Mouse Myeloid Leukemic M1 Cells Research Articles

Leukemia Inhibitory Factor and Human Embryo Implantation

The success of embryonic implantation relies on an ideal cross-talk between the embryo and the receptive endometrium. This article focuses on the role of leukemia inhibitory factor (LIF) and its receptors in human embryo implantation. LIF is a secreted glycoprotein first described as a factor that induced the differentiation of mouse myeloid leukemic M1 cells into macrophages and later proposed as a marker of the embryo implantation process. An important role for LIF in implantation was shown on LIF knockout mice, when embryo implantation did not occur. In endometrium of healthy women, LIF and LIF mRNA are expressed throughout the menstrual cycle with a striking increase in the midsecretory phase, coinciding with a supposed window of implantation. Correlation in the expression of LIF and some other markers of implantation has been reported. LIF acts on cells by binding to the LIF receptor (LIFR) and gp130. Human blastocysts express mRNAs for LIFR and gp130, participating actively in establishing contact with the endometrium. In the endometrium, LIFR and gp130 are expressed in the endometrial epithelium throughout the cycle with strong increase in the midsecretory phase. Endometrium of infertile women produces significantly less LIF during the period of receptivity. The role of LIF gene mutations in unexplained infertility and implantation failures in IVF patients is not clear yet. Infertile patients showed reduced secretion of LIFR and gp130 compared with fertile controls during the implantation window. Recombinant human LIF might help to improve the implantation rate in women with unexplained infertility.

Role of MgcRacGAP/Cyk4 as a regulator of the small GTPase Rho family in cytokinesis and cell differentiation.

To identify the key molecules that regulate differentiation of hematopoietic cells, we carried out retrovirus-mediated functional screening for cDNAs whose expression suppresses IL-6-induced differentiation of mouse myeloid leukemic M1 cells. From this screening, we obtained a full length cDNA encoding a mouse homologue of human MgcRacGAP. Overexpression of the anti-sense MgcRacGAP profoundly inhibited IL-6-induced macrophage-differentiation of M1 cells. On the other hand, overexpression of the full-length form of MgcRacGAP alone enhanced macrophage differentiation of M1 cells in response to IL-6, and induced macrophage differentiation of HL-60 leukemic cells. To determine how this protein regulates differentiation and proliferation, an antibody against MgcRacGAP was prepared. Immunohistochemical studies revealed that MgcRacGAP mainly localizes in the nucleus in interphase, accumulates on the mitotic spindle in metaphase, and is condensed in the midbody during cytokinesis. Overexpression of an N-terminal domain deletion mutant, which lacks the ability to localize to the midbody through association with tubulins, or a GAP-inactive mutant resulted in the formation of multinucleated cells in HeLa cells as well as in hemopoietic cells. Interestingly, MgcRacGAP in the midbody was phosphorylated probably on serine and threonine residues. These results indicate that MgcRacGAP regulates cytokinesis and cellular differentiation as a regulator of Rho family of GTPase and suggest that this process is controlled by some serine/threonine kinases.

Role of mgcracgap as a regulator of the small gtpase rho family in differentiation and cytokinesis

Abstract To identify the key molecule which regulats proliferation and differentiation of hematopoietic cells, we carried out retrovirus-mediated functional cDNA screening based on the ability to suppress IL-6 induced differentiation of mouse myeloid leukemic M1 cells, and an antisense cDNA encoding a full-length mouse MgcRacGAP was isolated. We also isolated a full-length of human MgcRacGAP cDNA, which was found to encode additional N terminal 105 amino acid residues compared with the previously published human MgcRacGAP. Overexpression of the full-length form of MgcRacGAP in human HL-60 leukemic cells induced growth suppression and macrophage differentiation. To determine how this protein regulats differentiation and proliferation, an antibody against MgcRacGAP was prepared. Immunohistochemical study revealed MgcRacGAP exhibits nuclear localization in interphase, spreads throughout cytoplasm in prometaphase, and condenses in the central spindle in metaphase to telophase. Overexpression of the N-terminal domain deletion mutant, which lacks the ability to condense into the central spindle, or a GAP-inactive mutant resulted in the formation of multinucleated cells without affecting nuclear division. These results suggest that this GAP regulats differentiation and cytokinesis of hematopoietic cells as a regulator of Rho family of GTPase.

