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We measured plasma nm23-H1 level (nm23-H1), a differentiation inhibitory factor, by an enzyme-linked immunosorbent assay (ELISA) in patients with aplastic anemia (AA) and myelodysplastic syndrome (MDS). The nm23-H1 in AA was not significantly elevated when compared to normal subjects (6.66 +/- 1.20 ng/ml vs 5.13 +/- 0.81 ng/ml; P = 0.274). In contrast, MDS patients had significantly high levels of nm23-H1 compared not only to normal subjects (11.16 +/- 1.42 vs 5.13 +/- 0.81 ng/ml; P = 0.0004) but also to those of the AA group (11.16 +/- 1.42 ng/ml vs 6.66 +/- 1.20 ng/ml; P = 0.018). In the MDS group of patients, no significant difference was observed in the nm23-H1 levels between patients with refractory anemia (RA) and RA with excess blasts (RAEB)/RAEB in transformation (10.71 +/- 1.61 ng/ml vs 9.24 +/- 2.66 ng/ml; P = 0.672). Of the patients with RA, patients with low risk according to the International Prognostic Scoring System (IPSS) had significantly low levels of nm23-H1 compared to those of IPSS INT-1 level cases (6.40 +/- 1.36 ng/ml vs 13.05 +/- 2.50 ng/ml; P = 0.0028), suggesting that nm23-H1 may be useful as a prognostic marker for MDS, especially in low risk patients.

Mouse myeloid leukemic M1 cell lines can be induced to differentiate into monocyte/macrophage pathways by various inducers. We have isolated resistant M1 cells that cannot be induced to differentiate into mature cells from the parent M1 cells. 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 the 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 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 16 000–17 000 Da (16K I-factor) and the amino acid sequences of all fragments of the 16K I-factor are identical with Nm23/nucleoside diphosphate kinase (EC2.7.4.6) protein involved in tumor metastasis. The findings indicate that the I-factor is Nm23/nucleoside diphosphate kinase protein.

The outcome of patients with non-Hodgkin's lymphoma has been improved by current approaches to treatment. Nevertheless, many patients either do not have a complete remission or ultimately relapse. To identify such patients, it is important to be able to predict the outcome. We previously found that the differentiation inhibitory factor/nm23 was correlated with the prognosis of acute myeloid leukemia. To examine the prognostic effect of nm23 on non-Hodgkin's lymphoma, we established an enzyme-linked immunosorbent assay procedure to determine nm23-H1 protein levels in plasma and assessed the association of this protein level with the response to chemotherapy, overall survival, and progression-free survival in patients with aggressive non-Hodgkin's lymphoma. The plasma concentration of nm23-H1 was significantly higher in patients with malignant lymphoma than in normal controls, especially in aggressive non-Hodgkin's lymphoma. The complete remission rate in patients with higher nm23-H1 levels was significantly worse than that in patients with lower nm23-H1 levels. Overall survival and progression-free survival were also lower in patients with higher nm23-H1 levels than in those with lower levels. The 3-year survival rates in patients with low and high nm23-H1 levels were 79.5% and 6. 7% (P =.0001). A multivariate analysis of prognostic factors showed that the plasma nm23-H1 level was independently associated with the survival and progression-free survival. An elevated plasma nm23-H1 concentration predicts a poor outcome of advanced non-Hodgkin's lymphoma. Therefore, nm23-H1 in plasma may be useful for identifying a distinct group of patients at very high risk.

