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The NM23 gene is overexpressed in many hematological malignancies and its overexpression predicts poor treatment outcomes. NM23 overexpression is thought to suppress myeloid differentiation of leukemia cells, but the molecular mechanism is unknown. In breast cancer cells, the lysophosphatidic acid (LPA) receptor EDG2/lpa1 was downregulated by NM23-H1 overexpression, and this reciprocal expression pattern was associated with suppressed or induced cell motility/metastasis. Here, we examined the relationship between EDG2 and NM23 expression during myeloid differentiation of leukemia cells. NM23 expression decreased and EDG2 expression increased during all-trans retinoic acid (ATRA)-induced myeloid differentiation of HL-60, NB4, and THP-1 leukemia cells. Moreover, this inverse correlation was more evident when myeloid differentiation was enhanced by ellagic acid, an inhibitor of NM23 activity. In contrast, there was no inverse correlation between EDG2 and NM23 expression during erythroid differentiation of HEL and K562 cells. ATRA plus LPA enhanced the motility of leukemia cells as well as breast cancer cells in an EDG2-dependent manner. These results suggest a common molecular mechanism between myeloid differentiation of leukemia cells and migration of breast cancer cells depending on NM23 and EDG2 expression levels., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

We previously found that expression of the Mm-1 cell-derived transplantability-associated gene 1b (MmTRA1b)/phospholipid scramblase 1 gene was markedly induced during the granulocytic differentiation of human myeloid leukemia cells. To evaluate the role of MmTRA1b expression in human myeloid leukemia, we investigated the relative levels of MmTRA1b transcripts in 81 patients with acute myelogenous leukemia (AML) by the reverse transcriptase polymerase chain reaction. The expression of MmTRA1b in AML-M1, -M5a and -M5b was significantly lower than that in normal bone marrow cells. The levels of MmTRA1b expression in AML-M2 and -M4 varied among patients. Higher MmTRA1b mRNA levels were associated with significantly longer overall survival in AML, especially in AML-M4 patients, independent of chromosomal aberrations such as t(8;21) and inv(16). The present results suggest that the MmTRA1b mRNA level is a new prognostic factor for AML, especially the AML-M4 subtype.

Cell surface expression of NM23 protein is only observed on tumor cell lines, but not on normal cells. To examine what types of tumor cell line express the cell surface NM23 protein, we measured the cell surface NM23-H1 and NM23-H2 proteins of leukemia line cells on various cellular lineage and differentiation stages. The NM23-H1 was expressed on myeloid leukemia lines but not lymphoid lines, while NM23-H2 was only expressed on erythroleukemia lines. The complement-dependent cytolysis confirmed the expression of these proteins on the surface. Surface NM23-H1 and NM23-H2 proteins were decreased during in vitro erythroid and granulocyte differentiation. These results show that the surface expression of NM23 proteins is related to cellular lineage and differentiation stage of leukemia line cells.

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.

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 microgram/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 1 alpha, 25-dihydroxyvitamin D3 (VD3), retinoic acid or tumor necrosis factor-alpha (TNF alpha). 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).

Mouse monocytic leukemia Mm cells are a line of spontaneously differentiated cells obtained from mouse myeloblastic leukemia M1 cells. The effect of interleukin 4(IL-4) on the proliferation of Mm cells in the presence or absence of growth inhibitory substances was investigated. In semi-solid agar culture, IL-4 markedly inhibited colony formation by Mm cells, reducing the number of colonies to 50% of that in control cultures at concentration of 3 U/ml. In contrast, IL-4 did not inhibit colony formation by the parent M1 cells. In liquid culture, IL-4 alone inhibited the proliferation of Mm cells only slightly. However, a combination of IL-4 and 1 alpha,25-dihydroxyvitamin D3 (VD3), which alone did not inhibit growth significantly, markedly inhibited the growth of Mm cells. This combination also increased the lysozyme activity of Mm cells significantly. On the other hand, IL-4 suppressed the antiproliferative effects of interferon alpha, beta and IL-6, which are growth inhibitory cytokines for these Mm cells. These results indicate that IL-4 can modulate the growth of monocytic leukemia Mm cells and that its modulatory effects depend on growth inhibitory substances.

