Masatsugu Ohta

Expression Cloning and Functional Characterization of Human cDNA for Ganglioside GM3 Synthase

Ganglioside GM3 is a major glycosphingolipid in the plasma membrane and is widely distributed in vertebrates. We describe here the isolation of a human cDNA whose protein product is responsible for the synthesis of GM3. The cloned cDNA spanned 2,359 base pairs, with an open reading frame encoding a protein of 362 amino acids with a predicted molecular mass of 41.7 kDa. The deduced primary structure shows features characteristic of the sialyltransferase family, including a type II transmembrane topology and the sialylmotifs L at the center and S at the C-terminal region. An amino acid substitution from aspartic acid to histidine was demonstrated at a position invariant in sialylmotif L of all the other sialyltransferases so far cloned. The best acceptor substrate for the gene product was lactosylceramide, and cells transfected with the cloned cDNA clearly exhibited de novo synthesis of GM3, with a measurable decrease in the precursor lactosylceramide. Despite the ubiquitous distribution of ganglioside GM3 in human tissues, a major 2.4-kilobase transcript of the gene was found in a tissue-specific manner, with predominant expression in brain, skeletal muscle, and testis, and very low expression in liver.

Prognostic significance of WT1 mRNA and anti-WT1 antibody levels in peripheral blood in patients with myelodysplastic syndromes

Wilms tumor gene (WT1) mRNA expression in peripheral blood cells was examined in 80 patients with myelodysplastic syndrome (MDS) or acute myeloid leukemia (AML) transformed from MDS. Serum anti-WT1 antibody titers were also determined in 45 patients. Their long-term follow-up showed that the survival rate became worse as the WT1 mRNA level increased. In particular, a high WT1 mRNA level was a strong predictor of a short time to AML transformation even if adjusted by the International Prognostic Scoring System category. Moreover, high values of anti-WT1 antibody were an independent predictor of longer survival. These data may justify therapeutic strategies targeting WT1 molecules in MDS.

Interleukin‐1 producing ability of leukaemia cells and its relationship to morphological diagnosis

Interleukin‐1 (IL‐1) is a low molecular weight polypeptide produced by monocyte–macrophage lineage cells. IL‐1 production by primary‐cultured leukaemic cells of several FAB subtypes was estimated and compared with in vitro and in vivo lysozyme production.

Differentiation of human leukemia cells and its usefulness for clinical diagnosis

Various chemical inducers have effects on the induction of terminal differentiation of human myelogenous leukemia cell lines. We studied morphological and functional changes of human leukemia cells freshly obtained from patients using 12-0-tetradecanoyl phorbol-13-acetate (TPA), retinoic acid (RA) or dimethyl sulphoxide (DMSO). The myeloid leukemia cells cultured with TPA became adherent to plastic culture dishes, and then developed macrophage-like morphology with long filamentous pseudopods within 48 h incubation. They showed marked enhancement of the ability to phagocytose latex particles. But these acquired properties did not always parallel each other, suggesting that the mechanism of functional maturation of leukemic cells induced by chemical agents was not identical with that of morphological changes. On the other hand, the lymphoid leukemia cells did not show morphological and functional changes when cultured with the above inducers. It is suggested that exposure of leukemic cells to TPA for relatively short times (12-24 h) may be useful for determining whether they are of myeloid or lymphoid origin. These characteristic changes were also observed in leukemic cells from the myeloid or lymphoid crisis of chronic myelogenous leukemia.

Preferential Production of Interleukin‐1β over Interleukin‐1 Receptor Antagonist Contributes to Proliferation and Suppression of Apoptosis in Leukemic Cells

Normal human monocytes were isolated in a nascent state by centrifugal elutriation and used for the study of interleukin‐1 (IL‐1) and interleukin‐1 receptor antagonist (IL‐1ra) expression. Neither IL‐1β nor IL‐1ra mRNA was present in monocytes just after the isolation, but they were induced simultaneously in response to various stimulants. In contrast, only IL‐1β mRNA was expressed in monocytic leukemia cell line JOSK‐1, while little or no IL‐1ra mRNA was detected even after stimulation. Dominant expression of IL‐1β over IL‐1ra was also observed in fresh leukemia cells including monocytic leukemias, i.e., IL‐1βmRNA was constitutively expressed in 26 out of 36 cases (72.2%), whereas IL‐1ra mRNA was present only in 8 cases (22.2%). The signal intensity of IL‐1βmRNA was stronger than that of IL‐1ra even in IL‐1ra‐positive cases. Apoptotic cell death of monocytes was significantly inhibited by IL‐1β, and it was enhanced by IL‐1ra. In fresh leukemia cells, 3H‐thymidine uptake was generally higher in IL‐1‐producing cases than in IL‐1ra‐producing cases, and was increased by the addition of IL‐1β in all cases tested. Cell proliferation was inhibited by either IL‐1ra or anti‐IL‐1β antibody in IL‐1‐producing cases, while it was enhanced by anti‐IL‐1ra antibody in IL‐1ra‐producing cases. These results suggest that the balance between IL‐1 and IL‐1ra is necessary for homeostasis of the mononuclear phagocytosis system. The imbalance between these two counter‐acting cytokines might contribute to the altered growth and accumulation of leukemic cells.

