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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 alpha and beta, 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.

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.

Synthesis of the biologically active metabolite(s) from 1 alpha-hydroxyvitamin D3 (1 alpha(OH)D3) was examined in various types of human leukemia cell lines. Untreated monocytoid leukemia cells (U937 and HEL/S) metabolized 1 alpha (OH)D3 to the active metabolite(s), possibly 1 alpha, 24- and/or 1 alpha, 25-dihydroxyvitamin D3, and these cells were efficiently induced to differentiate by treatment with 1 alpha (OH)D3. However, the other types of leukemia cells did not efficiently metabolize it and were not induced to differentiate by 1 alpha (OH)D3. The possible therapeutic advantage of 1 alpha (OH)D3 in the treatment of monocytic leukemia is discussed.

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 of 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 1 alpha,25-dihydroxyvitamin D3. These results indicate that modulation of differentiation of M1 cells by IL-4 depends on the differentiation inducer.

Inhibitors of myosin light chain kinase, 1-(5-chloronaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine hydrochloride (ML-9) and 1-(5-iodonaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine hydrochloride (ML-7), induced Nitroblue tetrazolium reducing activity, lysozyme activity and morphological maturation of human monoblastic U937, THP-1 and promyelocytic HL-60 cells, but not of erythroblastic K562 cells. However, three analogs of ML-9, which are an inhibitor and an activator of protein kinase C, and a calmodulin antagonist, respectively, did not induce differentiation of the cells.

Differentiation-stimulating factor (D factor)/leukemia inhibitory factor (LIF) and IL-6 are reported to be cytokines having multifaced functions including the induction of differentiation in mouse myeloid leukemia M1 cells. We here report that both D factor/LIF and IL-6 inhibit the differentiation 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 concluded that D factor/LIF and IL-6 are functionally closely related in the induction of differentiation in M1 cells and in the inhibition of F9 differentiation.

Differentiation-stimulating factor (D-factor)/leukemia inhibitory factor is a cytokine inducing differentiation of mouse myeloid leukemic 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 alpha-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-PK1 cells were found to have high-affinity binding sites (831 per cell) for D-factor with a dissociation constant of 197 pM.

A factor inducing differentiation of mouse myeloid leukemic cells (MI) into macrophages was purified to apparent homogeneity from 168 1 of CM of Ehrlich ascites tumor cells. The purified factor was half-maximally active at 2 X 10(-11) M. The factor was analyzed by radioiodination, SDS-polyacrylamide gel electrophoresis and autoradiography. Its Mr was 40 000-50 000. On reduction, the factor lost activity, but showed no subunit structure. Treatment of the factor with endo-beta-N-acetylglucosaminidase F, but not endo-beta-N-acetylglucosaminidase H, gave rise to a molecule of Mr 20 000-28 000. The activity of the factor from Ehrlich cells was completely neutralized by antiserum to the factor of Mr 50 000-70 000 from mouse fibroblast L929 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.