Shigeru Tomoyasu

Iron deprivation‐induced apoptosis in HL‐60 cells

Iron deprivation of HL‐60 cells with deferoxamine B mesylate (DFO) induced apoptosis. DNA fragmentation became apparent with 10−6 M DFO after 48 h treatment. The apoptosis peak according to the DNA histogram on flow cytometory and typical nuclear collapse and were observed microscopically after 48 h treatment with 10−4 M DFO. Cells treated with 10−4 M DFO for as little as 24 h were shown to be committed to apoptosis, as chromatin condensation progressed gradually thereafter.

Spontaneous cytokine overproduction by peripheral blood mononuclear cells from patients with myelodysplastic syndromes and aplastic anemia

We studied spontaneous cytokine production by peripheral blood mononuclear cells (PBMC) obtained from 14 patients with aplastic anemia (AA) and 28 various myelodysplastic syndromes (MDS). The levels of interleukin-6, interleukin-1 beta, and tumor necrosis factor-alpha in cultured PBMC were measured by ELISA. The average levels of these cytokines were higher in AA or in refractory anemia (RA) than in RA with excess of blasts (RAEB) or in RAEB in transformation (RAEB-T). Marked cytokine overproduction was observed in RA as well as in AA. High cytokine levels were observed in hypocellularity and low blast cell counts in the bone marrow. These results may suggest that the increase of cytokines may be a reactive response in hypocellular bone marrow.

Immunodeficiency and IL‐6 production by peripheral blood monocytes in multicentric Castleman's disease

Summary. To study the pathogenesis of multicentric Castlemans disease (MCD), IL‐6 producing cells and immune function were investigated in four MCD patients. The expression of IL‐6 mRNA in one MCD lymph node was analysed by in situ hybridization. IL‐6 mRNA expressing cells were scattered in the interfollicular areas and did not resemble plasma cells. Spontaneous IL‐6 production was detected in the culture supernatants of peripheral blood mononuclear cells (PBMNC) from four patients. The IL‐6 producing cells among the PBMNC were found to be monocytes by both in situ hybridization and immunohistochemistry. We evaluated immune function in four MCD patients. These studies show: (1) a negative PPD skin test in 3/4 patients. (2) decreased IL‐2 production in 3/4 patients, (3) decreased T cell colony formation in 3/4 patients, (4) decreased NK activity and NK cell number in 2/4 patients, (5) increased soluble IL‐2 receptor in 4/4 patients, and (6) decreased CD4/CD8 ratio in 3/4 patients. These results show that MCD resembles, in several ways, acquired immunodeficiency syndrome (AIDS).

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.

Molecular analysis of the Cip1/Waf1 (p21) gene in diverse types of human tumors.

We have screened for mutations in the Cip1/Waf1 gene using Southern blot analysis and the polymerase chain reaction single-strand conformation polymorphism (PCR-SSCP) method in diverse human tumors. Seven of 102 (7%) human tumor samples were identified to have point mutations within the coding region of the Cip1/Waf1 gene. Two of the seven mutated cases showed gene rearrangements. These results suggest that the frequency of genetic alterations in the Cip1/Waf1 gene is relatively low in comparison with several known tumor suppressor genes.

Disappearance of CD21-positive Follicular Dendritic Cells Preceding the Transformation of Follicular Lymphoma: Immunohistological Study of the Transformation Using CD21, p53, Ki-67, and P-glycoprotein

Some follicular lymphomas histologically transform into diffuse aggressive lymphomas, the prognosis of which is poor. There are, however, no reliable histological criteria for predicting which cases will later undergo such transformation. In low-grade B-cell lymphomas, follicular dendritic cells form dense mesh-like networks that contain accumulating neoplastic B-cells. These are rare in high-grade lymphomas. We immunohistochemically analyzed CD21-positive follicular dendritic cells in 32 follicular lymphomas, including 3 transformed lymphomas, in addition to immunohistological study using P-glycoprotein, p53, and Ki-67. We found that the mesh-like networks in follicles are more clearly defined in low-grade lymphomas than in high-grade lymphomas (p = 0.015). Neoplastic follicles in 2 transformed lymphomas lost the networks of follicular dendritic cells before transformation despite the existence of morphologically clear follicles. This differed from the non-transformed cases of the same cytological grades. Prognosis was statistically better for patients with low-grade tumor than for those with high-grade tumor (p = 0.026), and there was a trend toward poorer survival among CD21-negative cases (p = 0.186). P-glycoprotein, p53, and Ki-67 expressions did not provide sufficient information to predict the transformation of follicular lymphoma. The presence of CD21-positive follicular dendritic cells in neoplastic follicles might help predict the potential of follicular lymphoma to transform to diffuse large B-cell lymphoma.

Phosphatidylinositol 3-kinase inhibitors, Wortmannin or LY294002, inhibited accumulation of p21 protein after γ-irradiation by stabilization of the protein

Expression of the cyclin kinase inhibitor, p21, is regulated both transcriptionally and posttranscriptionally by the ubiquitin-proteasome degradation pathway. Recently, we reported that DNA damage is required for efficient p21 expression by demonstrating that enhanced p21 mRNA expression induced by DNA damage results in increased p21 protein, but enhanced p21 mRNA without DNA damage does not. In addition, we demonstrated that DNA damage suppressed the ubiquitination of p21. In this study, we analyze the link between p21 stabilization and DNA damage. Enhanced p21 protein expression in ML-1 cells resulting from 15 Gy gamma-irradiation was diminished by Wortmannin or LY294002 pretreatment of cells. However, the levels of p21 mRNA were not affected by inhibitor pretreatment. Wortmannin or LY294002 pretreatment reduces p53 expression after gamma-irradiation to a lesser degree than that of p21. In addition, we examined the involvement of DNA-PK, whose activity is inhibited by Wortmannin or LY294002, in p21 stabilization using the SCID fibroblast cell line and a DNA-PK targeting ML-1 cell line. Accumulation of p21 protein by gamma-irradiation was similar to that of DNA-PK intact cells and was reduced by Wortmannin or LY294002 pretreatment. Involvement of another DNA damage detecting enzyme, the ATM gene product, whose activity is also inhibited by Wortmannin or LY294002, was evaluated. ATM deficient cells induced p21 after gamma-irradiation, gamma-irradiation-induced p21 protein was diminished by pretreatment of cells with Wortmannin or LY294002. We conclude that the p21 stabilization mechanism functions after gamma-irradiation, was sensitive to Wortmannin or LY294002, and required neither DNA-PK nor ATM gene product for activity.

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.