Effects of combinations of transforming growth factor-beta 1 and tumor necrosis factor on induction of differentiation of human myelogenous leukemic cell lines

Effects of transforming growth factor-beta 1 (TGF-beta 1), either alone or in combination with TNF, on the induction of differentiation of human myelogenous leukemic cell lines were examined. TGF-beta 1 alone induced differentiation of a human monocytic leukemia U-937 line into the cells with macrophage characteristics. When combined with TNF, TGF-beta 1 synergistically or additively induced differentiation associated properties. A human myeloblastic leukemia cell line, ML-1, differently responded to TGF-beta 1 in induction of differentiation. FcR activity and phagocytic activity induced by TNF were suppressed by TGF-beta 1. However, nitroblue tetrazolium reducing activity was synergistically induced by combinations of TGF-beta 1 and TNF. Scatchard analysis of TNF receptors indicated that the number of binding sites and dissociation constant of TNF for its receptors on U-937 or ML-1 cells were not changed by treatment with TGF-beta 1. Although IFN-gamma, IL-6, granulocyte CSF, and granulocyte-macrophage CSF-induced nitroblue tetrazolium reducing activity of U-937 cells, only IFN-gamma, and TNF induced it synergistically in combination with TGF-beta 1. Synergism between TGF-beta 1 and TNF was also observed in inhibition of growth of U-937 and ML-1 cells. Although TGF-beta 1 induction of differentiation of other monocytoid leukemic THP-1 cells was similar to that of U-937 cells, TGF-beta 1 only slightly induced differentiation of promyelocytic leukemic HL-60 cells, either alone or in combination with TNF. Our observations indicate that TGF-beta 1 strongly modulates differentiation and proliferation of human myelogenous leukemia cells, macrophage precursors.     

Regulatory properties and cellular redistribution of zinc during macrophage differentiation of human leukemia cells

Many proteins require the binding of trace metals such as Ca, Fe, Cu, or Zn, which may modulate their structure, function, or activity. To determine if there were any overall changes in metalloprotein distribution or metal concentration during the process of macrophage differentiation we induced human myeloid HL-60 leukemia cells with phorbol 12-myristate 13-acetate (PMA) and quantitatively mapped their metal content using hard X-ray fluorescence micro-analysis. We found a transient increase in the zinc content of HL-60 cell nuclei during the early stages of differentiation induction. This finding was confirmed by spectrofluorometry in HL-60 cells and extended to U-937 leukemia cells. A role for protein kinase C-beta (PKC-beta) in this process was established by examining zinc content in an HL-60 variant, HL-525, which is PKC-beta deficient, and in HL-525 cells in which PKC-beta was restored by stable overexpression. Chemical chelation of both Cu and Zn served to inhibit macrophage differentiation in HL-60 cells, indicating a requirement for these metals during this process. Finally, we demonstrate that growth of HL-60 cells in a low-zinc environment removes their susceptibility to PMA-induced differentiation, and that this capacity can be partially restored by the addition of exogenous zinc.     

Differential regulation of 4E-BP1 and 4E-BP2, two repressors of translation initiation, during human myeloid cell differentiation

Human myeloid differentiation is accompanied by a decrease in cell proliferation. Because the translation rate is an important determinant of cell proliferation, we have investigated translation initiation during human myeloid cell differentiation using the HL-60 promyelocytic leukemia cell line and the U-937 monoblastic cell line. A decrease in the translation rate is observed when the cells are induced to differentiate along the monocytic/macrophage pathway or along the granulocytic pathway. The inhibition in protein synthesis correlates with specific regulation of two repressors of translation initiation, 4E-BP1 and 4E-BP2. Induction of HL-60 and U-937 cell differentiation into monocytes/macrophages by IFN-gamma or PMA results in a dephosphorylation and consequent activation of 4E-BP1. Dephosphorylation of 4E-BP1 was also observed when U-937 cells were induced to differentiate into monocytes/macrophages following treatment with retinoic acid or DMSO. In contrast, treatment of HL-60 cells with retinoic acid or DMSO, which results in a granulocytic differentiation of these cells, decreases 4E-BP1 amount without affecting its phosphorylation and strongly increases 4E-BP2 amount. Taken together, these data provide evidence for differential regulation of the translational machinery during human myeloid differentiation, specific to the monocytic/macrophage pathway or to the granulocytic pathway.     

