reported to catalyze biotransformation of hydrazines. Procarbazine and iproniazid were rapidly metabolized in these cell lines to methyl and isopropyl radicals, respectively. However, in HL60 cells, procarbazine was metabolized by myeloperoxidase while iproniazid was metab olized mostly by the cytochrome P-450 system. In the hepatic cells, both of these compounds were metabolized by the P-450 system.     

Undermethylation and DNase I hypersensitivity of myeloperoxidase gene in HL-60 cells before and after differentiation.

Methylation and DNase I-hypersensitive sites of the myeloperoxidase gene in human myeloid leukemia HL-60 cells were studied by Southern blot hybridization using the myeloperoxidase gene probes. Digestion of DNA with a methylation-sensitive restriction endonuclease indicated that a CpG in the CCGG sequence located 3.53 kbp upstream of the myeloperoxidase gene was unmethylated in HL-60 cells expressing the gene, whereas it was methylated in K562 cells and human placenta not expressing the gene. The site in HL-60 cells remained unmethylated after retinoic acid- or 12-O-tetradecanoyl-phorbol-13-acetate-induced differentiation that arrests myeloperoxidase synthesis. Digestion of isolated nuclei with various amounts of DNase I indicated that four DNase I-hypersensitive sites were in an upstream region of the myeloperoxidase gene in HL-60 cells and three sites were within the gene. In retinoic acid-induced cells, the bands of the hypersensitive site near the 5' side of the gene and that in the first intron became weak, while that of the site in the fifth intron became strong. The bands of these hypersensitive sites were weak in K562 cells. The implications of these changes in tissue-specific expression and developmental down-regulation of the myeloperoxidase gene are discussed.     

Cytochrome P-450- and Peroxidase-Dependent Activation of Procarbazine and Iproniazid in Mammalian Cells

Metabolism of hydrazine derivatives, procarbazine and iproniazid, to reactive free radical intermediates has been studied using spin-trapping techniques in intact human promyelocytic leukemia (HL60) and mouse hepatic cell lines. While HL60 cells have been shown to contain both myeloperoxidase and cytochrome P-450 enzymes, the hepatic cell line shows only cytochrome P-450 activity. Both peroxidases and cytochrome P-450 have been reported to catalyze biotransformation of hydrazines. Procarbazine and iproniazid were rapidly metabolized in these cell lines to methyl and isopropyl radicals, respectively. However, in HL60 cells, procarbazine was metabolized by myeloperoxidase while iproniazid was metab olized mostly by the cytochrome P-450 system. In the hepatic cells, both of these compounds were metabolized by the P-450 system.     

Defective chemiluminescence response in differentiated HL60 cells due to impaired degranulation

In the presence of dimethyl sulfoxide, the promyelocytic leukemic cell line, HL60, differentiates into apparently mature polymorphonuclear leukocytes. When correlating the superoxide production from HL60 cells with the number of phagocytozing and NBT-positive cells, no difference was observed in comparison with normal peripheral blood leukocytes. In contrast, the luminol-dependent chemiluminescence was greatly impaired in the differentiated HL60 cells. Analysis of degranulation, i.e., release of myeloperoxidase and N-acetyl-beta-glucosaminidase- and myeloperoxidase-mediated iodination by HL60 cells, suggested that the defective chemiluminescence response observed in HL60 cells may be due to impaired release of myeloperoxidase from azurophilic granulae. This may lead to impaired microbicidal activity in these cells.     

Retinoic acid treated HL60 cells express CEACAM1 (CD66a) and phagocytose Neisseria gonorrhoeae

Neisseria gonorrhoeae (gonococci, GC) are phagocytosed by neutrophils through the interaction between opacity proteins (Opa) and the CEA (CD66) family of antigens. In order to study this interaction, we used the human myeloid leukemia HL60 cell line, which differentiates into granulocyte-like cells upon treatment with dimethylsulfoxide (DMSO) or retinoic acid (RA). We found that RA-, but not DMSO- or untreated-HL60 cells, can phagocytose OpaI-expressing gonococci as well as Escherichia coli. The interaction of OpaI E. coli with RA-treated HL60 cells was inhibited by antibodies against CEACAM1. Phagocytosis of OpaI E. coli was found to be a result of the expression of CEACAM1 in RA-treated HL60 cells. Our results indicate that the level of expression of CEACAM1 in HL60 cells can be regulated by treatment with RA in a differentiation-dependent manner, and that this is important for phagocytosis of OpaI-expressing gonococci or E. coli.     

