Hiroto Ishikura

Requirement of AP-1 for Ceramide-induced Apoptosis in Human Leukemia HL-60 Cells

Ceramide has emerged as a novel lipid mediator in cell proliferation, differentiation, and apoptosis. In this work, we demonstrate that the levels of c-jun mRNA, c-Jun protein, and DNA binding activity of a nuclear transcription factor AP-1 to 12-o-tetradecanoylphorbol 13-acetate responsive elements all increased following treatment with the cell-permeable ceramide, N-acetylsphingosine in human leukemia HL-60 cells. N-Acetylsphingosine (1-10 μM) increased the levels of c-jun mRNA in a dose-dependent manner, and maximal expression was achieved 1 h after treatment. Increase of c-jun expression treated with 5 μMN-acetyldihydrosphingosine, which could not induce apoptosis, was one third of that with 5 μMN-acetylsphingosine. Ceramide-induced growth inhibition and DNA fragmentation were both prevented by treatment with curcumin, 1,7-bis[4-hydroxy-3-methoxy-phenyl]-1,6-heptadiene-3,5-dione (an inhibitor of AP-1 activation), or antisense oligonucleotides for c-jun. These results suggest that the transcription factor AP-1 is critical for apoptosis in HL-60 cells and that an intracellular sphingolipid mediator, ceramide, modulates a signal transduction inducing apoptosis through AP-1 activation.

Antinuclear antibodies in healthy aging people: a prospective study

In order to evaluate the expression of antinuclear antibodies (ANA) in normal elderly individuals over time and clinical significance, a cross-sectional ANA testing in healthy Japanese was performed, followed by annual evaluations of ANA positive aged (> or = 65 years) and a control group. ANA was more prevalent in the aged (11.4% vs. 3.8%) and most were persistent after 4 years. Anti-ssDNA and anti-histone antibodies were increased in aged ANA positive as compared to ANA negative controls. Except for a history of spontaneous abortion, there was no differences in clinical findings. HLA DRB1*0901 and the DQB1*0602 + 0302 + 0303 set of alleles were increased in ANA positive. Therefore, ANA in the aged were persistent, apparently directed toward chromatin elements, and shared MHC associations with autoimmune diseases. Longer follow-up may be necessary to improve the evaluation of clinical significance of ANA in the aged.

The effect of low dose Ara-C in acute non-lymphoblastic leukaemias and atypical leukaemia

Nine patients with nonlymphocytic leukaemia and one patient with refractory anaemia with excess of blasts (RAEB) were treated subcutaneously with Ara‐C at a low dose of 10 mg/m2/12 h; complete remission was obtained in seven patients. In all cases severe pancytopenia was observed during treatment. We measured the concentration of Ara‐C in serum, and found that the maximum concentration reached a peak (52‐132 ng/ml; mean 84‐2 ng/ml) at 15 min following injection. These concentrations can be considered sufficient to inhibit DNA synthesis. Primary short‐term culture of human leukaemic cells with low dose Ara‐C was also performed, and differentiation of leukaemic cells was observed for several types of leukaemic cells. The in vitro findings corresponded to the observed clinical effects. From the above results, the action of low dose Ara‐C may have resulted from a combination of two different mechanisms, its cytostatic effect and its differentiation‐inducing effect.

c-Myc and Bcl-2 Protein Expression During the Induction of Apoptosis and Differentiation in TNFα-Treated HL-60 Cells

We examined c-Myc and Bcl-2 protein expressions during the induction of apoptosis and differentiation in TNF alpha-treated HL-60 cells using a two-color flow cytometric method. We found that c-Myc protein was rapidly down-regulated in the apoptotic cells while Bcl-2 protein was expressed at relatively high levels. Concomitantly with terminal differentiation Bcl-2 protein was down-regulated in differentiating cells as well as c-Myc protein. We also showed that c-myc antisense oligonucleotides could induce apoptosis in HL-60 cells whereas bcl-2 antisense did not induce apoptosis during the early time of treatment. These results suggest that the down-regulation of c-Myc protein expression is a primary event to induce apoptosis and neither consistent expression of c-Myc protein nor rapid down-regulation of Bcl-2 protein is necessary for the initial processing of apoptosis in HL-60 cells. Furthermore, concomitant down-regulation of c-Myc and Bcl-2 is closely associated with terminal differentiation and apoptotic cell death of HL-60 cells treated with TNF alpha.

