Differentiation-associated changes in membrane proteins of mouse myeloid leukemia cells.

The mouse myeloid leukemia cell line (M1) is known to differentiate in vitro into macrophages and granulocytes upon treatment with various inducers including mouse ascitic fluid. Changes of cell surface proteins during differentiation of M1 cells were analyzed by the lactoperoxidase-catalyzed radioiodination method and SDS-polyacrylamide slab gel electrophoresis. Treatment of the cells with ascitic fluid changed the electrophoretic pattern of the iodinated proteins, the prominent change being the appearance of a new protein with a molecular weight of 180 000 (P180). Iodinated P180 was also detected in normal macrophages in granulocytes, which are similar to differentiated M1 cells. This protein was metabolically labeled with L-[14C]fucose, increasing with the period of the treatment. P180 was not expressed on ascitic fluid-treatment of a resistant clone of M1 cells that could not be induced to differentiate. These results indicate that P180 is a glycoprotein that is exposed on the outer surface of differentiated M1 cells, and that its expression is associated with differentiation of the cells. P180 was solubilized from 125I-labeled macrophages with detergents bound to concanavalin A-Sepharose. This suggests that P180 is one of the receptors for concanavalin A. Therefore, P180 may contribute partly to the increases in agglutinability by concanavalin A and in the number of concanavalin A binding sites on the surface of M1 cells, which are known to be associated with differentiation of M1 cells.     

Changes in protease during differentiation of mouse myeloid leukemia cells.

The protease activities of mouse myeloid leukemia cells Ml were examined using fluorescein isothiocyanate-labeled albumin as substrate. Protease activity in Ml cells was greatest at alkaline pH values with a maximum at pH 11.0, and only slight activity was seen at neutral and acidic pHs. When Ml cells were induced to differentiate into mature cells by lipopolysaccharide, their alkaline protease activity decreased greatly with marked increase in acid protease activity. Moreover, in a variant cell line Mml with the properties of differentiated Ml cells, no protease activity was found at alkaline pH values.     

Changes in neutral amino acid transport activity in myeloid leukemia cells differentiated by lipopolysaccharide

M1 cells derived from mouse myeloid leukemia have been reported to differentiate to macrophage-like cells upon treatment with substances such as lipopolysaccharide. Previously we found that in mouse peritoneal macrophages most of the neutral amino acids were taken up through a unique Na+-independent system. In this paper we have investigated the neutral amino acid transport in M1 cells and in those treated with lipopolysaccharide. In M1 cells serine, alanine and proline were taken up mainly by Na+-dependent transport systems, and leucine was largely transported by a Na+-independent system. By treating the cells with lipopolysaccharide, the activities of the Na+-dependent systems markedly decreased, whereas the activity of the Na+-independent system was little affected. The amino acid concentrations in the cells and the culture medium were measured. As a whole, the intracellular to extracellular distribution ratios for neutral amino acids that are preferred substrates for Na+-dependent systems were decreased on lipopolysaccharide treatment, whereas those for amino acids that are mainly transported by a Na+-independent system were slightly increased. From these results we conclude that M1 cells treated with lipopolysaccharide tend to differentiate to macrophage-like cells with respect to the neutral amino acid transport.     

Induction of differentiation of human and mouse myeloid leukemia cells by camptothecin

Low concentrations of camptothecin induced differentiation of human and mouse myeloid leukemia cells including human HL60, U937, ML1, and K562 cells and mouse M1 cells as measured by various differentiation-associated properties. When K562 cells were pretreated with 20 nM camptothecin for 2 h, 53% of the cells were induced to differentiate as measured by NBT staining. Significant single strand breaks in DNA of K562 cells were caused by this treatment. Most single strand breaks were accompanied by protein-DNA cross linking. The combination of camptothecin and rTNF synergistically induced differentiation of human ML1, U937, and M1 cells. These results suggest that topo I may be important in some differentiation of myeloid leukemia cells.     

Induction of differentiation of mouse myeloid leukemia cells by poly(ADP-ribose).

