Yuri Yamamoto-Yamaguchi

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.

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.

Combined Effects of Differentiation-inducing Factor and Other Cytokines on Induction of Differentiation of Mouse Myeloid Leukemic Cells

Mouse myeloid leukemic M1 cells are induced to differentiate into macrophage‐like cells by differentiation‐inducing factors (D‐factors) and granulocyte colony‐stimulating factor. We examined the effects of recombinant human tumor necrosis factor (rTNF), lymphotoxin (rLT) and interleukin 1 (rIL‐1) on the induction of differentiation of M1 cells, compared with the effects of D‐factor purified from the conditioned medium of mouse Ehrlich as cites tumor cells and recombinant human granulocyte colony‐stimulating factor (rG‐CSF). rIL‐1 induced phagocytic activity, a typical marker of cell differentiation, in at most 30% of M1 cells at concentrations ranging from 10‐10M to 10‐7M. The differentiation‐inducing activity of rIL‐1 was similar to that of rG‐CSF and less than that of D‐factor. rTNF induced phagocytic activity in 14% of M1 cells only at a high concentration (10‐7M). rLT did not induce differentiation of the cells even at 10‐7M. rTNF stimulated induction of differentiation of M1 cells by D‐factor, rG‐CSF or rIL‐1 by two or three fold. The combination of any two of the cytokines D‐factor, rG‐CSF and rIL‐1 induced differentiation of M1 cells more efficiently than any of these cytokines alone. Moreover, the combination of three cytokines rG‐CSF, rIL‐1 and rTNF, all of which are known to be produced by macrophages, was more effective than the combination of any two of these cytokines in induction of differentiation of M1 cells.

Prolongation by differentiation-stimulating factor/leukemia inhibitory factor of the survival time of mice implanted with mouse myeloid leukemia cells.

Mouse myeloid leukemic M1 cells can be induced to differentiate into macrophages by differentiation-stimulating factor (D-factor)/leukemia inhibitory factor (LIF). We examined the effect of D-factor on the survival times of syngeneic mice implanted with two different clones (T-22 and R-4) of M1 cells. D-factor induced differentiation and suppressed DNA synthesis of sensitive T-22 cells but not resistant R-4 cells in vitro. For in vivo experiments, we used recombinant mouse D-factor (rmD-factor) produced in mammalian cells, which is glycosylated and is more stable in vitro and in vivo than unglycosylated rmD-factor produced in Escherichia coli. Treatment with rmD-factor prolonged the survival times of mice implanted with T-22 cells but not R-4 cells.

Both D factor/LIF and IL-6 inhibit the differentation of mouse teratocarcinoma F9 cells

Differentation‐stimulating factor (D factor)/leukemia inhibitory factor (LIF) and IL‐6 are reported to be cytokines having multifaced functions including the induction of differentation in mouse myeloid leukemia MI cells. We here report that both D factor/LIF and IL‐6 inhibit the differentation of mouse teratocarcinoma F9 cells induced by retinoic acid alone or combined with dibutyryl cAMP. From the microscopic observation as well as Northern blot analysis using cDNA probes encoding several marker proteins for differentiation of F9 cells, we conclude that D factor/ LIF and IL‐6 are functionaly closely related in the induction of differentation in MI cells in the inhibition of F9 differentiation.

Contrasting effect of IFN-γ and IFN-α/gb on differentiation of some clones of mouse myeloid leukemic cells☆

Mouse myeloid leukemic M1 cells are induced to differentiate into macrophage-like cells by a differentiation-inducing factor (D-factor) and various agents. IFN-gamma alone did not induce differentiation of clone T22-3 of M1 cells but inhibited their differentiation by D-factor. That is, IFN-gamma at 4 U/ml inhibited 50% of phagocytic activity of T22-3 cells induced by 7 x 10(-11) M D-factor. In addition, it inhibited the induction of lysozyme activity and morphological differentiation of these cells by D-factor. IFN-gamma also inhibited dexamethasone-induced differentiation of T22-3 cells. Previously interferon-alpha/beta was shown not to induce differentiation of M1 cells itself, but to enhance induction of their differentiation by D-factor. The present study showed that IFN-alpha/beta and IFN-gamma had opposite effects on induction of differentiation of T22-3 cells by D-factor. The effect of IFN-gamma on the differentiation of M1 cells varied with the clone of M1 cells used: IFN-gamma inhibited D-factor-induced differentiation of cells of clones T22-3 and S2, but induced differentiation of cells of clones B24 and S1.