Ken-ichiro Hino

Epstein–Barr virus-encoded poly(A)– RNA supports Burkitt’s lymphoma growth through interleukin-10 induction

Akata and Mutu cell lines are derived from Burkitts lymphoma (BL) and retain the in vivo phenotype of Epstein–Barr virus (EBV) expression that is characterized by expression of EBV‐determined nuclear antigen 1 (EBNA1), EBV‐encoded RNAs (EBERs) and transcripts from the BamHI A region (BARF0). We found that EBV‐positive Akata and Mutu cell clones expressed higher levels of interleukin (IL)‐10 than their EBV‐negative subclones at the transcriptional level. Transfection of an individual EBV latent gene into EBV‐negative Akata cells revealed that EBERs were responsible for IL‐10 induction. Recombinant IL‐10 enabled EBV‐negative Akata cells to grow in low (0.1%) serum conditions. On the other hand, growth of EBV‐positive Akata cells was blocked by treatment either with an anti‐IL‐10 antibody or antisense oligonucleotide against IL‐10. EBV‐positive BL biopsies consistently expressed IL‐10, but EBV‐negative BL biopsies did not. These results suggest that IL‐10 induced by EBERs acts as an autocrine growth factor for BL. EBERs, EBER1 and EBER2, are non‐polyadenylated RNAs and are 166 and 172 nucleotides long, respectively. The present findings indicate that RNA molecules could regulate cell growth.

Differentiation of human acute myeloid leukaemia cells in primary culture in response to cotylenin A, a plant growth regulator.

Cotylenin A, which has a diterpenoid tricarbocyclic skeleton, has been isolated as a plant growth regulator, has been shown to affect several physiological processes of higher plants and have differentiation‐inducing activity in several myeloid leukaemia cell lines. We examined the effect of cotylenin A on the differentiation of leukaemic cells that were freshly isolated from acute myeloid leukaemia (AML) patients in primary culture. Cotylenin A significantly stimulated both functional and morphological differentiation of leukaemia cells in 9 out of 12 cases. This differentiation‐inducing activity was more potent than those of all‐trans retinoic acid and 1α,25‐dihydroxyvitamin D3 (VD3). Treatment with a combination of cotylenin A and VD3 was more effective than cotylenin A or VD3 alone at inducing the monocytic differentiation of AML cells.

Enhancement by bufalin of retinoic acid-induced differentiation of acute promyelocytic leukemia cells in primary culture

Bufalin, a cardiotonic steroid isolated from the Chinese toad venom preparation Chansu, has differentiation-inducing activity in several myeloid leukemia cell lines. We examined the effect of bufalin on differentiation of leukemic cells from acute myeloid leukemia (AML) patients in primary culture. Bufalin significantly stimulated functional and morphologic differentiation of leukemia cells in four of 20 cases, suggesting that bufalin alone is only a modest inducer of differentiation of AML cells in primary culture. In contrast, acute promyelocytic leukemia (APL) cells showed synergistic differentiation after treatment with all-trans retinoic acid (RA) and bufalin. In some cases, bufalin restored RA sensitivity to previously resistant APL cells. The effective concentration of bufalin for differentiation-inducing activity in APL cells was lower than for its cardiac action. Combined treatment with bufalin and RA may be more effective than RA alone in differentiation therapy of APL.

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.

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.

Effects of herbimycin A and its derivatives on growth and differentiation of Ph1-positive acute lymphoid leukemia cell lines.

The molecular basis of the Philadelphia chromosome (Ph1) is a structurally altered c-abl (bcr/abl) gene which encodes an abnormally large protein with protein tyrosine kinase activity. Herbimycin A, an inhibitor of tyrosine kinase, preferentially inhibited the growth of Ph1-positive acute lymphoid leukemia (ALL) cell lines, as well as Ph1-positive chronic myeloid leukemia (CML) cell lines. Although noncytotoxic concentrations of herbimycin A induced erythroid differentiation of two CML-derived cell lines, K562 and KU812, in a previous study, the differentiation-inducing effect of herbimycin A on Ph1-positive ALL cell lines was less strong. Herbimycin A enhanced some differentiation-associated properties of one Ph1-positive ALL cell line, L2, but the effect of herbimycin A on the other Ph1-positive ALL cell lines was cytotoxic rather than cytostatic (differentiation-inducing). Several derivatives of herbimycin A were synthesized and their effects on the cell proliferation of Ph1-positive CML and ALL cell lines were examined. The sensitivities of the Ph1-positive cell lines to herbimycin A derivatives were different from the data on the rat kidney cell line infected with Rous sarcoma virus (v-src) derived from a previous study, suggesting bcr/abl kinase may differ in sensitivity from other tyrosine kinases. Moreover, the sensitivities of the ALL cell lines were not the same as those of the CML cell lines. These results suggest that a specific inhibitor of bcr/abl kinase could be an effective antileukemic agent against Ph1-positive CML or ALL.