The effects of poly(ADP-Rib) on the differentiation of mouse myeloid leukemia cells were studied. The myeloid leukemia cells differentiated into cells with phagocytic activity, Fc receptors, and lysozyme activity on treatment with poly(ADP-Rib). Cells with morphological characteristics of macrophages and granulocytes also appeared on incubation with poly(ADP-Rib). Dextran sulfate and polyvinylsulfate were also effective for the induction of phagocytic cells, but poly(A), poly(U), poly(C), poly(I), poly(I) · poly(C), and poly(A) · poly(U) were not. The uptake of poly(ADP-Rib) by the myeloid leukemia cells is discussed in relation to their differentiation.     

Production of differentiation-stimulating factor in cultured mouse myeloid leukemia cells treated by glucocorticoids

Mouse myeloid leukemic cells (Ml) could be induced by glucocorticoids to produce a factor(s) stimulating their own differentiation to macrophage- and granulocyte-like cells. The differentiation-stimulating factor(s) (DSF) was not due to a contaminating steroid, although glucocorticoids were effective to induce differentiation of the cells. DSF seemed to be a glycoprotein(s) with a molecular weight of 20 000–40 000 D, since it was susceptible to a treatment of proteases of glycosidases. The inducing ability of corticoids to produce DSF was correlated with their glucocorticoid activity, which was in parallel with the inducing activity of cell differentiation. Moreover, glucocorticoids were unable to stimulate the production of DSF in a dexamethasoneresistant Ml cells which could not differentiate even in a high concentration of dexamethasone. These results suggest that production of DSF in Ml cells was closely associated with differentiation of the cells. Differentiation of Ml cells by DSF was confirmed by morphological, functional and biochemical evidences.     

Global dynamics of hematopoietic stem cells and differentiated cells in a chronic myeloid leukemia model

We consider a mathematical model describing evolution of normal and leukemic hematopoietic stem cells (HSC) and differentiated cells in bone marrow. We focus on chronic myeloid leukemia (CML), a cancer of blood cells resulting from a malignant transformation of hematopoietic stem cells. The dynamics are given by a system of ordinary differential equations for normal and leukemic cells. Homeostasis regulates the proliferation of normal HSC and leads the dynamics to an equilibrium. This mechanism is partially efficient for leukemic cells. We define homeostasis by a functional of either hematopoietic stem cells, differentiated cells or both cell lines. We determine the number of hematopoietic stem cells and differentiated cells at equilibrium. Conditions for regeneration of hematopoiesis and persistence of CML are obtained from the global asymptotic stability of equilibrium states. We prove that normal and leukemic cells can not coexist for a long time. Numerical simulations illustrate our analytical results. The study may be helpful in understanding the dynamics of normal and leukemic hematopoietic cells.     

Differentiation-associated changes in membrane proteins of mouse myeloid leukemia cells

The mouse myeloid leukemia cell line (M1) is known to differentiate in vitro into macrophages and granulocytes upon treatment with various inducer including mouse ascitic fluid. Changes of cell surface proteins during differentiation of M1 cells were analyzed by the lactoperoxidase-catalyzed radioiodination method and SDS-polycrylamide slab gel electrphoresis. Treatment of the cells with ascitic fluid changed the electrophoretic pattern of the iodinated proteins, the prominent change being the appearance of a new protein with a molecular weight of 180 000 (P180). Iodinated P180 was also detected in normal macrophages in granulocytes, which are similar to differentiated M1 cells. This protein was metabolically labeled with l-[¹⁴C]fucose, increasing with the period of the treatment. P180 was not expressed on ascitic fluid-treatment of a resistant clone of M1 cells that could not be induced to differentiate. These results indicate that P180 is a glycoprotein that is exposed on the outer surface of differentiated M1 cells, and that its expression is associated with differentiation of the cells.P180 was solubilized from ¹²⁵I-labeled macrophages with detergents bound to concanavalin A-Sepharose. This suggests that P180 is one of the receptors for concanavalin A. Therefore, P180 may contribute partly to the increases in agglutinability by concanavalin A and in the number of concanavalin A binding sites on the surface of M1 cells, which are known to be associated with differentiation of M1 cells.     

Induction of lysozyme activity by adenosine 3':5' cyclic monophosphate in cultured mouse myeloid leukemic cells.

Mouse myeloid leukemic cells(Ml) could be induced by glucocorticoids to form Fc receptors, phagocytize, migrate in agar, induce lysosomal enzyme activities, and change into forms that were morphologically similar to macrophages and granulocytes. Adenosine 3′:5′ cyclic monophosphate also induced lysosomal enzyme activities, but not the other differentiation-associated properties. The induction of lysozyme activity was marked, the activity reaching about 400 times the initial activity at 5 days after treatment. This suggests that adenosine 3′:5′ cyclic monophosphate may be important in induction of lysozyme activity during differentiation of the cells.     