The role of calcium in differentiation of leukemic cell lines

Increased calcium influx associated with differentiation of four human myeloid leukemic cell lines: HL-60, KG-1, U-937 and K-562, to either monocytic or granulocytic direction was demonstrated. Calcium influx was measured employing two methods; measurement of radioactive calcium influx rate at 4 degrees C and employing the fluorescent probe, fura-2 acetoxymethyl ester. The increase in Ca2+ influx was demonstrated with three chemically unrelated differentiation inducers: retinoic acid, 1 alpha, 25 dihydroxy vitamin D3 and dimethyl sulfoxide. Inhibitors of calcium uptake such as verapamil diltiazem and cromolyn, partially reduced differentiation, suggesting that differentiation of myeloid leukemic cell lines is dependent on the availability of extracellular calcium.     

Regulation of progranulin expression in myeloid cells

Progranulin (pgrn; granulin-epithelin precursor, PC-cell-derived growth factor, or acrogranin) is a multifunctional secreted glycoprotein implicated in tumorigenesis, development, inflammation, and repair. It is highly expressed in macrophage and monocyte-derived dendritic cells. Here we investigate its regulation in myeloid cells. All-trans retinoic acid (ATRA) increased pgrn mRNA levels in myelomonocytic cells (CD34(+) progenitors; monoblastic U-937; monocytic THP-1; progranulocytic HL-60; macrophage RAW 264.7) but not in nonmyeloid cells tested. Interleukin-4 impaired basal expression of pgrn in U-937. Differentiation agents DMSO, and, in U-937 only, phorbol ester [phorbol 12-myristate,13-acetate (PMA)] elevated pgrn mRNA expression late in differentiation, suggestive of roles for pgrn in more mature terminally differentiated granulocyte/monocytes rather than during growth or differentiation. The response of pgrn mRNA to ATRA differs in U-937 and HL-60 lineages. In U-937, ATRA and chemical differentiation agents greatly increased pgrn mRNA stability, whereas, in HL-60, ATRA accelerated pgrn mRNA turnover. The initial upregulation of pgrn mRNA after stimulation with ATRA was independent of de novo protein synthesis in U-937 but not HL-60. Chemical blockade of nuclear factor-kappaB (NF-kappaB) activation impaired ATRA-stimulated pgrn expression in HL-60 but not U-937, whereas in U-937 it blocked PMA-induced pgrn mRNA expression, suggestive of cell-specific roles for NF-kappaB in determining pgrn mRNA levels. We propose that: 1) ATRA regulates pgrn mRNA levels in myelomonocytic cells; 2) ATRA acts in a cell-specific manner involving the differential control of mRNA stability and differential requirement for NF-kappaB signaling; and 3) elevated pgrn mRNA expression is characteristic of more mature cells and does not stimulate differentiation.     

Differential changes in lipid metabolism of myeloid and lymphoid cell lines induced by treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA)

Treatment of the myeloid cell lines, U-937 or HL-60, with 10 nM of the phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), for 24 h increased the rate of incorporation of [3H]glycerol into total chloroform extracts. A proportionally greater labeling of the non-polar lipid (NL) fraction compared to the polar, phospholipid (PL), fraction was observed. Chromatographic analysis showed a 6-fold increase in the labeling of triacylglycerols (TAG), a 2-fold increase in diacylglycerols, and no changes in monoacylglycerols. PL labeling showed a 3-fold increase in phosphatidylcholine (PC). The effect of TPA on TAG labeling was selectively observed in myeloid cell lines. No such a change was found in the lymphoid cell line. MOLT-3, which did respond to TPA with increased PC labeling. Incorporation of [3H]arachidonic acid (AA) into TAG by U-937 cells was selectively increased (2.5-fold) after treatment with TPA for 24 h. Treatment of U-937 cells with TPA in serum-free medium resulted in no increased labeling of TAG. These studies suggest that changes in TAG metabolism may be characteristic of myeloid differentiation and depend on the presence of serum factor(s).     