IN VITRO ANTIOXIDANT ACTIVITIES OF THE FERMENTED MARINE MICROALGA PAVLOVA LUTHERI (HAPTOPHYTA) WITH THE YEAST HANSENULA POLYMORPHA1

Microalgae are major primary producers of organic matter in aquatic environments through their photosynthetic activities. Fermented microalga (Pavlova lutheri Butcher) preparation (FMP) is the product of yeast fermentation by Hansenula polymorpha. It was tested for the antioxidant activities including lipid peroxidation inhibitory activity, free‐radical‐scavenging activity, inhibition of reactive oxygen species (ROS) on mouse macrophages (RAW264.7 cell), and inhibited myeloperoxidase (MPO) activity in human myeloid cells (HL60). FMP exhibited the highest antioxidant activity on free‐radical scavenging, inhibitory intracellular ROS, and inhibited MPO activity. MTT [3‐(4,5‐dimethyl‐2‐yl)‐2,5‐diphenyltetrazolium bromide] assay showed no cytotoxicity in mouse macrophages (RAW264.7 cell), human myeloid cells (HL60), and human fetal lung fibroblast cell line (MRC‐5). Furthermore, the antioxidative mechanism of FMP was evaluated by protein expression levels of antioxidant enzyme (superoxide dismutase [SOD] and glutathione [GSH]) using Western blot. The results obtained in the present study indicated that FMP is a potential source of natural antioxidant.     

Receptor binding and mitogenic effects of insulin and insulinlike growth factors I and II for human myeloid leukemic cells

Insulin and insulinlike growth factors I and II (IGF-I and IGF-II) influence mesodermal cell proliferation and differentiation. As multiple growth factors are involved in hemopoietic cell proliferation and differentiation, we assessed the receptor binding and mitogenic effects of these peptides on a panel of mesodermally derived human myeloid leukemic cell lines. The promyelocytic cell line HL60 had the highest level of specific binding for these 125I-labeled ligands, with lower binding to the less differentiated myeloblast cell line KG1 and undifferentiated blast variants of these cell lines (HL60blast, KG1a). Insulin binding affinity and receptor numbers were reduced significantly by chemically induced granulocytic differentiation of HL60 cells and was unchanged following induced monocytic differentiation. No substantial alteration in IGF-I or -II binding occurred with induced HL60 cell differentiation. Insulin and IGF-I demonstrated cross competition for receptor binding and down-regulated their homologous receptors without detectable cross modulation of the heterologous receptors on HL60 cells. IGF-I and insulin increased HL60 cell proliferation, as assessed by 3H-thymidine uptake, IGF-I greater than insulin. IGF-I binding and mitogenic effects were blocked by the monoclonal anti-IGF-I receptor antibody IR3, indicating that IGF-I-induced proliferative effects were mediated via its homologous receptor. In contrast, insulin binding and mitogenesis displayed blocking by both anti-IGI-I and anti-insulin receptor antibodies, indicating mediation of its activity through both receptors. These data demonstrate specific binding and mitogenic interactions between insulin, IGFs, and hemopoietic cells which are associated with their state of differentiation.     

Inhibition of free radical-mediated oxidation of cellular biomolecules by carboxylated chitooligosaccharides

The objective of this study was to identify the cellular antioxidant effects of carboxylated chitooligosaccharides (CCOS), a chemically modified derivative of chitooligosaccharides (COS), by assessing oxidation inhibition potential on cellular biomolecules such as lipids, proteins, and direct scavenging of reactive oxygen species (ROS). Radical-mediated oxidation of cell membrane lipids and proteins was dose-dependently inhibited by CCOS, assessed by amount of lipid hydroperoxides and carbonyl carbon content in mouse macrophages, RAW264.7 cells. Further, CCOS inhibited myeloperoxidase (MPO) activity in human myeloid cells (HL60) suggesting indirect possibility of inhibiting generation of reactive oxygen species (ROS) such as superoxide radicals, H(2)O(2) and HOCl. Direct radical scavenging studies carried out with DCFH-DA fluorescence probe concluded that CCOS can act as a potent radical scavenger in cells.