Biosynthesis of glycosphingolipids by human myeloid leukemia cells

We have performed comparative studies of the neutral glycosphingolipids synthesized by three human myeloid leukemia cell lines, K562, KG1, and HL-60, which were metabolically labeled with [14C]galactose, to evaluate changes in neutral glycosphingolipid synthesis with myeloid cell differentiation. Individual neutral glycosphingolipids containing one to four sugars were purified by a combination of the following methods: diethylaminoethyl-Sephadex column chromatography, acetylation-Florisil column chromatography, and high-performance liquid chromatography using an Iatrobead column. Compounds with one sugar were analyzed by thin-layer chromatography on borate plates. This analysis showed that HL-60 cells synthesize only glucosylceramide, whereas K562 and KG1 cells synthesize predominately glucosylceramide, but also a small amount of galactosylceramide. Compounds with two to four sugars were characterized by treatment with exo- and endoglycosidases. The results showed that K562 and KG1 cells are similar to cells from patients with acute leukemia in expressing two series (globo and neolacto) of natural glycosphingolipids, whereas the HL-60 cells are similar to mature human myeloid cells in expressing only one series (neolacto). Therefore, human myeloid leukemia cells blocked at different stages of differentiation vary in their ability to synthesize neutral glycosphingolipids.     

Regulation of prostaglandin synthesis during differentiation of cultured mouse myeloid leukemia cells

Mouse myeloid leukemia cells (Ml) were induced to differentiate into mature macrophages and granulocytes by various inducers. The differentiated Ml cells synthesized and released prostaglandins, whereas untreated Ml cells did not. When the cells were prelabelled with [¹⁴C]arachidonate, the major prostaglandins released into the culture media were found to be prostaglandin E₂, D₂, and F_2α in an early stage of differentiation, but the mature cells produced predominantly prostaglandin E₂. The synthesis and release of prostaglandins were completely inhibited by indomethacin. Dexamethasone, a potent inducer of differentiation of Ml cells, did not induce production of prostaglandins in resistant Ml cells that could not differentiate even with a high concentration of dexamethasone. These results suggest that production of prostaglandins in Ml cells is closely associated with differentiation of the cells. Homogenates of dexamethasone-treated Ml cells converted arachidonate to prostaglandins, but this conversion was scarcely observed with homogenates of untreated Ml cells. Dexamethasone and the other inducers stimulated the release of arachidonate from phospholipids. Therefore, induction of prostaglandin synthesis during differentiation of Ml cells may result from induction of prostaglandin synthesis activity and stimulation of the release of arachidonate from cellular lipids. Lysozyme activity, which is a typical biochemical marker of macrophages, was induced in Ml cells by prostaglandin E₂ or D₂ alone, as well as by inducers of differentiation of the cells, but it was not induced by arachidonate or prostaglandin F_2α. These results suggest that prostaglandin synthesis is important in differentiation of myeloid leukemia cells.     

Changes in phospholipid composition during differentiation of cultured mouse myeloid leukemia cells

Mouse myeloid leukemia cells(M1) could be induced by various inducers to form Fc receptors, phagocytize, migrate in agar, produce lysosomal enzyme activities, and change into forms that were morphologically similar to macrophages and granulocytes. When M1 cells were cultured with inducer, the ratio of the percentage of phosphatidylethanolamine to that of phosphatidylcholine was increased about 2-fold. This ratio of the differentiated M1 cells was similar to that of peritoneal macrophages of normal mice or Mm-1 cells, which were established from spontaneously differentiated macrophage-like cells from M1 cells. These changes in phospholipid may be involved in the mechanisms of expression of the differentiation-associated phenotypic properties.     

Membrane potential in human myeloid leukemia cell line ML-1: responsiveness of granulocytic and monocytic differentiated cells.

