Motoo Hozumi

Effects of inhibitors of protein tyrosine kinase activity and/or phosphatidylinositol turnover on differentiation of some human myelomonocytic leukemia cells

The activities of protein tyrosine kinase and phosphatidylinositol turnover have been found to be associated with cell growth and differentiation. We examined the effects of some inhibitors for these biochemical activities in human myelogenous leukemia cells. Genistein, which is known to inhibit the activities of protein tyrosine kinase, phosphatidylinositol turnover and topoisomerase II, induced nitroblue tetrazolium (NBT) reduction and lysozyme activity in ML-1, HL-60 and U937 cells. Morphological studies showed that genistein-induced differentiation of myeloblastic ML-1 cells into promyelocytes and of promyelocytic HL-60 cells into mature granulocytes. The differentiation-inducing effect of genistein was augmented by addition of 1 alpha,25-dihydroxyvitamin D3 (VD3) or retinoic acid, VD3 being more effective than retinoic acid. Methyl 2,5-dihydroxycinamate, a protein tyrosine kinase inhibitor, had only a weak effect in inducing differentiation of ML-1 cells. On the other hand, psi-tectorigenin was more effective than genistein in inducing the differentiations of ML-1 and HL-60 cells. Psi-tectorigenin is reported to inhibit phosphatidylinositol turnover without inhibiting protein tyrosine kinase. Thus modulation of phosphatidylinositol turnover might be more important than that of protein tyrosine kinase activity for differentiation of some myelogenous leukemia cells.

Granulocyte-colony Stimulating Factor and Retinoic Acid Cooperatively Induce Granulocytic Differentiation of Acute Promyelocytic Leukemia Cells in vitro

The interaction of granulocyte‐colony stimulating factor (G‐CSF) and retinoic acid (RA) in proliferation and differentiation of acute promyelocytic leukemia (APL) cells was examined. G‐CSF stimulated proliferation of APL cells at concentrations of 0.1 to 50 ng/ml in a dose dependent manner. More than 10−8M RA induced granulocytic differentiation of APL cells. Although G‐CSF induced lysozyme activities in APL cells, it alone did not induce terminal differentiation of APL cells. G‐CSF significantly enhanced the RA‐induced granulocytic differentiation of APL cells in vitro. Enhancement by G‐CSF was not due to the prolongation of survival of RA‐induced differentiated cells, but the differentiation‐inducing effects of G‐CSF might be evident only in the presence of RA. Since G‐CSF has a potential to induce the granulocytic differentiation of myeloid leukemia cells, G‐CSF in combination with RA may be applicable in differentiation induction therapy for some types of myeloid leukemia.

Induction of differentiation of human myeloid leukemia HL-60 cells by novel pyrimidine nucleoside analogs

New pyrimidine nucleoside analogs (18 compounds) were synthesized and their growth-inhibiting and differentiation-inducing activities on human myeloid leukemia HL-60 cells were examined. Some of the analogs were found to induce nitroblue tetrazolium (NBT) reducing activity in the HL-60 cells. The inducing activities of these compounds were compared at their concentrations for 50% inhibition of cell growth. TI-79 (3-benzyl-5-methyl-3-(beta-D-ribofuranosyl)pyrido[2,3-d]pyrimidine- 2,4(1H,3H)-dione) was a very effective inducer of NBT-reduction and of differentiation of the cells into mature granulocytes. The induction of NBT-reducing activity by TI-79 was inhibited by high concentrations of the natural nucleoside, adenosine. Other differentiation inducers, such as retinoic acid, 1 alpha,25-dihydroxyvitamin D-3 and staurosporin markedly enhanced the induction of differentiation of HL-60 cells by TI-79. Nucleoside analogs such as TI-79 should be useful for differentiation therapy of some types of myelogenous leukemia.

Differentiation of human monoblastic leukemia U937 cells induced by inhibitors of myosin light chain kinase and prevention of differentiation by granulocyte-macrophage colony-stimulating factor

Inhibitors of protein kinase activities are useful for the study of intracellular signal transduction and some of these inhibitors are reported to induce differentiation of human leukemia cells. We examined effects of granulocyte-macrophage colony-stimulating factor (GM-CSF) in combination with several kinase inhibitors on differentiation of human leukemia U937 cells. Nitroblue tetrazolium (NBT)-reducing activity, a typical marker of myelomonocytic differentiation, of U937 cells was induced by genistein and GM-CSF enhanced this activity. GM-CSF also induced the NBT-reducing activity of the cells in combination with 2,5-dihydroxycinnamic acid methyl ester, psi-tectorigenin and staurosporine, although each of them did not induce the activity. 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 in U937 cells NBT-reduction, and lysozyme and alpha-naphthyl acetate esterase activities. GM-CSF inhibited this differentiation and counteracted the anti-proliferation effect of the kinase inhibitors. These results suggest that some protein kinases are involved in differentiation of U937 cells and the kinases inhibited by ML-9 and ML-7 are associated with signal transduction of GM-CSF.

Difference in effects of interferon-α and interferon-γ on the induction of differentiation of retinoic acid-treated acute myeloid leukemia cells in primary culture

Abstract We studied differentiation inducing effects of retinoic acid (RA), 1α,25-dihydroxyvitamin D 3 (D 3 ) and interferons (IFNs), alone and in combination, on fresh myeloid leukemic cells from 8 patients. RA not only induced the differentiation of leukemic cells in 5 8 cases, but potentiated differentiation by IFNs either in granulocytic or monocytic pathways. In particular, interferon-α enhanced granulocytic differentiation and interferon-γ induced mono-macrophage differentiation of promyelocytic leukemic cells in the presence of RA. Differentiation induced by D 3 , alone or in combination with IFNs, was limited in all cases. RA plus IFNs might be an effective combination for differentiation therapy for some types of myeloid leukemia.

Characterization of growth inhibitory factors for mouse monocytic leukemia cells

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.