Functional Role for Protein Kinase Cβ as a Regulator of Stress-Activated Protein Kinase Activation and Monocytic Differentiation of Myeloid Leukemia Cells

Human myeloid leukemia cells respond to 12-O-tetradecanoylphorbol-13-acetate (TPA) and other activators of protein kinase C (PKC) with induction of monocytic differentiation. The present studies demonstrated that treatment of U-937 and HL-60 myeloid leukemia cells with TPA, phorbol-12,13-dibutyrate, or bryostatin 1 was associated with the induction of stress-activated protein kinase (SAPK). In contrast, TPA-resistant TUR and HL-525 cell variants deficient in PKCβ failed to respond to activators of PKC with the induction of SAPK. A direct role for PKCβ in TPA-induced SAPK activity in TUR and HL-525 cells that stably express PKCβ was confirmed. We showed that TPA induced the association of PKCβ with MEK kinase 1 (MEKK-1), an upstream effector of the SAPK/ERK kinase 1 (SEK1)→SAPK cascade. The results also demonstrated that PKCβ phosphorylated and activated MEKK-1 in vitro. The functional role of MEKK-1 in TPA-induced SAPK activity was further supported by the demonstration that the expression of a dominant negative MEKK-1 mutant abrogated this response. These findings indicate that PKCβ activation is necessary for activation of the MEKK-1→SEK1→SAPK cascade in the TPA response of myeloid leukemia cells.     

Recombinant human interferon sensitizes resistant myeloid leukemic cells to induction of terminal differentiation

Recombinant human leukocyte interferon (IFN-alpha A) inhibits growth of the human promyelocytic leukemic cell line HL-60 without inducing these cells to differentiate terminally. When IFN-alpha A is combined with agents capable of inducing differentiation in HL-60 cells, such as 12-O-tetradecanoyl-phorbol-13-acetate (TPA), cis or trans retinoic acid (RA) or dimethylsulfoxide (DMSO), growth suppression and induction of differentiation are dramatically increased. By growing HL-60 cells in increasing concentrations of TPA, RA, or DMSO, a series of sublines have been developed which are resistant to the usual growth inhibition and induction of differentiation seen when wild type HL-60 cells are exposed to these agents. Treatment of these resistant HL-60 cells with the combination of IFN-alpha A and the appropriate inducer results, however, in a synergistic suppression in cell growth and a concomitant induction of terminal differentiation. The ability of interferon to interact synergistically with agents which promote leukemic cell maturation may represents a novel means of reducing resistant leukemic cell populations.     

Identification of the differentiation-associated p93 tyrosine protein kinase of HL-60 leukemia cells as the product of the human c-fes locus and its expression in myelomonocytic cells

A differentiation-associated 93-kDa tyrosine kinase (p93) was purified previously from the human promyelocytic leukemia cell line HL-60. The present study conclusively identifies p93 as the c-fes proto-oncogene product and shows that expression of p93c-fes and its associated tyrosine kinase activity are marked in mature granulocytes, monocytes, and human myeloid leukemia cell lines. Antisera to peptides obtained by expression of c-fes cDNA fragments in Escherichia coli reacted strongly with p93 purified from HL-60 cells. Western blots using one of these antisera demonstrated high levels of p93c-fes protein in normal human granulocytes and monocytes, as well as the cell lines KG-1, THP-1, HEL, and U-937, all of which can be induced to differentiate along the myelomonocytic pathway. Conversely, in cell lines resistant to myeloid differentiation, p93c-fes expression was either very low or absent. Expression of immunoreactive p93c-fes in these cell lines showed a strong positive correlation with p93c-fes tyrosine kinase activity, which was measured in cell extracts using a nondenaturing gel assay. Finally, the expression of p93c-fes, its tyrosine kinase activity, and the binding of 125I-granulocyte-macrophage colony-stimulating factor (GM-CSF) were all coordinately increased in HL-60 cells treated with the granulocytic differentiation inducer dimethyl sulfoxide, while all three parameters were low in untreated or differentiation-resistant HL-60 cells. These results suggest that expression of p93c-fes tyrosine kinase activity may be an essential component of myeloid differentiation and responsiveness to granulocyte-macrophage colony-stimulating factor.     