Differentiation- and Apoptosis-inducing Activities of Phospholipids Containing Docosahexaenoic Acid for Mouse Myeloid Leukemia M1 Cells

Differentiation- and Apoptosis-inducing Activities of Phospholipids Containing Docosahexaenoic Acid for Mouse Myeloid Leukemia M1 Cells

A Janus Kinase Inhibitor, JAB, Is an Interferon-γ–Inducible Gene and Confers Resistance to Interferons

It has been shown that interferons (IFNs) exert their signals through receptor-associated Janus kinases (JAKs) and signal transducers and activators of transcription (STATs). However, molecular mechanism of regulation of IFN signaling has not been fully understood. We have reported novel cytokine-inducible SH2 protein (CIS) and JAK binding protein (JAB) family genes that can potentially modulate cytokine signaling. Here we report that JAB is strongly induced by IFN-γ but not by IFN-β in mouse myeloid leukemia M1 cells and NIH-3T3 fibroblasts. NIH-3T3 cells ectopically expressing JAB but not CIS3 lost responsiveness to the antiviral effect of IFN-β and IFN-γ. M1 leukemic cells stably expressing JAB were also resistant to IFN-γ and IFN-β–induced growth arrest. In both NIH-3T3 and M1 transformants expressing JAB, IFN-γ did not induce tyrosine phosphorylation and DNA binding activity of STAT1. Moreover, IFN-γ–induced activation of JAK1 and JAK2 and IFN-β–induced JAK1 and Tyk2 activation were inhibited in NIH-3T3 JAB transformants. These results suggest that JAB inhibits IFN signaling by blocking JAK activity. We also found that IFN-resistant clones derived from LoVo cells and Daudi cells expressed high levels of JAB without stimulation. In IFN-resistant Daudi cells, IFN-induced STAT1 and JAK phosphorylation was partially reduced. Therefore, overexpression of JAB could be, at least in part, a mechanism of IFN resistance. © 1998 by The American Society of Hematology.

A Janus Kinase Inhibitor, JAB, Is an Interferon-γ–Inducible Gene and Confers Resistance to Interferons

It has been shown that interferons (IFNs) exert their signals through receptor-associated Janus kinases (JAKs) and signal transducers and activators of transcription (STATs). However, molecular mechanism of regulation of IFN signaling has not been fully understood. We have reported novel cytokine-inducible SH2 protein (CIS) and JAK binding protein (JAB) family genes that can potentially modulate cytokine signaling. Here we report that JAB is strongly induced by IFN-γ but not by IFN-β in mouse myeloid leukemia M1 cells and NIH-3T3 fibroblasts. NIH-3T3 cells ectopically expressing JAB but not CIS3 lost responsiveness to the antiviral effect of IFN-β and IFN-γ. M1 leukemic cells stably expressing JAB were also resistant to IFN-γ and IFN-β–induced growth arrest. In both NIH-3T3 and M1 transformants expressing JAB, IFN-γ did not induce tyrosine phosphorylation and DNA binding activity of STAT1. Moreover, IFN-γ–induced activation of JAK1 and JAK2 and IFN-β–induced JAK1 and Tyk2 activation were inhibited in NIH-3T3 JAB transformants. These results suggest that JAB inhibits IFN signaling by blocking JAK activity. We also found that IFN-resistant clones derived from LoVo cells and Daudi cells expressed high levels of JAB without stimulation. In IFN-resistant Daudi cells, IFN-induced STAT1 and JAK phosphorylation was partially reduced. Therefore, overexpression of JAB could be, at least in part, a mechanism of IFN resistance.© 1998 by The American Society of Hematology.