Transforming growth factor beta (TGF-beta) enhanced the growth-inhibitory activities of dexamethasone (Dex) and 1alpha,25-dihydroxyvitamin D3 (VD3) on human monocytoid leukemia U937 cells. TGF-beta and VD3 synergistically increased the expression of differentiation-associated markers such as the CD11b and CD14 antigens, whereas TGF-beta and Dex did not. On the other hand, TGF-beta and Dex synergistically increased the number of Apo2.7-positive cells, which represents the early stage of apoptosis, whereas TGF-beta and VD3 did not, suggesting that TGF-beta enhanced apoptosis with Dex and enhanced monocytic differentiation with VD3. In the presence of TGF-beta, the retinoblastoma susceptibility gene product, pRb, was synergistically dephosphorylated by Dex as well as VD3. TGF similarly enhanced the expression of the p21Waf1 gene in U937 cells treated with Dex and VD3. TGF-beta dose-dependently increased the expression of Bcl-2 and Bad and decreased the expression of Bcl-X(L) in U937 cells. Dex enhanced the down-regulation of Bcl-X(L) expression in TGF-beta-treated cells, whereas VD3 blocked this down-regulation of Bcl-X(L). However, the down-regulation of Bcl-X(L) by treatment with the antisense oligomer did not affect the apoptosis or differentiation of U937 cells. The apoptosis of CD14-positive cells was suppressed in the VD3 plus TGF-beta-treated cultures. These results suggest that the expression of CD14 is involved in the survival of differentiated cells.

Differentiation inhibitory factor nm23 gene has been found to be expressed in high quantities in acute myelogenous leukemia (AML), especially in acute monocytic leukemia (AML-M5) and is suggested as a new prognostic factor in AML-M5. We report an example of elevated expression of nm23 mRNA in a patient with chronic myelogenous leukemia (CML) who developed monoblastic crisis. Relative levels of nm23-H1 and -H2 mRNA extracted from the patient's peripheral blood mononuclear cells and bone marrow mononuclear cells were measured by quantitative reverse transcriptase polymerase chain reaction. The level of nm23-H1 mRNA in CML cells at the chronic phase was as high as that in bone marrow cells from healthy volunteers. The mRNA level of nm23-H2 was slightly below the normal level. At blastic crisis, however, expression of both nm23-H1 and -H2 mRNA was elevated to about three to nine times of that at the chronic phase. Proliferated blastic cells were positive for non-specific esterase, and the serum lysozyme level was elevated and diagnosed as monoblastic crisis. The patient received combined chemotherapy but response was partial. These findings are compatible with our previous report that nm23 gene is overexpressed in monocytic leukemia.

Glucocorticoids inhibit the proliferation of lymphoid leukemia cells, whereas most myeloid leukemia cells are resistant to glucocorticoids. However, this study showed that glucocorticoids significantly and preferentially inhibited growth of monocytoid leukemia cells in combination with a low concentration of transforming growth factor beta (TGF beta). Combined 1 alpha,25-dihydroxyvitamin D3 and TGF beta markedly induced monocytic differentiation of U937 cells, whereas dexamethasone (Dex) and TGF beta essentially did not, although both combinations similarly inhibited the growth of U937 cells. The growth inhibition was accompanied by a block in the cell cycle progression from G1 to S phase (G1 arrest). Expression of glucocorticoid receptors was not affected by TGF beta, although they are induced during the monocytic differentiation of myelogenous leukemia cells and have increased sensitivity to glucocorticoids. The expression of TGF beta receptors also was not enhanced by Dex. TGF beta significantly stimulated glucocorticoid responsive element-mediated transcription activity. Combined Dex and TGF beta stimulated the expression of c-jun and c-fos early responsive genes in U937 cells, although Dex or TGF beta alone did not. The combination synergistically induced expression of c-jun gene, reaching a maximum level at 24 h. On the other hand, expression of c-fos gene was induced by TGF beta alone and increased additively in combination with Dex. Treatment with antisense oligonucleotide complementary to the first exon of c-jun mRNA reduced the growth-inhibitory effect of Dex and TGF beta in a dose-dependent manner. However, exposure of U937 cells to the sense oligomer of c-jun mRNA or an antisense oligomer of c-fos mRNA did not affect the growth inhibition. These results suggested that the preferential expression of c-jun and stimulation of glucocorticoid responsive element-mediated transactivation are closely associated with the growth arrest of U937 cells incubated with Dex and TGF beta.

