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.     

Changes in myosin during differentiation of myeloid leukemia cells

Changes in cellular myosin were followed during the differentiation into macrophages of a myeloid leukemia cell line (Ml) which can be induced by conditioned medium (CM) from a rat embryo culture. To extract the myosin, we used three different procedures, all of which gave a lower yield of myosin for the differentiated than for the undifferentiated Ml cells. This low extractability we attributed to increased binding of the myosin to the plasma membrane. Taking the different extractabilities into consideration, we calculated the myosin contents in the total cellular protein from the densitometry of SDS-polyacrylamide electrophoresis, 0.6% for the untreated Ml cells and 1.0% for the differentiated ones. The three ATPase activities of the Ml cell myosin were in the order, K+-EDTA-=Ca2+- much greater than Mg2+-ATPase in the presence of 0.6 M KCl, whether or not there was treatment with CM. Myosin was purified through fractionation with 25-55% saturated ammonium sulfate, then gel filtration with Sepharose 4B followed by affinity chromatography on F actin-Sepharose 4B. The Ml cell myosin consists of 1 heavy chain (H) and 3 light chains (L1, L2, L3), with molecular ratios of L1 + L2/H not equal to and L3/H not equal to 1. The ratio of L1/L2 was about 1.2 for the untreated Ml cells, but it decreased to about 0.7 after differentiation.     

Purification and characterization of a vimentin-specific protease in mouse myeloid leukemia cells. Regulation during differentiation and identity with cathepsin G.

Strong vimentin-degrading activity was found in a mouse myelomonocytic leukemic cell line, M1. When M1 cells were induced to differentiate into macrophage-like cells, this degrading activity decreased, while expression of the vimentin gene increased as reported previously [Tsuru, A., Nakamura, N., Takayama, E., Suzuki, Y., Hirayoshi, K. and Nagata, K. (1990) J. Cell Biol. 110, 1655-1664]. This activity was not due to calpain, which was reported to degrade vimentin, because it was independent of the presence or absence of Ca2+. This activity was revealed to be strongly associated with membranes by differential-centrifugation experiments. To identify this protease, purification of the degradation enzyme was performed. A membrane fraction was prepared and extracted with a buffer containing Triton X-100, then subjected to column chromatography using carboxymethyl-Sepharose and heparin-Sepharose. Quantitative analysis using the purified protease revealed that the specificity of this protease was more than 1000-fold higher for vimentin than for bovine serum albumin, ovalbumin and actin. Four protein bands expressing the activity were finally identified by SDS/PAGE. Amino-terminal sequences of these four proteins were identical, suggesting lower-molecular-mass proteins were degradative products. Furthermore, it was revealed that the sequence had the highest similarity with that of human cathepsin G. This result was consistent with the cathpsin-G-like properties of the purified protease, such as the optimum pH and the specificities for inhibitors. The purified protease degraded a synthetic substrate for cathespin G, succinyl-alanyl-alanyl-prolyl-phenylalanyl-p-nitroanilide, with a comparable specific activity to human cathespin G and was specifically detected with anti-(human cathepsin G) serum in immunoblot analysis. The purified protease thus belongs to the 'cathepsin G family', and perhaps is a mouse homologue of human cathepsin G.     

Changes in contractile proteins during differentiation of myeloid leukemia cells. I. Polymerization of actin

Quantitative and qualitative changes in cellular actin were followed during differentiation of a myeloid leukemia cell line, namely Ml, which was inducible with conditioned medium (CM). During 3 d of incubation with CM, when the Ml cells differentiated to macrophages and lost their mitotic activity, the actin content, F-actin ratio in total actin, and the actin synthesis showed an increase. A greater difference before and after differentiation was found in the ability of G-actin to polymerize. Actin harvested from CM-treated cells showed a greater ability to polymerize, depending on the increased concentration of MgCl2 and/or KCl and proteins, as compared with the actin from untreated Ml cells. Actin harvested from the Mml cell line, a macrophage line, had a particularly high polymerizability with or without CM treatment. In contrast, the actin from the D- subline, which is insensitive to CM, showed almost no polymerization.     

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 actin-related gelation of crude cell extracts during differentiation of myeloid leukemia cells

Gelation of extracts of a myeloid leukemia cell line (Ml) was compared before and after differentiation induced with conditioned medium (CM) from rat embryo cells. Although an extract of Mml cells, a macrophage line derived from Ml line, gelled when warmed in the presence of 2 mM MgCl2, undifferentiated Ml cells gelled only after dialysis and a supplement of exogenous actin. After differentiation had been induced, an addition of exogenous actin, but not dialysis, was needed for gelation. Small amounts of KCl always inhibited the gelation of the control Ml cell extracts, but they promoted gelation of the CM-treated Ml and Mml cell extracts. Thus, the dialysis required for gelation of the control Ml cell extract appears to be necessary for the exclusion of endogenous KCl. Several possible mechanisms for the KCl control of gelation, as well as different requirements of exogenous actin needed for gelation are discussed based on the results of our experiments.     

