Junko Okabe-Kado

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.

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‐H2His 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.

Ellagic acid, a natural polyphenolic compound, induces apoptosis and potentiates retinoic acid-induced differentiation of human leukemia HL-60 cells

All-trans retinoic acid (ATRA) is a standard drug used for differentiation therapy in acute promyelocytic leukemia. To potentiate this therapy, we examined the effect of ellagic acid (EA), a natural polyphenolic compound with antiproliferative and antioxidant properties, on the growth and differentiation of HL-60 acute myeloid leukemia cells. EA was found to induce apoptosis, which was blocked by pan-caspase inhibitor, Z-VAD-FMK. EA activated the caspase-3 pathway and enhanced the expressions of myeloid differentiation markers (CD11b, MRP-14 protein, granulocytic morphology) induced by ATRA treatment. These results indicate that EA is a potent apoptosis inducer and also effectively potentiates ATRA-induced myeloid differentiation of HL-60 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 leukemic 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-H2His 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.

Induction of differentiation of human myeloid leukemia cells by immunosuppressant macrolides (rapamycin and FK506) and calcium/calmodulin-dependent kinase inhibitors

OBJECTIVE Potent immunosuppressants, such as rapamycin, FK506, and ascomycin, are known to regulate the phosphorylation of proteins. The purpose of this study was to investigate the effects of these immunosuppressants on differentiation of several human myeloid leukemic cell lines. MATERIALS AND METHODS Human myeloid leukemic cell lines were cultured with each immunosuppressant, and several differentiation markers were assayed. RESULTS Rapamycin effectively induced granulocytic differentiation of human myeloid leukemic HL-60 and ML-1 cells. In addition to morphologic differentiation, it also induced nitroblue tetrazolium reduction, lysozyme activity, and expression of CD11b in HL-60 cells. The commitment to differentiation was observed after treatment with rapamycin for 1 day, indicating that the effect of rapamycin was irreversible. FK506 and ascomycin induced differentiation of HL-60 cells, but at higher concentrations than rapamycin. A calcium/calmodulin-dependent kinase (CaMK) was copurified with FKBP52 immunophilin, a binding protein of immunosuppressants. We also found that the CaMK inhibitors KN62 and KN93 induced differentiation of HL-60 cells. Rapamycin and CaMK inhibitors induced differentiation of human myeloid leukemia ML-1 and K562, but not of other cell lines such as NB4, U937, or HEL. CONCLUSION Immunosuppressants and CaMK inhibitors induced differentiation of HL-60, ML-1, and K562 cells.

Extracellular NM23 protein promotes the growth and survival of primary cultured human acute myelogenous leukemia cells.

An elevated serum level of NM23‐H1 protein is found in acute myelogenous leukemia (AML), and predicts a poor treatment outcome in AML patients. To investigate the potential pathological link between the elevated serum level of this protein and poor prognosis, we examined the extracellular effects of recombinant NM23‐H1 protein on the in vitro growth and survival of primary cultured AML cells at concentrations equivalent to the levels found in the serum of AML patients. Extracellular NM23‐H1 protein promoted the in vitro growth and survival of AML cells and this activity was associated with the cytokine production and activation of the MAPK and signal transducers and activators of transcription signaling pathways. Inhibitors specific to MAPK signaling pathways inhibited the growth‐ and survival‐promoting activity of NM23‐H1. These findings indicate the novel biological action of extracellular NM23‐H1 and its association with poor prognosis, and suggest an important role for extracellular NM23‐H1 in the malignant progression of leukemia and a potential therapeutic target for these malignancies. (Cancer Sci 2009; 100: 1885–1894)

Clinical Significance of Intracytoplasmic nm23-H1 Expression in Diffuse Large B-Cell Lymphoma

Purpose: Recently, we established an ELISA technique for measuring nm23-H1 protein in serum and found that the serum nm23-H1 level is a potential prognostic factor for patients with non-Hodgkin’s lymphoma. Experimental Design: We used immunohistochemistry to examine the expression of nm23-H1 by the lymphoma cells in patients with diffuse large B-cell lymphoma (DLBCL). Results: By analyzing a consecutive series of 172 untreated DLBCL patients, we found that 100 (58.1%) were strongly positive. The cytoplasmic nm23 expression in lymphoma cells correlated significantly with the serum nm23-H1 level. There was a significant correlation between patients with cytoplasmic nm23-positive lymphoma and those with performance status 2–4, stage III/IV, bulky mass, B symptoms, elevated serum level of soluble interleukin 2 receptor, and elevated serum level of C-reactive protein. Overall and progression-free survival rates were significantly lower in patients with nm23-H1-positive lymphomas than in those with nm23-H1-negative lymphomas. Similar difference was seen between patients with high and low serum levels of nm23-H1. Thus, the correlation between presence or absence of cytoplasmic nm23-H1 expression and serum nm23-H1 levels suggests that serum nm23-H1 is produced directly by lymphoma cells. Conclusion: We suggest that nm23-H1 expression is a prognostic factor for DLBCL, and that it is as important as serum nm23-H1, both of which are useful for planning a treatment strategy.