Angelmicin b, a new inhibitor of oncogenic signal transduction, inhibits growth and induces myelomonocytic differentiation of human myeloid leukemia HL-60 cells

Angelmicin B is a new microbial substance which inhibits src tyrosine kinase activity and oncogenic signal transduction. We investigated the effect of angelmicin B on the proliferation and differentiation of the HL-60 human myeloid leukemia cell line. Angelmicin B caused the dose-dependent inhibition of cell proliferation and induction of differentiation along the myelomonocytic pathway, as determined by morphological changes, nitroblue tetrazolium (NBT) reduction, and non-specific esterase and lysozyme activities at concentrations ranging from 0.1 to 0.5 μg/ml. Also, it induced significantly the differentiation of mouse myeloid leukemia M1 cells. A similar concentration of angelmicin B inhibited the growth of the myeloid leukemia cell lines K562, HEL, KU812, ML-1, U937 and THP-1, but did not induce differentiation of these cells significantly. The differentiation of HL-60 cells was enhanced by combined treatment with angelmicin B and 1α,25-dihydroxyvitamin D 3 (VD 3), retinoic acid or tumor necrosis factor-α (TNFα). Angelmicin analogs (A1, A2, B, C and D) had almost equivalent effects on the differentiation of HL-60 cells, although angelmicins C and D inhibited src tyrosine kinase activity less than the other analogs. The effective concentrations of angelmicin B in src kinase inactivation was about 100-fold higher than those required for the growth inhibition and differentiation induction. These findings indicate that the differentiation-inducing activity of angelmicins is not associated with their src kinase-inhibiting activity, and may be associated with the modulation of other signal pathway(s).

Transcriptional regulation of the vimentin-encoding gene in mouse myeloid leukemia M1 cells

To investigate the regulatory mechanisms controlling expression of the vimentin-encoding gene ( Vim) during mouse myeloid leukemia M1 cell differentiation, mouse Vim was cloned and the transcriptional activity of its 5′ promoter region was analysed by chloramphenicol acetyltransferase (CAT) assay. Analyses of various deletion mutants revealed that a 188-bp fragment of the proximal Vim promoter ( pVim) was sufficient for effective transcription in M1 cells. This 188-bp sequence is highly conserved between mouse, hamster and human. Further deletion analyses revealed that a minimum promoter element (−44 to + 26) is essential for basic promoter function and could respond to cell differentiation. Detailed analyses of mutant and chimeric pVim constructs defined a CCAAT box at −89 to −84 to be an essential positive regulatory element. A G + C-rich element between the CCAAT and TATA boxes was found to act as a strong negative regulatory element in Vim transcription.

Analysis of recombinant soluble mouse D-factor/LIF receptor.

The recombinant soluble mouse D-factor/LIF receptor (sD-factor-R) was expressed in COS-7 cells. Scatchard analysis of the bindings of mouse 125I-D-factor and human 125I-D-factor to the sD-factor-R indicated dissociation constants (Kd) of 12 and 0.267 nM, respectively, which were comparable to those of the binding protein in mouse serum. The apparent molecular masses of the sD-factor-R and human D-factor observed by gel filtration chromatography were 150 and 50 kDa, respectively. The size of the sD-factor-R.human D-factor complex was approximately 200 kDa, indicating that D-factor forms a 1:1 complex with the sD-factor-R. The sD-factor-R inhibited the induction of differentiation of mouse myeloid leukemic M1 cells by mouse D-factor by blocking the binding of this factor to the cells.