Growth inhibitory (GI) factors for mouse monocytic leukemia cells were previously found in conditioned medium (CM) of a clone of mouse myeloblastic leukemia Ml cells (R1-GI factor) and in CM of mouse lung tissue (L-GI factor). In the present study, the effects of these GI factors on the growth of variant cell lines (Mm-A, Mm-P and Mm-S2) of mouse monocytic line Mm-1 cells were examined. Mm-A are highly leukemogenic to syngeneic SL mice, Mm-P cells are moderately leukemogenic, while Mm-S2 cells have little or no leukemogenicity. The R1-GI factor markedly suppressed the growth of Mm-A cells, whereas it inhibited the growth of Mm-P and Mm-S2 cells only moderately. Recombinant mouse interferon beta (IFN beta) had similar target specificities to those of the R1-GI factor on these variant cells. Moreover, anti-mouse IFN beta antibody completely neutralized the GI activity of the R1-GI factor on Mm-A cells. These results show that the R1-GI factor and mouse IFN beta are very similar and probably identical proteins. The L-GI factor had different target specificities from the R1-GI factor: it showed strongest GI activity on Mm-P cells, moderate GI activity on Mm-S2 cells and weak GI activity on Mm-A cells. Recombinant human interleukin 6 (IL-6) had similar target specificities to the L-GI factor on these Mm-1 variant cells. Furthermore, the L-GI factor could support the proliferation of IL-6-dependent MH60.BSF2 cells. Anti-mouse IL-6 antiserum neutralized the GI activity of the L-GI factor on Mm-P cells. Thus the L-GI factor and mouse IL-6 seem to be closely related and possibly to be identical proteins.

Erythroid differentiation of human leukemic cell lines (HEL, KU812F and K562) was induced by their treatment with transforming growth factor beta (TGF-beta) or erythroid differentiation factor (EDF/Activin A). Their erythroid differentiation was markedly inhibited by vitamin D3, dexamethasone or 12-O-tetradecanoyl-phorbol-13-acetate, which are known to induce monocytic differentiation of leukemic cells. These results suggest that inducers of monocytic differentiation may switch off cellular regulatory mechanisms for induction by TGF-beta and EDF of erythroid differentiation of leukemia cells.

A near-tetraploid cell line (LL-1) was established from mouse myeloid leukemia Ml cells. This paper reports characterization of the LL-1 cells and the in-vivo detection of the leukemia cells transplanted in syngeneic mice. The LL-1 cells are myeloblastic and grow well in suspension culture. Morphological analysis showed that the nucleus of LL-1 cells was almost twice as large as that of the parent line cells. The modal chromosome number of LL-1 cells was 75, and the DNA index determined by flow cytometry was 2.3. The cells were unresponsive to the inducer of differentiation of M1 cells. Transplantation experiments showed that the LL-1 cells were leukemogenic in syngeneic SL mice: ten mice inoculated i.p. with LL-1 cells (4 X 10(6)) all died of leukemia within 6 weeks. The cells in the peritoneal cavity were collected at appropriate times during progression of the leukemia. On microscopic examination the LL-1 cells were clearly distinguishable from normal host cells in the peritoneal cavity by the size of their nucleus. Counts showed that their number decreased markedly during the first 2 weeks after their transplantation, and then increased about ten times in a week. By 4 or 5 weeks after transplantation these LL-1 cells filled the peritoneal cavity. These large-sized leukemia cells that grow in syngeneic mice will be useful for investigating the mechanisms of in-vivo responses of leukemia cells to various therapeutic treatments.