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.

Reversibility of monocytic differentiation of HL-60 cells by 1α,25 dihydroxyvitamin D3

Although 1alpha,25(OH)2D3 induces HL-60 cells to differentiate into monocytes, whether or not these monocytes revert to native promyelocytes is not clear. To investigate this question, HL-60 cells were treated with 1alpha,25(OH)2D3, and divided into CD14-positive and -negative cells with a cell sorter. These two populations were cultured with or without 1alpha,25(OH)2D3. Sorted CD14-positive HL-60 cells, treated with 1alpha,25(OH)2D3 for 7 days, reverted to CD14-negative cells and promyelocyte-like cells if 1alpha,25(OH)2D3 was removed from the medium. We conclude that the 1alpha,25(OH)2D3-induced differentiation of HL-60 cells into monocytes is reversible, and continuous administration of 1alpha,25(OH)2D3 is required for the differentiation of HL-60 cells.

Induction of Differentiation of Mouse Myeloid Leukemia M1 Cells by Serum of Patients with Chronic Myeloid Leukemia

We examined the capacities of sera from patients with myeloid leukemia to induce differentiation in mouse myeloid leukemic M1 cells. Higher differentiation‐inducing activity (D‐activity) was detected in sera of patients with chronic myelomonocytic leukemia or chronic myeloid leukemia (CML) than in sera of patients with acute myeloid leukemia and normal volunteers. The D‐activity in the sera was lost on heating the sera at 56° for 30 min. The major peak of D‐activity on Sephadex G‐200 gel filtration had an apparent molecular weight of 160,000. The origin of the D‐activity in sera of patients with CML was studied by culturing fractions of peripheral blood cells of patients with D‐activity for 3 days and then measuring the ability of the conditioned medium (CM) to induce differentiation of M1 cells. The cells in the myeloblast and promyelocyte fraction differentiated spontaneously into macrophage‐like cells during culture for 3 days and the cells in the late granulopoietic cell fraction differentiated into neutrophil‐like cells. Higher D‐activity was present in CM of cells in the myeloblast and promyelocyte fraction than in CMs of late granulopoietic cell fractions. These results suggest that human leukemic cells produce D‐activity for M1 cells during their differentiation into macrophage‐like cells.

A patient homozygous for a Gly354Cys mutation in factor VII that results in severely impaired secretion of the molecule, but not complete deficiency

We investigated the molecular basis of factor VII (FVII) deficiency in a Japanese woman who suffered occasional epistaxis. The patient had low levels of both FVII coagulant activity (FVII:C) and antigen (FVII:Ag) (5·0% and 7·3% of normal controls respectively). DNA sequence analysis of the FVII gene showed that the patient was homozygous for a mutation that resulted in a Cys for Gly354 substitution, a novel missense mutation in the catalytic domain. Haplotype analysis showed that this missense mutation was inherited from her consanguineous parents. Transient expression experiments showed that secreted FVII Cys354, FVII:C and FVII:Ag levels in conditioned media were reduced to 4% and 5%, respectively, of levels secreted from wild‐type FVII. However, the intracellular FVII Cys354 was 67% that of normal recombinant protein. Immunohistochemical analysis showed that intracellular FVII:Ag from FVII 354Cys was present diffusely throughout the cytoplasm. Substitution of FVII 354Gly with amino acids other than Cys (Arg, Asp, Ser and Phe), did not produce a phenotype similar to FVII Cys354Gly. Molecular modelling indicated that FVII Gly354 was located outside the FVII heavy chain, and that Cys135 in the EGF2 domain, Cys262 in the catalytic domain and Cys127 all exist within 10 Å of Gly354. Therefore, we propose that the introduction of an additional free cysteine residue in the FVII molecule results in the formation of illegitimate disulphide bonds and a mis‐folded domain, leading to defective secretion.