Inhibitory action of nm23 proteins on induction of erythroid differentiation of human leukemia cells

We recently identified a differentiation inhibitory factor (I-factor) in mouse myeloid leukemia M1 cells as a murine homolog of the human nm23-H2 gene product. nm23 genes encode proteins that participate in tumor metastasis regulation and in various fundamental cellular processes, although their mechanisms of action are still unknown. Although all nm23 proteins contain nucleoside diphosphate (NDP) kinase activity, it has not been established that the enzyme activity mediated the various functions of nm23 proteins. In the present experiment, we examined the effect of nm23 proteins on various differentiation induction systems of human leukemia cells including HL-60, U937, HEL/S, KU812F, K562, and HEL cells. Native human erythrocyte NDP kinase protein inhibited the induction of erythroid differentiation of HEL, KU812 and K562 cells, but not the induction of monocytic or granulocytic differentiation of HL-60, U937 and HEL/S cells. The erythroid differentiation of HEL cells was inhibited by recombinant human nm23-H1, -H2, mouse nm23-M1, and -M2 proteins. Moreover, both the mutant nm23-H2 His protein and truncated nm23-H2 protein containing N-terminal (1-60) peptide, which do not have NDP kinase activity, also inhibited erythroid differentiation of HEL cells. These results suggest that (1) the differentiation inhibitory activity of I-factor,/nm23 protein is not restricted to monocytic differentiation of M1 cells, (2) the inhibitory activity is exhibited without species specificity, and (3) the differentiation inhibitory activity of the nm23/NDP kinase protein is independent of its enzyme activity and requires the presence of N-terminal peptides.

A new function of Nm23/NDP kinase as a differentiation inhibitory factor, which does not require it's kinase activity

We recently identified a differentiation inhibiting factor (I-factor) in mouse myeloid leukemia M1 cells as a murine homolog of nm23-H2/nucleoside diphosphate kinase (NDPK)-B gene product. We examined the I-factor activities of several authentic nm23/NDPK proteins, i.e recombinant rat NDPK α and β, recombinant mouse nm23-M1 and -M2, and recombinant human nm23-H1 and -H2 containing a mutant nm23-H2 His protein lacing NDPK activity. Almost all these nm23/NDPK proteins showed I-factor activity. Moreover, to understand the active domain exhibiting I-factor activity of nm23-H2 protein lacking NDPK activity, we have investigated the I-factor activities of some truncated nm23-H2 proteins. The truncated nm23-H2 protein containing N-terminal peptide 1–60 retained the I-factor activity. These results provide the first evidence for a function of nm23/NDPK as a differentiation inhibiting factor in leukemic cells, that is independent of its NDPK activity and dependent on the presence of N-terminal peptide.

Induction of Differentiation of WEHI-3B D+ Leukemic Cells Transfected With Differentiation-Stimulating Factor/Leukemia Inhibitory Factor Receptor cDNA

Differentiation-stimulating factor (D-factor)/leukemia inhibitory factor can induce the differentiation of mouse myeloid leukemia M1 cells and also stimulate proliferation of the interleukin-3 (IL-3)-dependent cell line, DA-1a. To determine whether D-factor can induce the differentiation of leukemia cells other than M1 cells, WEHI-3B D+ mouse myelomonocytic leukemia cells were transfected with a plasmid containing mouse D-factor receptor cDNA. Expression of D-factor receptor in transfected cells was determined by binding of [125]D-factor and analyzed by Scatchard's method. The transfected cells had high-affinity D-factor receptors with a dissociation constant of 100 to 200 pmol/L and binding sites per cell varied from 67 to 1,500 among several clones. The cells expressing a high level of D-factor receptor were induced to differentiate by D-factor; about 60% of the cells exhibited the ability to reduce nitroblue tetrazolium and expression of the differentiation antigen Mac-1 (CD11b) on the cell surface increased. The effect of cytokines, which induce the differentiation of M1 cells, on the transfected WEHI-3B cells was examined. The sensitivity to oncostatin M was identical to that against D-factor in the cells of each clone. Expression of D-factor receptor in WEHI-3B cells promoted sensitivity to IL-6 and granulocyte colony-stimulating factor (G-CSF). Induction of differentiation of the cells accompanied the suppression of proliferation. Treatment of the cells with D-factor for longer than 5 days resulted in 50% inhibition of growth. These results indicate that the stimulating effect of D-factor on the differentiation of malignant myeloid cells is not unique to M1 cells.