Studies were made on whether differentiation and proliferation of antitumor drug-resistant leukemia cells could be controlled by specific inducers of terminal differentiation. Leukemia subclones resistant to daunomycin and/or cytosine arabinoside were isolated from differentiation-inducible mouse myeloid leukemia M1-B24 cells by selection with these antitumor drugs. The drug-resistant cells were found to retain their potential for terminal differentiation induced by various inducers, such as a protein inducer in the conditioned medium of mouse L929 cells, dexamethasone, 1 alpha,25-dihydroxyvitamin D3, 2-[2-(dodecyloxy)ethoxy]ethyl 2-pyridinioethyl phosphate, and poly I. Differentiated cells showed morphological changes to mature macrophage-like cells, increase in phagocytic activity, and decrease in proliferative activity. Clonal analysis of M1-B24 cells showed that the cellular responses to the protein inducer of differentiation were not significantly different between drug-resistant clones selected with anti-tumor drugs and control (drug-sensitive) clones randomly isolated without selection. These results suggest that induction of differentiation of leukemic cells with the specific inducers is another approach to control the drug-resistant leukemia.

K562/VCR cells, which are resistant to the cytotoxicity of vincristine, were isolated from human erythroleukemia K562 cells. Various compounds that induce erythroid differentiation of K562 cells were tested on K562/VCR cells. Differentiation of K562/VCR cells was not induced by actinomycin D or adriamycin alone, but the resistance of these cells to the inducers was overcome by verapamil. In contrast, mitomycin C, butyric acid and hemin induced differentiation of K562/VCR cells as effectively as that of K562 cells. These results suggest that therapy by induction of differentiation of leukemic cells is effective for leukemic cells that have acquired resistance to therapeutic drugs.

The differentiation of mouse myeloid leukemia line cells (M1) into macrophages or granulocytes has been reported. Resistant M1 cells which did not differentiate even with a high concentration of inducer of differentiation were isolated from M1 cells. The conditioned medium of the resistant M1 cells (RCM) inhibited the induction of differentiation of M1 cells and the formation of macrophage and granulocyte colonies of normal mouse bone marrow cells. Acidic isoferritins known as negative regulators of normal bone marrow cells (CFU-GM) failed to inhibit the induction of differentiation and growth of M1 cells. The RCM treated with anti-acidic isoferritin serum could inhibit the induction of differentiation of M1 cells as did the untreated RCM. The activities, in RCM, inhibiting growth and differentiation of the normal bone marrow cells were partly neutralized by treatment with the antiserum but most of the activities remained. These results suggest that growth and differentiation of the mouse myeloid leukemia M1 cells are not regulated by acidic isoferritins and other inhibitory activities affecting normal bone marrow colony formation, in addition to acidic isoferritins, are released from M1 cells.

We developed an experimental model to investigate in vivo differentiation and proliferation of leukemia cells using mouse myeloid leukemia aneuploid cells (LL-2) and syngeneic SL mice. The LL-2 cells were near-tetraploid cells isolated from mouse myeloid leukemia cell line M1 (clone D501). In suspension culture, the LL-2 cells were myeloblastic and grew well like parent D501 cells, but were distinct from the parental cells due to the large size of their nucleus, double chromosome number and DNA content. The LL-2 cells as well as D501 cells could be induced to differentiate in vitro into mature macrophage-like cells by a protein inducer of differentiation. After transplantation of 4 X 10(6) LL-2 cells into the intraperitoneal cavity of syngeneic SL mice, most of them died of leukemia within 10 weeks. On microscopic examination of the peritoneal cells of the mice, the transplanted LL-2 cells were clearly distinguishable from normal host cells by the size of their nucleus. We determined the increase in the LL-2 cells in the peritoneal cavity by morphological examination of the large-sized LL-2 cells. Survival times of the mice inoculated with the LL-2 cells were prolonged by administrations of an inducer of differentiation, poly(I).poly(C). We found morphological changes in the peritoneal blastic LL-2 cells to mature macrophage-like cells after the serial administrations of poly(I).poly(C) to the recipient mice. Thus the aneuploid LL-2 cells that grow in syngeneic mice may be useful to study in vivo differentiation and proliferation of leukemia cells, and to develop a therapeutic strategy of leukemia using various treatments including differentiation inducers.