The human myelomonocytic cell line U-937 as a model for studying alterations in steroid-induced monokine gene expression: marked enhancement of lipopolysaccharide-stimulated interleukin-1 beta messenger RNA levels by 1,25-dihydroxyvitamin D3.

The active metabolite of vitamin D, 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3], is a potent regulator of human monocyte/macrophage function in vitro. To establish a model for 1,25-(OH)2D3 regulation of human monocyte monokine synthesis, three human cell lines (U-937, THP-1, and HL-60) were examined for: 1) the presence of functional 1,25-(OH)2D3 receptors; 2) the accumulation of interleukin-1 beta (IL-1 beta) mRNA and IL-1 beta protein in response to lipopolysaccharide (LPS); and 3) the regulation of this response by 1,25-(OH)2D3. All three cell lines expressed vitamin D receptor and had increased levels of IL-1 beta mRNA in response to LPS. Preincubation of cells with 1,25-(OH)2D3 augmented IL-1 beta mRNA levels only in U-937 and HL-60 cells. From these data, and taking into consideration their state of differentiation and relative ease of culture, U-937 was chosen over HL-60 and THP-1 as the cell line we further characterized. In U-937 cells, optimum time and dose of pretreatment with 1,25-(OH)2D3 were determined to be 12-24 h at a receptor saturating concentration of 1,25-(OH)2D3 (10 nM). Preincubation of cells with 1,25-(OH)2D3 had no effect on the time course of IL-1 beta mRNA appearance in response to LPS. However, exposure of U-937 cells to 1,25-(OH)2D3 increased by 200% the level of IL-1 beta mRNA detected and decreased by three orders of magnitude the concentration of LPS required to achieve steady state mRNA levels equivalent to those observed in U-937 cells not preincubated with the hormone.2+o     

Properties of a nuclear protein marker of human myeloid cell differentiation

The Mr 55,000 nuclear antigen present in the human promyelocytic cell line HL-60 is a basic protein that is extracted from nuclei or chromatin by 0.35 M NaCl. The antigen is confined to the nucleus of the interphase HL-60 cell as judged by immunocytochemical localization but disperses throughout the cell during mitosis. The antigen was not detected in leukemic cell lines with blast cell properties or in cell lines representing other lineages. Additional cell lines (ML-1, ML-2, and U937) with myeloid cell characteristics similar to those of the HL-60 cells, which also differentiate in vitro, express the antigen. The presence of antigen in normal human myeloid cells in peripheral blood and bone marrow is consistent with its proposed role in nuclear events associated with normal human myeloid cell differentiation.     

Characteristics of U-937 and HL-60 leukemia cells differentiated by spinach extract

Some characteristics of U-937 and HL-60 leukemia cell lines treated with a fraction of non-dialyzable extract of spinach are reported. The absorbed fraction separated by a DEAE-Tyopearl 650 column chromatography of the non-dialyzable extract induced NBT reducing activity of U-937 and HL-60 cells. This fraction also induced substrate adhesion of U-937 cells, and the non-specific esterase activity of HL-60 cells. The expression of CD11b, CD11c and CD36 antigens on the U-937 cell surface was enhanced by the treatment with the fraction, whereas CD24 antigen was not. The treatment of HL-60 cells with the fraction also induced the expression of CD11b and CD11c antigens, but CD24 and CD36 were not expressed. These results indicated that the non-dialyzable extract of spinach induced immature differentiation of U-937 and HL-60 cells into monocyte/macrophages.Abbreviations NBT nitroblue tetrazolium - TPA 12-O-tetradecanoyl-phorbol-13-acerate - PBS phosphate buffered saline - FITC fluorescein isothiocyanate