Induction of differentiation of myelogenous leukemia cells by humulone, a bitter in the hop.

The active form of vitamin D, 1alpha,25-dihydroxyvitamin D3 (VD3), inhibits proliferation and induces differentiation of myelomonocytic leukemia cells, but its clinical use is limited by the adverse effect of hypercalcemia. VD3 mobilizes calcium stores from bone by inducing the dissolution of bone mineral and matrix. We have recently found that humulone, a bitter in the hop extract for beer brewing, effectively inhibits bone resorption. In this study we examined the effect of humulone on the differentiation of human myelogenous leukemia cells. Humulone alone inhibited the growth of monoblastic leukemia U937 cells while only slightly increasing differentiation markers such as nitroblue tetrazolium (NBT)-reducing and lysozyme activities. Humulone effectively enhanced the differentiation-inducing action of VD3. Other myelomonocytic leukemia cells were induced to differentiate by VD3 and this was also enhanced by humulone. Since humulone is a less-toxic inhibitor of bone resorption, the combination of humulone and VD3 may be useful in differentiation therapy of myelomonocytic leukemia.

Differentiation inhibitory factor Nm23 as a prognostic factor for acute myeloid leukemia.

The differentiation inhibitory factor nm23 inhibits the differentiation of murine and human myeloid leukemia cells. The inhibition of differentiation may be associated with the aggressive behavior of leukemia. To clarify the role of nm23 in human myeloid leukemia, we investigated the relative levels of nm23-H1, nm23-H2 and c-myc transcripts in bone marrow and blood samples from 110 patients with acute myeloid leukemia (AML) using the reverse transcriptase polymerase chain reaction. The expression levels of nm23-H1 and nm23-H2 in these AML samples were significantly higher than in normal blood cells, and a higher level of nm23-H1 expression was correlated with poor prognosis for AML patients. Analysis of the correlation between nm23 expression and clinical parameters demonstrated that increased nm23-H1 mRNA levels were associated with resistance to initial chemotherapy and reduced overall survival. Multivariate analysis of putative prognostic factors revealed that elevated nm23-H1 mRNA levels significantly influenced the prognosis of patients with AML, particularly in AML-M5.

Role of MmTRA1b/phospholipid scramblase1 gene expression in the induction of differentiation of human myeloid leukemia cells into granulocytes

OBJECTIVE We previously cloned a human normal counterpart (MmTRA1b/phospholipid scramblase 1) of the mouse leukemogenesis-associated gene MmTRA1a. MmTRA1b gene expression was increased during differentiation of human monoblastic leukemia U937 cells using some differentiation inducers but not 1alpha,25-dihydroxyvitamin D(3) (a typical monocytic differentiation inducer). To further elucidate the role of human MmTRA1b gene expression in the differentiation of myelogenous leukemia cells, we measured MmTRA1b gene expression in several myeloid leukemia cell lines and primary leukemia cells. MATERIALS AND METHODS The expression of MmTRA1b mRNA was determined by semiquantitative reverse transcriptase polymerase chain reaction. RESULTS Expression of the MmTRA1b gene was markedly induced during granulocytic differentiation of promyelocytic leukemia NB4 and HT93 cells induced by all-trans retinoic acid (ATRA). The level of MmTRA1b mRNA was significantly increased during differentiation toward granulocytes, but not monocytes/macrophages, in bipotential myeloid leukemia HL-60 cells. The level of MmTRA1 mRNA was not increased during erythroid differentiation induced by hemin in erythroid leukemia K562 and HEL cells or during megakaryocytic differentiation induced by 12-O-tetradecanoylphorbol-13-acetate in K562 cells. Expression of the MmTRA1b gene also was not induced when apoptosis of NB4 cells was induced by antileukemic drugs. ATRA-induced differentiation of antisense MmTRA1b-transfected NB4 cells was significantly suppressed. On the other hand, ATRA induced the differentiation of MmTRA1b-transfected NB4 cells more efficiently than that of mock-transfected cells. MmTRA1b mRNA also was clearly induced in ATRA-treated primary acute promyelocytic leukemia cells during granulocytic differentiation. CONCLUSION MmTRA1b mRNA was specifically induced during granulocytic differentiation of acute promyelocytic leukemia cells and was associated with induction of their differentiation.