Activation of Protein Kinase C Enhances TNF-α-induced Differentiation by Preventing Apoptosis via Rapid up-regulation of c-Myc Protein Expression in HL-60 Cells

Tumor necrosis factor- α (TNF- α ) induces both rapid onset of apoptosis and monocytic differentiation in HL-60 human myeloid leukemia cells. In this study, we examined the effect of activation of protein kinase C (PKC) in c-Myc protein expression in association with the induction of apoptosis and differentiation in TNF- α -treated HL-60 cells. Pretreatment with phorbol 12-myristate 13-acetate (PMA), an activator of PKC, prevented TNF- α -induced rapid onset of apoptosis, which occurs at 3 h culture with TNF- α, concomitantly with the up-regulation of c-Myc protein expression. In addition, PMA enhanced TNF- α -induced differentiation at 24 h treatment. This was documented by the expression of integrin Mac-1 molecule (CD11b) on the cell surface and the cellular adhesion to the plastic bottom of the flask, indicating the differentiation along with the monocyte/macrophage lineage. These results indicate that activation of PKC not only counteracts apoptosis but also enhances differentiation in TNF- α -treated HL-60 cells. Up-regulation of c-Myc protein evoked by pretreatment with PMA for a short time could disturb the signaling pathway of the ceramide and sphingosine, which are known to function as the endogenous modulators mediating the apoptotic signal of TNF- α. Our results strongly suggest the role of c-Myc protein as a mediator of cytoprotective effect of PKC pathway, and PKC pathway opposes apoptosis and consequently undergo differentiation via rapid up-regulated c-Myc protein expression during TNF- α signaling of HL-60 cells. Our findings provide a new insight for a role of PKC and c-Myc protein with special reference to the regulatory mechanisms in the decision of cellular fate, differentiation or apoptosis.

c-myc Protein Expression During Cell Cycle Phases in Differentiating HL-60 Cells

We examined c-myc protein expression in cell cycle phases during differentiation induction of HL-60 cells by flow cytometry using an indirect immunofluorescence method. In exponentially proliferating HL-60 cells, c-myc protein was expressed in a cell cycle dependent manner. During the differentiation induction of HL-60 cells with dimethylsulfoxide (DMSO), c-myc protein was rapidly down-regulated in the G1/0 specific phase prior to the appearance of differentiation associated markers. Our results indicate that c-myc protein functions in the G1/0 specific phase in cellular differentiation, and the rapid down-regulation of c-myc protein in G1/0 phase is closely associated with initial differentiation programs.

A favourable effect of long-term α-interferon therapy in refractory idiopathic thrombocytopenic purpura

Summary We successfully treated two patients with refractory idiopathic thrombocytopenic purpura (ITP) with a weekly or monthly administration of α‐interferon followed by short‐course treatment with α‐interferon. In both cases an increase in platelet count was brought about within 1 week after administration of α‐interferon and the favourable response in platelet count was sustained over a few years with the weekly or monthly administration of α‐interferon in the absence of any noticeable side‐effects except for fever. So. α‐interferon therapy seems to be beneficial in some cases of refractory ITP in terms of both remission induction and maintenance therapy.

Elevated Anti-Neutrophil Cytoplasmic Antibody Titer in a Patient with Atypical Orbital Pseudotumor

We recently examined a patient with ophthalmic manifestations of Wegeners granulomatosis in whom antineutrophil cytoplasmic antibody (cANCA) titers provided helpful diagnostic information. A 40-year-old man who had suffered from bilateral exophthalmos for 9 months was diagnosed initially as having idiopathic inflammatory pseudotumor. The patient exhibited purulent nasal discharge and microhematuria. A histopathologic study revealed vasculitis. His ANCA titer for cANCA was found to be evaluated, and our patient was subsequently diagnosed as having Wegeners granulomatosis. His ocular symptoms resolved and did not recur after treatment with corticosteroid in combination with cyclophosphamide. We believe that cANCA levels should be investigated in patients with orbital pseudotumor as a possible sign of Wegeners granulomatosis.