We examined the effect of vesnarinone, an oral cardiotonic, on the growth and differentiation of human myeloid leukemia cells. Vesnarinone alone markedly induced erythroid differentiation of HEL cells. All-trans-retinoic acid also induced erythroid differentiation of the cells, and the differentiation was greatly enhanced by combined treatment with vesnarinone and retinoic acid. HEL cells are highly resistant to some anticancer drugs, including vincristine, but treatment with vesnarinone greatly increased the sensitivity of HEL cells to vincristine. Enhancement of vincristine sensitivity by vesnarinone was not as significant for other leukemia cells. Expression of P-glycoprotein in HEL cells was effectively inhibited by vesnarinone, suggesting that the restoration of vincristine sensitivity is associated with decrease of P-glycoprotein expression in HEL cells. The plasma level of vesnarinone required to induce differentiation of leukemia cells is 30 micrograms/mL, which could be achieved with oral administration. These results suggest that vesnarinone should be useful in differentiation therapy for some types of myelogenous leukemia.

An animal leukemia model was developed to investigate in vivo induction of differentiation of myeloid leukemia cells. An aneuploid cell line (C15) was isolated from mouse myelomonocytic leukemia WEHI-3B D+ cells. The C15 cells contained twice as much DNA as the parental cells but retained the morphology of myelomonocytic cells and the ability to differentiate into macrophage-like cells in response to all-trans retinoic acid (ATRA) in vitro. When the C15 cells were inoculated into the peritoneal cavity of syngeneic Balb/c mice (10(6) cells/mouse), the mice died of leukemia within 19 days. The DNA content and differentiation antigen (Mac-1) of the cells in the peritoneal cavity were determined by dual-parameter flow cytometry. On day 12 after inoculation, the C15 cells were distinguishable from normal host cells in the peritoneal cavity by their different DNA content. The administration of recombinant human granulocyte colony-stimulating factor (rhG-CSF) (10 micrograms/day) to mice bearing C15 cells induced the leukemia cells to express Mac-1 antigen and to change morphologically into mature granulocytic cells. Because the C15 cells were not responsive to G-CSF in suspension culture in vitro, this result suggests that the cytokine's actions on the cells in vivo and in vitro are different. This experimental model for analyzing in vivo differentiation of leukemia cells will be useful for studying the therapeutic effects of potential differentiation-inducing agents.

Interleukin 4 (IL-4) inhibited the differentiation of mouse myeloid leukemia M1 cells induced by leukemia inhibitory factor (LIF), interleukin 6, or dexamethasone and conversely enhanced the induction of M1 cell differentiation by 1 alpha,25-dihydroxyvitamin D3. IL-4 blocked LIF-induced differentiation of M1 cells when it was added to the culture medium within 10 h after LIF, but IL-4 did not block differentiation when it was added 12 h after LIF. These results indicate that IL-4 inhibited a critical intermediate step in myeloid leukemia cell differentiation. LIF markedly stimulated the expression of junB mRNA within 2 h but suppressed the expression of c-myb and c-myc after 2- and 12-h treatment, respectively. IL-4 did not significantly affect LIF-induced junB expression or suppression of c-myb expression. However, it interfered significantly with the LIF-induced suppression of c-myc gene expression. Similar results were obtained when interleukin 6 was used to induce differentiation of M1 cells. Dexamethasone and 1 alpha,25-dihydroxyvitamin D3 did not induce junB gene expression but suppressed the expression of c-myb and c-myc. IL-4 also interfered with dexamethasone-induced suppression of c-myc gene expression. On the other hand, IL-4 enhanced 1 alpha,25-dihydroxyvitamin D3-induced down-regulation of c-myc gene expression, consistent with its enhancement of differentiation. These results indicate that the change in c-myc expression induced by IL-4 in M1 cells is closely associated with the effect of IL-4 on the induction of differentiation of M1 cells.

In severe infection, the host responds to foreign agents and produces cytokines to activate lymphocytes and macrophages. Some of these cytokines can modulate growth and differentiation of myeloid leukemia cells. We examined differentiation-inducing activities in the sera from 9 patients with leukemia or lymphoma. These results indicate that some sera from infected patients, even with acute leukemia, have significant differentiation-inducing activities on both mouse and human leukemia cells, and that cytokines having differentiation-inducing activities varied for different specimens.

