Mikio Tomida

Stimulation by interferon of induction of differentiation of human promyelocytic leukemia cells.

Abstract Treatment of human promyelocytic leukemia cells (HL-60) for 6 days with conditioned medium of mouse myeloid leukemic M1 cells which had been treated with phytohemagglutinin resulted in their morphological differentiation into macrophages and induction of ability to reduce nitroblue tetrazolium dye in the cells. Human IFN-α and IFN-β enhanced induction of differentiation of the cells into macrophages by the conditioned medium of M1 cells or 12-O-tetradecanoylphorbol-13-acetate (0.1 ng/ml). IFNs also enhanced induction of differentiation of the cells into granulocytes by retinoic acid.

Hyaluronic acid synthetase in cultured mammalian cells producing hyaluronic acid Oscillatory change during the growth phase and suppression by 5-bromodeoxyuridine

Abstract A hybrid line B-6 synthesizes and secretes a large amount of hyaluronic acid and this differentiated function is suppressed by growth in the presence of the thymidine analog, 5-bromodeoxyuridine. In order to elucidate the mechanism of this analog inhibition, the levels hyaluronic acid synthetase and UDPglucose dehydrogenase in the cells were investigated. The results obtained were as follows. 1. 1.The addition of 5-bromodeoxyuridine at concentrations as low as 5 μg/ml to medium while having the effect on cell growth, caused a great drop in the activity of hyaluronic acid synthetase. By contrast, the specific activity of UDPglucose dehydrogenase was slightly increased. 2. 2. This suppression on the synthetase was detected 10 h after addition of the analog; when the culture treated for one generation (16 h) was transferred to the analog-free medium, the activity was recovered to control value during the next three to four generations. The analog effect was prevented by simultaneous addition of hydroxyurea, an inhibitor of DNA synthesis, with the analog. These results prove that incorporation of the analog into DNA is necessary for this inhibitory effect. 3. 3. In the presence of either actinomycin D or cycloheximide, cellular hyaluronic acid synthetase activity decayed with a half-life of 3.5 and 2 h, respectively, whereas UDPglucose dehydrogenase decayed with a half-life of 20 h. 4. 4. The activity of the synthetase, but not that of UDPglucose dehydrogenase, was correlated with high growth rate, falling sharply with decline of cell growth. Recovery from low activity in the cells transferred to fresh medium required new synthesis of both RNA and protein.

Characterization of a factor inducing differentiation of mouse myeloid leukemic cells purified from conditioned medium of mouse Ehrlich ascites tumor cells

A factor inducing differentiation of mouse myeloid leukemic cells (M1) into macrophages was purified to apparent homogeneity from 168 1 of CM of Ehrlich ascites tumor cells. The purified factor was halt‐maximally active at 2 × 10−11 M. The factor was analyzed by radioiodination, SDS‐polyacrylamide gel electrophoresis and autoradiography. Its M r was 40000–50000. On reduction, the factor lost activity, but showed no subunit structure. Treatment of the factor with endo‐β‐N‐acetylglucosaminidase F, but not endo‐β‐N‐acetylglucosaminidase H, gave rise to a molecule of M r 20000–28000. The activity of the factor from Ehrlich cells was completely neutralized by antiserum to the factor of M r 50000–70000 from mouse fibroblast L929 cells.

Induction by recombinant human granulocyte colony-stimulating factor of differentiation of mouse myeloid leukemic M1 cells

The effect of recombinant human granulocyte colony‐stimulating factor (G‐CSF) on induction of differentiation of mouse myeloid leukemic M1 cells was examined. Purified G‐CSF caused dose‐dependent induction of phagocytic activity and lysozyme activity in M1 cells. Its half‐maximally effective concentration was 10 ng/ml. On treatment of M1 cells with G‐CSF (100 ng/ml) for 4 days, 30–50% of the cells differentiated morphologically into macrophage cells; 30–40% of the cells were blast cells and 20–30% of the cells were forms intermediate between blastic cells and mature macrophages.

Pregnancy associated increase in mRNA for soluble D-factor/LIF receptor in mouse liver☆

We examined the distribution of mRNAs for differentiation‐stimulating factor (D‐factor)/leukemia inhibitory factor (LIF) receptor in various mouse tissues by Northern blotting. A mouse cDNA fragment encoding the D‐factor receptor was prepared by the RT‐PCR method using human cDNA sequences as primers. The smallest mRNA (3 kb) was present in the liver, but not detectable in other tissues examined. Larger mRNAs (5 and 10 kb) were present in the placenta and the M1 cells, and also detectable in the liver, kidney, heart, lung, brain and embryos. Expression of 3 kb mRNA in the liver increased during pregnancy, being 20 times the initial level on day 15. D‐factor receptor cDNAs were isolated from a cDNA library prepared from the liver of a pregnant mouse. Most of the cDNA clones encoded a soluble receptor. A cDNA probe specific for the cellular receptor did not hybridize with 3 kb mRNA in the liver. These results suggest that 3 kb mRNA encodes a soluble D‐factor receptor and that the liver is the primary site of synthesis of this soluble receptor.

