Masaro Tashima

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.

Ceramide Generation in Nitric Oxide-induced Apoptosis ACTIVATION OF MAGNESIUM-DEPENDENT NEUTRAL SPHINGOMYELINASE VIA CASPASE-3

Sodium nitroprusside (SNP), a NO donor, has been recognized as an inducer of apoptosis in various cell lines. Here, we demonstrated the intracellular formation of ceramide, a lipid signal mediator, in SNP-induced apoptosis in human leukemia HL-60 cells and investigated the mechanisms of ceramide generation. The levels of intracellular ceramide increased to, at most, 160% of the control level in a time- and dose-dependent manner when the cells were treated with 1 mm SNP. SNP also decreased the sphingomyelin level to ∼70% of the control level and increased magnesium-dependent neutral sphingomyelinase (N-SMase) activity to 160% of the control activity 2 h after treatment. Neither acid SMase nor magnesium-independent N-SMase was affected by SNP. Caspases are thought to be key enzymes in apoptotic cell death. Acetyl-Asp-Glu-Val-Asp-aldehyde, a synthetic tetrapeptide inhibitor of caspases, inhibited magnesiumdependent N-SMase, ceramide generation, and apoptosis. Moreover, recombinant purified caspase-3 increased magnesium-dependent N-SMase in a cell-free system. These results suggest that the findings that SNP increased ceramide generation and magnesium-dependent N-SMase activity via caspase-3 are interesting to future study to determine the relation between caspases and sphingolipid metabolites in NO-mediated signaling.

Diversity and complexity of ceramide signalling in apoptosis.

Sphingolipid ceramide has emerged as a lipid messenger of cell functions including differentiation and apoptosis. Diverse kinds of stresses (ultraviolet, irradiation, heat shock and hypoxia) and biological factors (TNF-alpha, IFN-gamma and Fas antibody) require ceramide generation to execute apoptosis. The review summarises the diversity and complexity of up- and downstream of ceramide signalling in apoptosis and clinical implications of ceramide-induced apoptosis.

Fulminant hepatitis B following bone marrow transplantation in an HBsAg-negative, HBsAb-positive recipient; reactivation of dormant virus during the immunosuppressive period

It is widely accepted that seroconversion of HBsAg to HBsAb indicates clearance of hepatitis B virus. We describe a 50-year-old man with chronic myelocytic leukemia who developed lethal hepatitis B 22 months after allo-BMT. He had been negative for HBsAg and positive for HBsAb before BMT. Hepatitis B virus latently existing in the liver cells before BMT proliferated during the immunosuppressed period causing fatal hepatitis. Recipients with positive HBsAb should be considered to have the potential for active hepatitis B to emerge after BMT. Bone Marrow Transplantation (2000) 25, 105–108.

Increase of Nuclear Ceramide through Caspase-3-Dependent Regulation of the “Sphingomyelin Cycle” in Fas-Induced Apoptosis

Regardless of the existence of ceramide-related molecules, such as sphingomyelin (SM), neutral sphingomyelinase (nSMase), and SM synthase, in the nucleus, the regulation of ceramide in the nucleus is poorly understood in stress-induced apoptosis. In Fas-induced Jurkat T-cell apoptosis, we found a time- and dose-dependent increase of ceramide content in the nuclear and microsomal fractions. Fas-induced increase of ceramide content in the nucleus also was detected by confocal microscopy using anticeramide antibody. Activation of nSMase and inhibition of SM synthase were evident in the nuclear fraction after Fas cross-linking, whereas nSMase was activated, but SM synthase was not affected, in the microsomal fraction. Pretreatment with d-609, a putative SM synthase inhibitor, enhanced Fas-induced increase of ceramide in the nucleus and induction of apoptosis along with increase of Fas-induced inhibition of nuclear SM synthase. Fas-induced activation of caspase-3 was detected in the nuclear fraction and in whole cell lysate. A caspase-3 inhibitor, acetyl-Asp-Glu-Val-Asp-chloromethyl ketone, blocked not only Fas-induced increases of apoptosis and ceramide content but also Fas-induced activation of nSMase and inhibition of SM synthase in the nuclear fraction. Taken together, it is suggested that the nucleus is a site for ceramide increase and caspase-3 activation in Fas-induced Jurkat T-cell apoptosis and that caspase-3-dependent regulation of the “SM cycle” consisting of nSMase and SM synthase plays a role in Fas-induced ceramide increase in the nucleus.

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.