Changes in contractile proteins during differentiation of myeloid leukemia cells. II. Purification and characterization of actin

A myeloid leukemia cell line, M1, differentiates to macrophage and gains locomotive and phagocytic activity when incubated with conditioned medium (CM) from a fibroblast culture and bacterial endotoxin. To characterize the actin molecules before and after differentiation, the actin was purified through three sequential steps: DEAE-sephadex A- 50, polymerization/depolymerization, and sephadex G-150 chromatography. There were no essential differences between the inhibitory activity of actins from control M1 cells and CM-treated M1 cells on both DNase I and heavy meromyosin (HMMM) K(+)-EDTA-ATPase; the same dose response as with skeletal muscle actin took place. After the treatment with CM, however, the specific activity for the activation of HMMM Mg(2+)- ATPase by actin became two-fold that of untreated M1 actin, which was one third of the value for skeletal muscle actin. The V(max) for the control and the CM-treated M1 cell, as well as the skeletal muscle actins, proved to be the same. By contrast, the K(app) values for the control and CM-treated M1-cell actins were 3- and 1.5-fold the value for skeletal-muscle actin. This means that CM treatment of the M1 actin produced a twofold affinity for the Mg(2+)-ATPase of skeletal-muscle myosin. The critical concentrations for polymerization were compared under different salt concentrations and temperatures. Although no marked difference was found for the presence of 2 mM MgCl(2), 0.1 M KCl in place of MgCl(2) at 5 degrees C gave the following values: 0.1 mg/ml for skeletal-muscle actin, 0.7 mg/ml for control M1 actin, 0,5 mg/ml for CM- treated M1 actin, and 1.0 mg/ml for the D(-) subline that is insensitive to CM. Although the critical concentration of D(-) actin is extraordinarily high, this actin showed normal polymerization above the critical concentration. This together with the data presented in our previous paper, that the D(-) actin in the crude extract did not polymerize, suggests that an inhibitor for actin polymerization is present in the subline. The kinetics experiment at 0.1 M KCl and 25 degrees C revealed a slower polymerization of untreated M1- and D(-)-cell actins as compared with CM-treated M1 actin. This delayed polymerization was due to a delay during the nucleation stage, not during the elongation stage. By isoelectric focusing, the ratios of β- to γ-actin showed a marked difference depending on the states of cells: about 4.9 for control M1, 2.8 for CM-treated M1, and 7.6 for D(-)-subline actins. Tryptic peptide maps also revealed the presence of different peptides. Thus, the functional differences of actin before and after the differentiation was accompanied by some chemical changes in actin molecules.     

Changes in ultrastructures and enzyme activities during differentiation of myeloid leukemia cells to normal macrophages

Changes in ultrastructures and in enzyme activities were investigated electron microscopically, cytochemically and biochemically when mouse myeloid leukemia cells, Ml cell line, successfully differentiated to normal macrophages after incubation with a conditioned medium harvested from secondary embryo fibroblasts, or a lipopolysaccharide from Salmonella typhosa. The number of mitochondria increased significantly accompanied by the enhanced activity of cytochrome oxidase per cell, although the activity in each mitochondrion remained unchanged. The rough-surfaced endoplasmic reticulum elongated and often exhibited a concentrically multilayered lamellae. Glucose-6-phosphatase activity, a marker enzyme for the endoplasmic reticulum, also increased. Primary lysosomes were newly formed where acid phosphatase activity was positively demonstrated. Ten-nm cytoplasmic microfilaments, mainly forming bundles, and other microfilaments less than 6 nm wide were formed newly and abundant. Budding of type C viruses from the plasma membranes was reduced strikingly. Another established cell line, Mm-1, which spontaneously differentiated from the Ml cell line, was characterized completely by a macrophage, in which azurophilic granules (primary lysosomes), secondary lysosomes possessing strong activity of acid phosphatase and 10-nm microfilaments were most remarkable. These non-transplantable Mm-1 cells sometimes exhibited budding of viruses.     

Dephosphorylation of "abp38" protein during the differentiation of mouse myeloid leukemia cells

We have characterized the phosphorylation of "abp38" before and after the differentiation of mouse myeloid leukemic cells (M1 cells). The abp38 of both undifferentiated and differentiated M1 cells contained phosphoserine and phosphotyrosine. The extent of phosphorylation of abp38 decreased to about 30% of the value of undifferentiated cells during cell differentiation; phosphoserine and phosphotyrosine decreased to 41 and 11% of the values before differentiation, respectively.     