Herbimycin A, a benzoquinonoid ansamycin antibiotic, reduces intracellular phosphorylation by some tyrosine kinases, including v-abl. The mouse megakaryoblastic cell line C1 expresses v-abl protein at high levels. Herbimycin A at about 20 ng/ml caused 50% inhibition of growth of C1 cells but at 100 ng/ml scarcely affected the growth of another mouse leukemia cell line, M1 cells, or of normal bone marrow cells. Injection of 10(6) C1 cells into nude mice resulted in death of all the mice within 30 days. Administration of herbimycin A significantly enhanced the survival of mice inoculated with C1 cells but scarcely affected the survival of mice inoculated with M1 cells. These results suggest that herbimycin A and/or related compounds may be useful for treatment of some types of leukemia in which tyrosine kinase activity is implicated as a determinant of the oncogenic state.

Leukemic cells are arrested in less differentiated stages of development. However, there are many reports that various leukemic cells can be induced to differentiate into mature cells with attenuation of proliferative and leukemogenic activity. Mouse myeloid leukemia (Ml) cells both in vitro and in vivo can be induced to differentiate into macrophages and granulocytes by various compounds. During long-term culture of Ml cells, however, some populations of the cells spontaneously become resistant to induces of cell differentiation. The isolated resistant variant cell lines have higher leukemogenicity than parent cells and produce differentiation inhibitory protein factors (I-factor). The variant cell lines are useful to studies on how leukemic cells are arrested in less differentiated stages even in the presence of differentiation inducers both in vitro and in vivo. We have purified and characterized the I-factor. We review herein the properties of the I-factor and other inhibitory factors of differentiation of myeloid leukemia cells.

The sensitivity of human myelogenous leukemia cells to 1-beta-D-arabinofuranosylcytosine (ara-C) during induction of differentiation was examined. Treatment with hemin greatly increased the sensitivity of erythroid leukemia cells to ara-C. The enhancement of ara-C sensitivity by hemin was not as remarkable in nonerythroid leukemia cells. Hemin altered the metabolism of ara-C in human erythroleukemia K562 cells by reducing ara-C deaminase activity, increasing intracellular accumulation of ara-C, and activating the nucleoside kinases. These alterations may be involved in the enhancing effect of hemin on sensitivity of ara-C. These results suggest that some inducers of differentiation potentiate the antileukemic effect of ara-C on human erythroleukemia cells.

Mouse C1 line cells are megakaryoblastic cells established by coinfection of Abelson murine leukemia virus and recombinant simian virus 40. We examined the effects of various compounds on growth and differentiation of these cells. Megakaryocytic differentiation of C1 cells was not induced by cytokines that stimulate megakaryocytic maturation of normal progenitor cells, such as interleukin 3 and 6 and granulocyte-macrophage colony-stimulating factor. However, the cells were induced to differentiate into megakaryocytes by treatment with some protein kinase inhibitors. The inhibition of v-abl tyrosine kinase activity preceded induction of differentiation of the cells treated with tyrosine kinase inhibitors such as genistein, herbimycin A, and erbstatin. Treatment of C1 cells with a v-abl antisense oligomer inhibited their proliferation and induced acetylcholinesterase activity, a typical marker of megakaryocytic differentiation. These results suggest that inhibition of v-abl function is associated with induction of megakaryocytic differentiation of C1 cells. Among the compounds tested, 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7), a potent inhibitor of cyclic nucleotide-dependent and Ca(2+)-phospholipid-dependent (protein kinase C) protein kinases, was the most potent inducer of differentiation of C1 cells. However, the differentiation-inducing effect of H-7 was unlikely to be mediated through inhibition of protein kinase C or cyclic nucleotide-dependent kinases, because other types of inhibitors of these kinases were not effective, and a protein kinase activator (phorbol ester) induced differentiation of C1 cells. Moreover, neither v-abl mRNA expression nor v-abl kinase activity in C1 cells was affected by treatment with H-7. These findings indicate that induction of megakaryocytic differentiation by H-7 is not related to inhibition of v-abl kinase, but rather to some novel function of H-7.