Pregnancy associated increase in differentiation-stimulating factor (D-factor)/leukemia inhibitory factor (LIF)-binding substance(s) in mouse serum

Differentiation-stimulating factor (D-factor)/leukemia inhibitory factor (LIF) is known to have multiple biological activities besides induction of differentiation of mouse myeloid leukemia M1 cells. Little is known about how its activities are regulated in vivo, but it has been suggested to play a regulatory role in the mechanisms involved in development of mice. In this study, we found that a single class of D-factor-binding substance is present in normal mouse serum and that it increases transiently in the late stage of pregnancy. It inhibits the induction of differentiation of mouse myeloid leukemic M1 cells by D-factor by blocking the binding of this factor to the cells. It is a heat-labile protein with an apparent molecular weight of 130,000–150,000. The binding of 125I-D-factor to the substance is specific since it was inhibited by excess unlabeled D-factor, but not by interleukin 6 or interferon γ. The dissociation constant of the binding substance for mouse D-factor in normal mouse serum is 6.6–9.4 nM. In the late stage of pregnancy, the amount of the D-factor-binding substance in the serum apparently increases about 30-fold. These results suggest that the D-factor-binding substance regulates the activity of D-factor during embryonic development of mice.

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 an the survival times of syngeneic mice implanted with two different clones (T-22 and R4) 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.

Identity of a differentiation inhibiting factor for mouse myeloid leukemia cells with NM23/nucleoside diphosphate kinase

Mouse myeloid leukemic line M1 cells can be induced to differentiate into the monocyte/macrophage pathway by various inducers. The induction of differentiation of M1 cells can be inhibited by protein inhibitors termed differentiation inhibiting factors (I-factors) in a cell lysate and conditioned medium of differentiation resistant M1 cells. Production of the I-factor activity in resistant M1 cells is well associated with development of resistance of M1 cells to differentiation inducers. We have now purified one of the I-factors from conditioned medium of differentiation resistant M1 cells. The purified I-factor has a relative molecular mass of approximately 16000–17000 Da (16K I-factor). The amino acid sequence of all fragments of the 16K I-factor we have found are identical with Nm23/nucleoside diphosphate kinase (EC2.7.4.6) protein involved in tumor metastasis. The findings indicate that the I-factor, a candidate suppressor protein for differentiation of leukemic cells, is Nm23/nucleoside diphosphate kinase protein.

Interleukin-4 inhibits the differentiation of mouse myeloid leukemia M1 cells induced by dexamethasone, D-factor/leukemia inhibitory factor and interleukin-6, but not by 1α,25-dihydroxyvitamin D 3

The effects of interleukin-4(IL-4) on the growth and differentiation of mouse myeloid leukemia M1 cells induced by various differentiation inducers were investigated. IL-4 alone did not have any significant effect on the growth or differentiation of M1 cells, but inhibited their differentiation induced by dexamethasone, D-factor/leukemia inhibitory factor, or interleukin 6. IL-4 also restored the proliferation or M1 cells after growth inhibition during their induction of differentiation by inducers. In contrast, IL-4 enhanced inhibition of growth and induction of differentiation of M1 cells by 1α,25-dihydroxyvitamin D 3. These results indicate that modulation or differentiation of M1 cells by IL-4 depends on the differentiation inducer.

Expression of arginase by mouse myeloid leukemic cell differentiation in vitro induced with tumor necrosis factor.