Meningeal involvement of chronic myelomonocytic leukemia

Sirs: Chronic myelomonocytic leukemia (CMML) is classified as a clonal disorder of hematopoietic stem cells featuring myelodysplastic syndromes (MDS) and myeloproliferative disorders. CMML is present predominantly in elderly patients and unfortunately most medications prove disappointing. Therefore, the treatment is palliative and the prognosis is poor [4]. There are some reports of CMML involved in extramedullary tissues [2, 3, 11], but few in the central nervous system (CNS). The sole case reported [8] so far had a fatal outcome. Here we report two cases of CMML involving meningeal infiltration which were treated with intrathecal injections of cytosine arabinoside (Ara-C), methotrexate (MTX) and methylprednisolone or MTX alone resulting in favorable responses. Patient 1. An 83-year-old man was admitted to our hospital for inguinal hernia. Postoperative peripheral blood examinations revealed sustained leukocytosis (the WBC count was 19.8–22.7 109/liter) with monocytosis (35–45 %) mild anemia (10.8–11.0 g/dl) and thrombocytopenia (91–100 109/liter). While the blast was not observed in the peripheral blood, 10 % blasts were counted in the bone marrow with hypercellularity. Bilineage dysplastic changes (micromegakaryocytes and hypersegmentation of neutrophils) were also noted. Chromosomal analysis revealed deletion of the Y chromosome. We considered this patient as CMML according to the FAB criteria [1] and did not perform any specific treatment because of the stable hematological condition. Six months later, he had double vision and right facial palsy. He showed left sixth, right fifth and seventh cranial nerve palsies and left Horner’s syndrome. Lumbar puncture revealed an elevated mature monocyte count (13/μl, Fig. 1), but no neoplastic cells, with 52 mg/dl of glucose and 87 mg/dl of protein in the cerebrospinal fluid (CSF). Head magnetic resonance imaging (MRI) showed normal findings. Therefore, we presumed the diagnosis was CMML with meningeal infiltration, and performed 3 intrathecal injections of Ara-C (40 mg), MTX (15 mg) and methylprednisolone (4 mg) in one month. The patient recovered gradually and the symptoms disappeared on the 25th day after the treatment ended. The monocyte count in the CSF gradually decreased with improvement. His hematological condition is still stable without any treatment for CMML more than 2 years after discharge. Patient 2. A 50-year-old man was admitted to our hospital because of fever, headache and disorientation. Lumber puncture revealed marked increase of cell numbers (296/μl) with dominant neutrophil population (96 %), 61 mg/dl of glucose and 49 mg/dl of protein. We could not detect any pathogen in CSF. The gadoliniumDTPA enhanced head MRI showed thickening of the meninges. He had double vision, right ptosis, in addition to hypesthesia and tingling of the bilateral lower extremities, after admission. MRI revealed a mass lesion which could not be enhanced by gadolinium-DTPA in the spinal cord at the L1 level (Fig. 2), this suggesting an intramedullary location. Unfortunately, we could not biopsy it. Then an infiltrative shadow appeared at the left lung. The persistent level of leukocytosis (the WBC count was 11.6–16.6 109/liter) with monocytosis (18–43 %), and the appearance of blastic cell (0.5–1 %) indicated the necessity for bone marrow examination. This showed hypercellular bone marrow occupied predominantly with myelomonocytic cells. The blast cell count was 25 % and dysplastic changes were observed in two lineages (myeloid and erythroid). These findings grossly accorded with CMML except for the percentage of blastic cells in the bone marrow. Cytogenetic abnormality with t[11, 19](q23;p13.1) was also recognized. Bronchoscopic biopsy of the pulmonary lesion revealed inflammatory change with monocytic infiltration. We diagnosed this patient as CMML with meningeal and pulmonary involveLETTER TO THE EDITORS

Phagocytosis of Terminally Differentiated Acute Promyelocytic Leukemia Cells by Marrow Histiocytes During Treatment with All-trans Retinoic Acid

It has been established that acute promyelocytic leukemia (APL) cells are induced to terminally differentiate by all-trans retinoic acid (ATRA), however, the clearance of differentiated APL cells in vivo has not been well understood. Here, we documented the elimination of terminally differentiated APL cells by histiocytes in bone marrow during differentiation induction therapy. In two ATRA-treated APL patients, bone marrow showed the striking phagocytosis of differentiated APL cells by histiocytes just before the achievement of complete remission. Histiocytes phagocytosed APL cells at the terminal stage of differentiation prior to the late apoptotic event of cell lysis. Engulfed APL cells then undergo morphological features of late apoptosis and finally fragmentation in the cytoplasm of histiocytes. This swift and efficient elimination of APL cells undergoing apoptosis by the histiocytes in bone marrow may be possible pathway, at least partially, for the clearance of differentiated APL cells.