The membrane potential responsiveness of human myeloid leukemia cells (ML-1 line) was studied with the voltage sensitive fluorescent dye diS-C3-(5). The experimental procedure used in this study enabled us to assess the magnitude of the membrane potential change in cells treated with ouabain, 12-0-tetradecanoylphorbol-13-acetate (TPA) and N-formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP), relative to the membrane potential in the untreated control. Inhibition of the Na, K-ATPase by ouabain was followed by a (20 +/- 4) mV depolarization. In undifferentiated homogeneous cell population TPA caused a (19.4 +/- 4.4) mV depolarization while FMLP had virtually no effect. Cells in which granulocytic or monocytic differentiation was induced by retinoic acid or 1,25-dihydroxyvitamin D3 exhibited under the effect of TPA a (57.8 +/- 7.1) mV and (34.8 +/- 10.9) mV depolarization, respectively. A very small transient depolarization was also observed up on treating of the cells with FMLP. The changes in the membrane potential responsiveness in the induced cells are obviously connected with the cell differentiation.     

Modification of membrane phospholipid composition by choline analogues induces differentiation of cultured mouse myeloid leukemia cells

Mouse myeloid leukemia M1 cells could be induced by various inducers to form Fc receptors, phagocytize, produce lysozyme, and change into forms that were morphologically similar to macrophages and granulocytes. Previous experiments showed that change in phospholipid metabolism was associated with cell differentiation. In the present experiment, culture of M1 cells with choline analogs such as N-monomethylethanolamine and N,N′-dimethylethanolamine resulted in accumulation of phosphatidyl-N-monomethylethanolamine and phosphatidyl-N,N′-dimethylethanolamine in the cell membranes. This change upon treatment with choline analogs was associated with morphological and functional differentiation of the M1 cells into macrophages and granulocytes. These results suggest that phospholipid metabolism is involved in the mechanism of differentiation of M1 cells.     

Induction of differentiation of cultured mouse myeloid leukemic cells by arginase.

Mouse myeloid leukemic cells (Ml) could be induced by a factor in ascitic fluid to phagocytize, migrate in agar, and change into forms that were morphologically similar to macrophages and granulocytes. Arginase also induced these differentiation-associated properties of the cells. The Ml cells did not differentiate in culture medium containing arginine, but they differentiated into macrophages and granulocytes during culture in arginine-deficient culture medium. Therefore, the effect of arginase may be attribute to arginase-mediated arginine depletion.     

Characterization of "abp38" in cytoskeleton of mouse myeloid leukemia cells and distribution of abp38 in various tissues

A specific antibody was prepared against "abp38," a 38 kDa-dimer protein purified from mouse myeloid leukemia cells (M1 cells), that induce gelation of actin filaments in a K+-dependent manner. Immunochemically, the total content of abp38 in undifferentiated M1 cells was found to be 0.89% of the total protein (10.7 micrograms/10(7) cells). This content increased about 8-fold (89.5 micrograms/10(7) cells) after the M1 cells had differentiated into macrophages. When the cells before differentiation were extracted with Triton solution containing 150 mM KCl, almost all abp38 in the cytoskeleton was removed, whereas in cells after differentiation, amount of abp38 remaining in the cytoskeleton was 4.5 micrograms per 10(7) cells. The amount of cytoskeleton-bound abp38 of M1 cells and mouse peritoneal macrophages decreased with increase in K+ concentration in the extraction solution. Immunoreactive molecules against abp38 antibody were present in various tissues and cultured cell lines except for skeletal muscle and erythrocytes. Furthermore, actin binding protein with a molecular size of 38 kDa was found in bovine brain. These data suggest that abp38 is a ubiquitous protein present in various tissues and species.     

Induction of differentiation and arrest of proliferation of mouse myeloid leukemia M1 cells

The relationship between differentiation and the cell cycle of mouse myeloid leukemia M1 cells was studied. The cells were induced to differentiate into macrophage-like cells by treatment with conditioned medium (CM) of hamster embryo cells. CM-treated cells traversed the S phase of the cell cycle at least once, then a fraction of the cells lost the ability to enter the S phase and accumulated in the G1 phase. Incorporation of [³H]thymidine in phagocytosis-induced cells decreased after 12–18 h of CM treatment. The morphology of the differentiated cells changed and the nucleus-cell ratio (NCR) of the individual cells decreased significantly between 12 h and 24 h of CM treatment. The decrease in NCR was well associated with arrest of proliferation in the G1 phase of the cells. The results suggest that G1 arrest of CM-treated M1 cells is an expression of cellular characteristics encoded in the differentiation program.