The K562/VCR cell line, exhibiting acquired multidrug resistance (MDR) with increased expression of a cell surface glycoprotein (P-glycoprotein), was isolated from human erythroleukemia K562 cells. Various compounds that induced erythroid differentiation of K562 cells were tested for their effects on growth and differentiation of these K562/VCR cells. Sodium butyrate, hemin, 1-beta-D-arabinofuranosylcytosine, and erythroid differentiation factor (EDF) induced erythroid differentiation of K562/VCR cells as well as K562 cells. The MDR of K562/VCR cells was partly overcome by treatment with EDF but not with the other inducers. Expression of P-glycoprotein by K562/VCR cells was measured by radioimmunoassay using MRK16 monoclonal antibody. Results showed that EDF caused down-regulation of P-glycoprotein in K562/VCR cells, whereas the other inducers did not cause its down-regulation. Thus, in addition to inducing erythroid differentiation, EDF enhanced the sensitivity of K562/VCR cells to multidrugs and suppressed expression of P-glycoprotein. These results suggest that differentiation inducers may be useful in chemotherapy of leukemic MDR cells.

We examined the effects of transforming growth factors beta(TGF-beta) alone and in combination with 1 alpha,25-dihydroxyvitamin D3 (VD3) on human leukemic cell lines (HEL/S, HL-60 and K562). TGF-beta 1 alone at higher concentrations induced monocytic differentiation of HEL/S cells. Combinations of TGF-beta 1 and VD3 synergistically inhibited cell proliferation and induced monocytic differentiation of HEL/S and HL-60 cells. TGF-beta 2 (a subtype of TGF-beta) and erythroid differentiation factor (EDF/Activin A, a member of the TGF-beta gene family) also inhibited growth and induced monocytic differentiation of HL-60 cells in synergy with VD3. Thus combinations of VD3 and TGF-beta (TGF-beta 1, -beta 2, or EDF) acted synergistically in inhibiting cell proliferation and inducing monocytic differentiation of human leukemia cells.

We have purified and characterized several protein factors that regulate the growth and differentiation of mouse myeloid leukaemia M1 cells. The differentiation factor (D-factor) from conditioned medium (CM) of Ehrlich ascites tumour cells is a glycoprotein of Mr 40,000-50,000. Its amino acid sequence was found to be almost identical to that of leukaemia inhibitory factor (LIF) from Krebs II ascites cells. The differentiation inhibitory factor (I-factor) from the CM of variant M1 cell clones which were resistant to several differentiation inducers is a basic protein of apparent Mr 68,000. The growth inhibitory factor (GI-factor) that specifically inhibits the partially differentiated and still growing monocytic leukaemia M1 cells was isolated from the CM of a clone of M1 cells resistant to the differentiation inducers. This GI-factor is a basic protein with an Mr of 25,000. Regulation by these protein factors together with other known cytokines of growth and differentiation of M1 cells is reported.

Mouse myeloid leukemic M1 cells are induced to differentiate by various differentiation inducers. Activity for inhibition of induction of differentiation of M1 cells (I-activity) has been found in conditioned medium of variant M1 cell clones resistant to differentiation inducers, and this I-activity has been shown to be closely associated with resistance of the cells to differentiation inducers. In this work, the I-activity in the conditioned medium of the resistant M1 cells was shown to bind to Carboxymethyl-Sepharose CL-6B and to be eluted with 0.27-0.4 M NaCl. The profile of gel filtration of I-activity from Sephadex G-200 indicated considerable heterogeneity in molecules with I-activity; the apparent molecular range of the main I-activity was 60,000-80,000. On chromatofocusing, the I-activity was eluted with Polybuffer 96-acetic acid at pH 8.8-9.0. The I-activity was inactivated by treatment with trypsin or by heating at 75 degrees C for 30 min. Therefore, the main I-activity seemed to be due to a basic protein(s).

