Hiroyoshi Sawada

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.

Ceramide-induced Translocation of Protein Kinase C-δ and -ϵ to the Cytosol IMPLICATIONS IN APOPTOSIS

Ceramide is now recognized as an intracellular lipid signal mediator, which induces various kinds of cell functions including apoptosis. Ceramide-induced apoptosis was reported to be blocked by 12-O-tetradecanoylphorbol 13-acetate, a protein kinase C (PKC) activator, but its mechanism remained unclear. Therefore, we investigated whether ceramide has any effects on PKC in the induction of apoptosis. We here report that N-acetylsphingosine (synthetic membrane-permeable ceramide) induced translocation of PKC-δ and -ε isozymes from the membrane to the cytosol within 5 min in human leukemia cell lines. Treatment with sphingomyelinase, tumor necrosis factor-α, or anti-Fas antibody, all of which can induce apoptosis by generating natural ceramide, similarly induced cytosolic translocation of PKC-δ and -ε. In Fas-resistant cells anti-Fas antibody did not induce cytosolic translocation of PKC-δ and -ε because of no generation of ceramide, whereas N-acetylsphingosine induced apoptosis with cytosolic translocation of PKC-δ and -ε. Furthermore, both 12-O-tetradecanoylphorbol 13-acetate and a nonspecific kinase inhibitor, staurosporine, prevented ceramide-induced apoptosis by inhibiting cytosolic translocation of PKC-δ and -ε. These data suggest that cytosolic translocation of PKC-δ and -ε plays an important role in ceramide-mediated apoptosis.

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.

Molecular heterogeneity of β-thalassaemia in the Japanese: identification of two novel mutations

Five unrelated Japanese β‐thalassaemia genes, from one homozygote and four heterozygotes, have been systematically characterized using DNA polymorphism analysis, polymerase chain reaction, dot‐blot hybridization and direct sequencing of amplified genomic DNA. Four different molecular defects were observed on three different β‐globin gene frameworks. One of these, the A→G mutation in the TATA box, a previously described mutation, was detected by dot‐blot hybridization in one homozygote and one heterozygote with the β‐globin gene of framework 2. The second mutation is a C→T substitution at position 654 of IVS‐2, the mutation commonly found in Chinese, which was associated with the framework 1 gene. Another two mutations, both associated with framework 3 genes, are novel ones; an amber mutation in codon 90 (GAG to TAG) and a frameshift (+G) insertion in codon 54, both of which cause a β0‐thalassaemia phenotype by premature termination of the β‐globin chain synthesis.

Molecular Basis of β-Thalassemia in Japan: Heterogeneity and Origins of Mutations

Characterization of β-thalassemia mutations was attempted for 13 unrelated Japanese patients heterozygous for β-thalassemia. We have systematically analyzed β-thalassemia genes using polymerase-chain-

Acute myeloid leukemia with t(5;11): two case reports

A case of acute monocytic leukemia (AMoL) with t(5;11)(q31;q23) and a case of acute myelomonocytic leukemia (AMMoL) with t(5;11)(q35;q13.1) are reported. The translocation between the long arm of chromosome 11q and that of chromosome 5q with leukemia have been rarely reported. Though breakpoint of both cases were subtlety different, they had morphologically monocytic character and showed hyperleukocytosis and chemoresistance.

Acquired hypochromic and microcytic sideroblastic anaemia responsive to pyridoxine with low value of free erythrocyte protoporphyrin: a possible subgroup of idiopathic acquired sideroblastic anaemia (IASA)

Patients with idiopathic acquired sideroblastic anaemia (IASA) usually show macrocytic or normocytic anaemia and increased free erythrocyte protoporphyrin (FEP). The mean cell haemoglobin concentration is normal or slightly low. Here we report a pyridoxine‐responsive IASA patient with microcytic and hypochromic anaemia and low FEL level; these features are usually seen in cases of hereditary sideroblastic anaemia. Microcytosis increased during therapy.

Terminal deoxynucleotidyl transferase activity in leukaemic T cells from Japanese adults.

Terminal deoxynucleotidyl transferase (TdT) is one of the important markers for studying the derivations of the malignant haemopoietic cells (Sarin, 1977). Since the activity of this enzyme is normally confined to the possible T cell precursors in the bone marrow and thymus cells, the more mature T cells in a post-thymic stage lack this activity (Kung et al, 1975; Barr et al , 1976). The cells of T cell acute lymphoblastic leukaemia in infants, children or adolescents always exhibit TdT activity and are supposed to represent T cells in the thymic stage (Brouet & Seligman, 1978). In contrast, T cell malignancies in adult or aged patients, not yet fully classified (Lutzner et al, 1975), have not yet been fully studied regarding TdT activity. The cells of Skzary syndrome and mycosis fungoides have been reported to lack TdT activity (Donlon et a l , 1977; Kung et al , 1978). In Japan, variable types of T cell malignancies are found among adults, and such have been summarized as ‘adult T cell leukaemia’ (Uchiyama et al , 1977). In most cases, bizarre, lobulated cells and skin lesions are present, and distinction from Stzary syndrome is often difficult. We have investigated TdT activity in the leukaemic T cells from three Japanese adults. Table I gives details of the patients, and Table I1 shows the results of surface marker study

Effect of 1,3-bis(2-chloroethyl)-1-nitrosourea on newly synthesized DNA in L-1210 cells

Abstract The synthesis of DNA in L-1210 cells was selectively inhibited by BCNU [1,3-bis-(2-chloroethyl)-1-nitrosourea, NSC 409962]. When the DNA chain growth in exponentially grown L-1210 cells was analyzed by alkaline sucrose gradient centrifugation, the initially synthesized DNA had short segments (approximately 5S) which increased in size to 30, 70 and to over 100S. When treated with BCNU, the short, newly synthesized DNA segments accumulated; the molecular weights were 5-30S. While it appears that BCNU inhibits the early elongation steps of newly synthesized DNA, the chasing experiments suggest that the drug does not affect the joining process from replicon-sized DNA (70S) to bottom peaks with a molecular weight of over 100S.

Low Levels of Free Endotoxin Result from Carbapenem Treatment of Gram-Negative Bacteria

We investigated the effects of imipenam (IPM), 3 new carbapenems (PAPM, BIPM, MEPM) and 2 cephems (CZX, CAZ) on the release of free endotoxin from 2 clinically isolated gram-negative bacteria (Escherichia coli andPseudomonas aeruginosa). On the growth curves we constructed ofE. coli in RPMI-1640 medium supplemented with 10% inactivated calf serum, the greatest amounts of free endotoxin were released into the medium, whenE. coli was treated with bacteriostatic concentrations of IPM or CZX.E. coli treated with bacteriostatic concentrations of IPM, PAPM and BIPM released less endotoxin into the medium than untreated cultures. MEPM induced a greater release of endotoxin than the control, although the statistical difference was only observed between the control and the treatment with MEPM for 6 hours (P<0.01). Bacteriostatic concentrations of CZX and CAZ induced the release of more free endotoxin than the control, and more endotoxin than all carbapenems tested (P<0.01). Similar results were obtained withP. aeruginosa. Endotoxin from IPM and CZX-treatedE. coli induced the release of TNF-α from differentiated HL-60 cells in a concentration dependent manner. These results indicate that carbapenems, which primarily bind penicillin-binding protein 2 (PBP2), induce the release of lower amounts of endotoxin than do cephems, which primarily bind PBP3.