Changes in diacylglycerol and cyclic GMP during the differentiation of human myeloid leukemia K562 cells

When the human myeloid leukemia cell line, K562, was induced to differentiate along the erythroid lineage by a 4 day treatment with 10 microM tiazofurin, the cellular content of diacylglycerol decreased to 35% of the value in untreated control cells. Under the same conditions the content of cGMP decreased to 61% of the control value. Tiazofurin inhibits guanine nucleotide biosynthesis and lowers cellular GTP. When guanosine and adenine were added together with tiazofurin, the differentiation of K562 was prevented, the concentration of diacylglycerol was maintained at control values, and the reduction in the concentration of cGMP was partially prevented. Other inducers of differentiation which acted by different mechanisms, caused similar changes in the concentrations of diacylglycerol and cGMP.     

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.     

Changes in K+,Na+-sensitive actin gelation factor during the differentiation of myeloid leukemia cells

A homodimer protein consisting of two 38,000 dalton peptides was isolated from a murine leukemia cell line (M1). The binding molar ratio of the 38K-dimer protein to purified skeletal muscle actin was saturated at 1:3, and when the 38K-dimer/actin ratio exceeded 1:12, gelation occurred. This gelation was completely inhibited by the presence of either 10 mM KCl or 20 mM NaCl. The protein induced actin filament bundling, which required a higher 38K-dimer/actin ratio and was not affected by the presence of monovalent cations. During the differentiation of Ml cells, the sensitivity of the 38K protein to monovalent cations was decreased; that is 20 mM KCl or 50 mM NaCl was required to inhibit the gelation by the 38K protein isolated from differentiated cells. On the other hand, the intracellular K+ content of Ml cells decreased from 70 +/- 5 mM to 18 +/- 3 mM, and Na+ increased from 10 +/- 5 mM to 40 +/- 10 mM during the differentiation. These findings suggest that the differentiation brought about conditions favourable for the 38K protein to induce actin gelation, and in turn, the locomotive and phagocytic activities which were induced only after differentiation in this cell line.     

Development of membrane-potential responsiveness by myeloid leukemia cells during neutrophilic differentiation

The ability of a myeloid leukemia cell line (HL-60) to undergo membrane electrical potential changes was followed during neutrophilic differentiation induced by 2 compounds. Membrane-potential changes were induced with 12-O-tetradecanoylphorbol 13-acetate (TPA) or formyl-methionyl-leucyl-phenylalanine (FMLP) and were monitored by flow cytometry. The magnitude of the membrane-potential response to TPA increased in a more uniform manner as the population of cells matured than did acquisition of mature morphology or ability to undergo the respiratory burst in response to TPA. The response to TPA and FMLP of HL-60 cells, maximally induced to differentiate by dimethylsulfoxide, closely resembled that of neutrophils. Thus, HL-60 cells may be a useful tool in the study of the relation between membrane depolarization and subsequent cellular activation.     

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.     

Membrane protein hMYADM preferentially expressed in myeloid cells is up-regulated during differentiation of stem cells and myeloid leukemia cells

We report here the molecular cloning and characterization of a novel human gene (hMYADM) derived from a human bone marrow stromal cell (BMSC) cDNA library, which shares high homology with mouse myeloid-associated differentiation marker (MYADM). hMYADM is also closely related to many other eukaryotic proteins, which together form a novel and highly conserved MYADM-like family. hMYADM with 322-residue protein contains eight putative transmembrane segments and confocal microscopic analysis confirmed its membrane localization by using anti-hMYADM monoclonal antibody. hMYADM mRNA was selectively expressed in human monocytes, dendritic cells, promyeloid or monocytic leukemia cell lines, but not in CD4+, CD8+, CD19+ cells, nor in T cell leukemia or lymphocytic leukemia cell lines. hMYADM expression was also found in normal human bone marrow enriched for CD34+ stem cells, and the expression was up-regulated when these cells were induced to differentiate toward myeloid cells. The mRNA expression level of hMYADM significantly increased in acute promyelocytic leukemia HL-60 and chronic myelogenous leukemia K562 cell line after phorbol myristate acetate (PMA)-induced differentiation. Our study suggests that hMYADM is selectively expressed in myeloid cells, and involved in the myeloid differentiation process, indicating that hMYADM may be one useful membrane marker to monitor stem cell differentiation or myeloid leukemia differentiation.     

Changes in the cytoskeletons during the differentiation of a myeloid leukemia cell line]

Cytoskeletons are supposed to play important roles in the cell growth, metastasis and invasion as well as the reduction of the adhesion to the matrix, which are characteristic of cancer cells. The dynamic changes of cytoskeletons were observed during the differentiation of mouse myelomonocytic leukemic cells, M1. We discuss an increase of the vimentin expression, which is correlated with the changes in the cell morphology, and a reduction of the vimentin degradation activity during the differentiation of M1 cells. And we also discuss about an increase of alpha-actinin synthesis, which is presumed to correlate with the cell adhesion and mobility.