The mouse myeloid leukemia cell line M1 can be induced by various substances, including dexamethasone, lipopolysaccharide and protein inducer (differentiation inducing factor, D-factor) to differentiate both in vitro and in vivo into macrophages and granulocytes losing leukemogenicity to syngeneic SL mice (Hozumi 1983, 1985).

Mouse myeloid leukemic cells (M1) could be induced to differentiate into macrophage-like and granulocyte-like cells by a lysine-rich, histone H1 fraction (10 to 100 microgram/ml). The differentiated M1 cells expressed phagocytic and lysozyme activity and were macrophage-like and granulocyte-like cells. The differentiation-inducing activity of histone H1 was found in histone H1 fractions isolated from calf thymus, rat liver, and mouse leukemia M1 cells. Histone H2A and H2B fractions did not induce differentiation of M1 cells at concentrations of 10 to 100 microgram/ml but did induce differentiation at a high concentration (200 microgram/ml). The histone H3 fraction, poly-L-lysine and poly-L-arginine, inhibited induction of differentiation of M1 cells.

Human promyelocytic leukemia cells (HL-60) were found to be induced by dimethylulfoxide and several other compounds to phagocytize, reduce NBT dye, and change into forms that were morphologically similar to granulocytes and macrophages. Arginase also induced these differentiation-associated properties of the cells. The induction of differentiation by arginase was significantly inhibited by addition of excess arginine, but not by lysine or leucine; therefore, the effect of arginase may be due to arginase-mediated arginine depletion.

The effects of conditioned media (CM) of various clones of mouse myeloid leukemia cells (M1) on colony formation of normal mouse bone marrow cells were examined under various conditions. The CM of sensitive M1 cells, which could be induced to differentiate into macrophage-like and granulocyte-like cells by various factors stimulating the differentiation (D-factor), slightly inhibited colony formation of normal bone marrow cells by colony stimulating factor (GM-CSF). The CM of resistant M1 cells, which were resistant to the induction of differentiation even with high concentrations of the D-factor, significantly inhibited the colony formation of bone marrow cells by GM-CSF. The inhibitory activity of the CM from resistant cells decreased on treatment of the cells with a low concentration of actinomycin D, which could sensitize the resistant cells to induction of differentiation. The CM of differentiated sensitive M1 cells induced by dexamethasone stimulated the colony formation of bone marrow cells without exogenously added GM-CSF. Most of the GM-CSF activity in the CM was separated from the D-factor of M1 cells by Sephadex G-75 gel filtration.

Herbimycin A, a benzoquinonoid ansamycin antibiotic, is found to reduce intracellular phosphorylation by tyrosine protein kinase. The human chronic myelogenous leukemia cell line K562 expresses a structurally altered c-abl protein with tyrosine kinase activity. When K562 cells are induced to undergo erythroid differentiation by hemin, reduction in the intracellular level of tyrosine phosphorylation occurs. In order to understand the relationship between induction of differentiation and reduction of tyrosine phosphorylation by the c-abl gene product, the effect that herbimycin A, a selective inhibitor of intracellular tyrosine kinase activity, exerts on the differentiation of K562 cells was examined. Reduction of tyrosine phosphorylation in K562 cells by herbimycin A was observed within 1 h. Noncytotoxic concentrations of herbimycin A induced erythroid differentiation of K562 cells but not of murine erythroleukemia 745A cells. The other human myeloid leukemia cell lines (HL-60, THP-1, and U937) tested were not induced to undergo cell differentiation by this antibiotic. Herbimycin A and the other well-known inducers such as hemin, butyric acid, Adriamycin, and 1-beta-D-arabinofuranosylcytosine had additive or more than additive effects on induction of erythroid differentiation of K562 cells. With respect to inhibition of cell growth, the sensitivity of K562 cells to herbimycin A was highest in the human leukemia cell lines we tested. Noncytotoxic concentrations of herbimycin enhanced the antiproliferative effect of Adriamycin or 1-beta-D-arabinofuranosylcytosine on K562 cells. Combination therapy with herbimycin A and its derivatives may be considered for use in the treatment of some types of leukemia where tyrosine kinase activities are implicated as determinants of the oncogenic state.