Induction of arginase activity in mouse myeloid leukemic M1 cells by treatment with recombinant human tumor necrosis factor (rH-TNF) or TNF-elicited mouse serum (TNS) were examined in vitro. M1 cells differentiated into macrophage-like cells by addition of rH-TNF or TNS. The differentiated cells expressed phagocytic function and did not grow anymore. Cytolytic effect of rH-TNF or TNS was not observed. The differentiation of M1 cells into phagocytic cells by the TNS treatment was more rapidly than that by the rH-TNF treatment though TNS contained 25-time less amounts of TNF indicating species specificity of TNF action on myeloid leukemic cells or TNS containing other differentiation factor(s). 3H-ornithine formation from 3H-arginine is catalyzed by arginase (EC. 3.5.3.1). The enzyme product increased in the M1 cell culture medium by the treatment with rH-TNF or TNS. The arginase activity statistically correlated with the appearance percentage of differentiated cells with phagocytic function. These results suggest that in vitro differentiation of M1 cells is accompanied by induction of arginase activity.

Macrophage polykaryons are capable of bone resorption

Mouse myeloid leukemic M1 cells are induced to differentiate into macrophage-like cells by a differentiation-inducing factor (D-factor) and various agents. IFN-γ alone did not induced differentiation of clone T22-3 of M1 cells but inhibited their differentiation by D-factor. That is, IFN-γ at 4 U/ml inhibited 50% of phagocytic activity of T22-3 cells induced by 7 × 10−11 M D-factor. In addition, it inhibited the induction of lysozyme activity and morphological differentiation of these cells by D-factor. IFN-γ also inhibited dexamethasone-induced differentiation of T22-3 cells. Previously interferon-α/β 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 IF-α/gb and IFN-γ had opposite effects on induction of differentiation of T22-3 cells by D-factor. The effect of IFN-γ on the differentiation of M1 cells varied with the clone of M1 cells used: IFN-γ inhibited D-factor-induced differentiation of cells of clones T22-3 and S2, but induced differentiation of cells of clones B24 and S1.

Partial Purification and Properties of Cathepsin G-like Proteinase of Mouse Myeloid Leukemia M1 Cells

A proteinase extracted with 1M NaCl from particulate fraction of the postnuclear fraction of mouse myeloid leukemia M1 cells was partially purified by Bio-Gel HTP treatment and Sephadex G-75 gel filtration. The apparent molecular mass of the proteinase was 26,000 Da and the isoelectric point was about pH 10. The enzyme activity was inhibited by phenylmethanesulfonylfluoride, chymostatin, and soy-bean trypsin inhibitor. It hydrolysed specifically Suc-Ala2-Pro-Phe-4-methylcoumaryl-4-amide (MCA). NaCl and KCl enhanced several times the activity for Suc-Ala2-Pro-Phe-MCA, but not that for fluorescein-labeled albumin and fibrinogen. These enzymic properties of the major proteinase are similar to those of chymotrypsin and cathepsin G. The role of a cathepsin G-like proteinase in relation to M1 cell differentiation is discussed.

Inhibitory Action of Transforming Growth Factor‐β on Induction of Differentiation of Myeloid Leukemia Cells

The effect of transforming growth factor‐β (TGF‐β) on induction of differentiation of mouse myeloid leukemia M1 cells was investigated. TGF‐β1 induced adherence of M1 cells to plastic dishes and inhibited their proliferation. However, it did not induce differentiation‐associated properties, such as phagocytic activity, lysozyme activity or morphological maturation. TGF‐β1 also caused dosedependent inhibition of dexamethasone‐induced differentiation of M1 cells. The inhibitory activity of TGF‐β1 was 20 times that of TGF‐β2 on M1 cells. These results suggest that TGF‐β1 inhibits proliferation and dexamethasone‐induced differentiation of M1 cells by interacting with receptors that can distinguish between TGF‐β1 and TGF‐β2. TGF‐β1 had a much lower inhibitory effect on the growth of a variant M1 cell clone, which was resistant to differentiation inducers, and it did not induce adherence of the resistant M1 cells.