Production of interleukin 6 and its relation to the macrophage differentiation of mouse myeloid leukemia cells (M1) treated with differentiation-inducing factor and 1α,25-dihydroxyvitamin D3

We have studied the production of interleukin 6 (IL-6) and its relation to the macrophage differentiation in murine myeloid leukemia cells (M1). As has been reported, differentiation-inducing factor (D-factor), 1 alpha, 25-dihydroxyvitamin D3 [1 alpha, 25(OH)2D3], and recombinant IL-6 similarly induced differentiation of M1 cells into macrophages. The three compounds also induced mRNA expression of IL-6 in M1 cells. M1 cells treated with D-factor or 1 alpha, 25(OH)2D3 produced biologically active IL-6, but the amounts of IL-6 secreted into culture media did not appear to be enough to induce differentiation of M1 cells. Furthermore, simultaneous addition of anti-IL-6 antibody did not suppress the differentiation of M1 cells induced by D-factor or 1 alpha, 25(OH)2D3. These results show that IL-6 production is an essential property associated with the macrophage differentiation of M1 cells, but it may not be responsible for the D-factor- and 1 alpha, 25(OH)2D3-induced differentiation.

Effect of tunicamycin on molecular heterogeneity of colony stimulating factor in cultured mouse mammary carcinoma FM3A cells.

Abstract Granulocyte and macrophage colony stimulating factors obtained from cultured mouse mammary carcinoma FM3A cells showed heterogeneity in molecular size giving rise to a major component with an apparent molecular weight of 80,000 and a minor one with that of 35,000 on Sephadex G-200 column chromatography. In the presence of tunicamycin, a specific inhibitor of asparagine-linked glycosylation, the colony stimulating factor was produced normally and consisted of a single component with an apparent molecular weight of 30,000. These data indicate that the sugar moiety is not essential for the production or activity of colony stimulating factor and that the heterogeneity in molecular size of the colony stimulating factor mainly resulted from tunicamycin-sensitive glycosylation.

Preparation and neutralization characteristics of an antibody to the factor inducing differentiation of mouse myeloid leukemic cells

Mouse myeloid leukemic M1 cells can be induced to differentiate into macrophages and granulocytes in vitro by a protein inducer (D‐factor) in body fluids and conditioned media from various cells and tissues. Antiserum to D‐factor was prepared by immunizing a rabbit with D‐factor partially purified from conditioned medium of mouse L929 cells. At 1:20 dilution, the antiserum nearly completely suppressed the activities of various D‐factor preparations from mice and partially suppressed the activities of preparations from rats and hamsters. It did not cross‐react with factors stimulating colony formation of macrophages and granulocytes from normal bone marrow cells.

Inhibition of development of Na(+)-dependent hexose transport in renal epithelial LLC-PK1 cells by differentiation-stimulating factor for myeloid leukemic cells/leukemia inhibitory factor.

Differentiation‐stimulating factor (D‐factor)/leukemia inhibitory factor is a cytokine inducing differentiation of mouse myeloid leukemie M1‐T22 cells. The effect of recombinant human D‐factor on growth and differentiation of pig kidney LLC‐PK1 cells was examined. LLC‐PK1 cells did not concentrate α‐methylglucoside during their early growth in culture but developed the capacity to concentrate this hexose as they reached confluence and their growth rate decreased. Purified D‐factor caused dose‐dependent inhibition of the development of this concentrative capacity. It did not affect the growth rate of the cells, but inhibited the formation of multicellular domes in confluent cultures. LLC‐PK, cells were found to have high‐affinity binding sites (831 per cell) for D‐factor with a dissociation constant of 197 pM.

Specific binding of a factor inducing differentiation to mouse myeloid leukemic M1 cells

A factor inducing differentiation of mouse myeloid leukemic M1 cells into macrophages (differentiation-inducing factor, D-factor), which was purified to homogeneity from conditioned medium of mouse Ehrlich ascites tumor cells, could be iodinated without detectable loss of biological activity. The binding of 125I-D-factor to M1 cells was specific; the binding was inhibited competitively by D-factor derived from Ehrlich cells and mouse fibroblast L929 cells, but not by other growth factors or D-factor derived from differentiated M1 cells. The latter differs from D-factor of Ehrlich cells and L929 cells in antigenicity and molecular weight. At 21 degrees C, the binding was saturated at 370 pM 125I-D-factor. M1 cells showed a high affinity for 125I-D-factor (dissociation constant, 1.0 X 10(-10) M) and expressed a small number of binding sites (170 per cell). Specific binding of 125I-D-factor was observed only to several clones derived from M1 cells, including those sensitive and resistant to induction of differentiation by D-factor.