The sensitivity of human leukemia K562 cells to cancer chemotherapeutic drugs during induction of erythroid differentiation of the cells by hemin was examined. Treatment with hemin greatly increased the sensitivity of the cells to 1-beta-D-arabinofuranosylcytosine (ara-C) but did not affect their sensitivities to other chemotherapeutic drugs, including Adriamycin, daunomycin, hydroxyurea, methotrexate, and vincristine. Thymidine and deoxyguanosine, which are known to potentiate the antileukemic effects of ara-C in K562 cells, also induced erythroid differentiation of K562 cells, but other inducers, such as sodium butyrate and delta-aminolevulinic acid, did not increase the sensitivity of K562 cells to ara-C. Hemin did not enhance the sensitivity to ara-C of other leukemia cell lines (Friend erythroleukemic cells, myeloid leukemic M1 cells, and promyelocytic leukemia HL-60 cells). These results indicate that some inducers of erythroid differentiation of K562 cells potentiate the antileukemic effect of ara-C on K562 cells.

Mouse myeloid leukemia cells (M1) could be induced to differentiate into mature macrophages and granulocytes with dexamethasone or proteinaceous inducer. Retinoic acid inhibited functional and morphological differentiation of M1 cells, but the pyridyl analog of retinoic acid had no effect. M1 cells could be induced to produce a factor(s) inhibiting their own differentiation to macrophage- and granulocyte-like cells by retinoic acid but not by its pyridyl analog. This factor(s) inhibited induction by inducers of phagocytic activity, locomotive activity, lysozyme activity, and morphological changes in M1 cells. The production of the inhibitory factor(s) by M1 cells incubated with retinoic acid was inhibited by a low concentrations (5--10 ng/ml) of actinomycin D. The inhibitory factor seemed to be a protein(s), since it was susceptible to heat treatment and proteases. The effect of retinoic acid in inducing production of the inhibitory factor(s) by M1 cells seemed to be reversible, since it was low on washing the cells with fresh medium. Therefore, induction of this inhibitory factor may be involved in the mechanism of inhibition of functional and morphological differentiation of M1 cells by retinoic acid.

Mouse monocytic Mm-A cell line is a highly leukemogenic variant cell line of the monocytic and non-leukemogenic Mm-1 cell line, which developed spontaneously from mouse myeloid leukemia M1 cells. Growth-inhibitory factor (GI factor) for Mm-A cells was found in conditioned medium (CM) of differentiation inducer-resistant myeloblastic M1 cells (clone R-1). The R-1 cells were cultured with or without 2% calf serum for 2 days, and the CM was fractionated with 50% ammonium sulfate and used as the GI factor preparation (termed R1CM). When Mm-A cells were cultured with 5% (v/v) R1CM for 3 days, their growth was inhibited about 80%. This inhibition of Mm-A cell growth by R1CM was irreversible. This GI factor also inhibited the growth of M1 cells that had been pretreated with inducer and had expressed some differentiation-associated properties but still retained a proliferative capacity. In contrast, it scarcely inhibited the growth of untreated M1 cells. The GI factor inhibited the growth of other mouse monomyeloblastic leukemic WEHI-3B D+ cells pretreated with a differentiation inducer, retinoic acid, and mouse monocytic leukemia J774.1 cells. However, it did not affect the growth of human monocytic (U937 and THP-1) or myeloid (KG-1, ML-1, and HL-60) cell lines. These results suggest that GI factor produced by parent myeloblastic and inducer-resistant M1 cells preferentially inhibits the growth of mouse monocytic leukemia cells in intermediate stages of differentiation from myeloblastic leukemia cells to mature macrophages.