Angiogenic mediators of the angiopoietin system are highly expressed by CD10‐positive lymphoma cells in angioimmunoblastic T‐cell lymphoma

Angioimmunoblastic T‐cell lymphoma (AILT) is a malignant disease of peripheral T‐cell origin that is characterized by a prominent proliferation of high endothelial venules in the lymph node. To investigate angiogenic mechanisms in AILT we measured the angiogenic mediator gene expression levels in the lymph nodes of 54 non‐Hodgkin lymphoma patients, by immunostaining and quantitative reverse transcription polymerase chain reaction. Angiogenic mediators angiopoietin (Ang) 1 (ANGPT1), Ang2 (ANGPT2) and their receptor, Tie2 (TEK), vascular endothelial growth factor (VEGF; VEGFA) and its receptor, VEGFR2 (KDR), and hepatocyte growth factor (HGF) and its receptor, c‐Met (MET) were all more highly expressed in AILT lymph nodes (16 cases) than in B‐cell lymphomas (24 cases). Moreover, significantly higher Ang1 and Tie2 expression was detected in AILT cases with CD10‐positive neoplastic T‐cells by comparison with unspecified peripheral T‐cell lymphoma (14 cases). Immunostaining confirmed the expression of Ang1 and VEGF by both neoplastic T‐cells and follicular dendritic cells. These results suggest that the angiopoietin system may play an important role in the development of high vascularity in AILT lymph nodes. Consequently, as neoplastic T‐cells and follicular dendritic cells are both increased in AILT and may represent an important source of angiogenic mediators, targeting these cells with anti‐angiogenic strategies might represent a novel therapy for AILT.

Treatment of acute myeloid leukemia patients aged more than 75 years: Results of the E-AML-01 trial of the Japanese Elderly Leukemia and Lymphoma Study Group (JELLSG)

The feasibility and effects of combination chemotherapy for very elderly patients with acute myeloid leukemia was examined in 65 patients (including previous myelodysplastic syndrome) aged 76 years or morewith a performance status of 0 – 3. Induction chemotherapy was performed with 30 mg/m2 daunorubicin on days 1 – 3, 150 mg/m2 behenoyl cytosine arabinoside on days 1 – 7, and 70 mg/m2 6-mercaptopurine with 300 mg allopurinol taken orally on days 1 – 7 (BHAC-DM). The complete remission (CR) rate was 38.5%, whereas overall survival at 2 and 5 years was 22.0% and 4.7%, respectively. Two- and 5-year survival of CR patients was 41.8% and 11.2%, respectively. The relapse rate of the 25 CR patients was 64.0% and disease-free survival at 2 and 5 years was 21.0% and 11.2%, respectively. The therapy-related mortality rate at induction was 13.8%. BHAC-DM is feasible and effective for selected very elderly acute myeloid leukemia patients.

Abnormal N-glycosylation of the immunoglobulin G κ chain in a multiple myeloma patient with crystalglobulinemia : Case report

Spontaneous crystallization of monoclonal immunoglobulins (crystalglobulin) is a rare complication of multiple myeloma. We describe a 64-year-old Japanese man with skin ulcers and renal failure associated with immunoglobulin G κ multiple myeloma. Crystallized immunoglobulin was detected in his serum at room temperature. Analysis of the patient’s crystalglobulin by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and mass spectrometry suggested that the crystallization was due to abnormal glycosylation of the immunoglobulin light chain. Treatment with thalidomide and dexamethasone improved the severe skin ulcers on the patient’s extremities and partially reversed his renal failure. This report is the first of abnormal glycosylation of immunoglobulin possibly caused by modification ofN-glycans in the light chain. We concluded that abnormal glycosylation of the immunoglobulin light chain might be the cause of the patient’s skin ulcers and renal dysfunction. cr 2007 The Japanese Society of Hematology

Prognosis of elderly patients with acute myelogenous leukemia: analysis of 126 AML cases.

We retrospectively analyzed 126 acute myelogenous leukemia (AML) patients aged ≥60 years who had all been referred to the same hematological department between 1989 and 1999. In 76 de novo AML cases, 53 patients (median age, 72 years) were treated with combination chemotherapy (CT) for remission induction. Complete remission (CR) rate was 57.1%. The median overall survival (OS) was 16 months, and the rate of 3-year OS was 28%. The favorable prognostic factors were performance status ≥2, cholinesterase ≥100 IU, and intermediate or favorable karyotype (P < .01). Seventeen patients (median age, 78 years) with hypocellular bone marrow or poor general condition were treated with low-dose cytosine arabinoside (LDAraC). In these patients, the CR rate was 50% and the median OS was 11 months, with an OS estimate at 3 years of 14%. All patients with hypocellular bone marrow who received LDAraC for 21 days achieved CR. In 50 patients who developed AML following a myelodysplastic syndrome (MDS/AML), 22 patients (median age, 74 years) were treated with CT, and 14 (median age, 74 years) patients were treated with LDAraC. The CR rates were 22.7% and 21.4%, respectively, and the median OS durations were 8 months and 11 months, respectively. There were no significant factors that would indicate a good prognosis in MDS/AML patients.

Over-expression and amplification of the CDC2 gene in leukaemia cells

The expression and structure of the cdc2 gene, one of the master regulators of the eukaryotic cell cycle, were investigated in fresh leukaemic cells from 51 cases of various types of leukaemia. Cdc2 mRNA transcripts were detectable in approximately 40% (21/51) of cases by Northern blotting. Over‐expression of cdc2 mRNA as compared to normal bone marrow cells was noted in 10/21 cases with detectable cdc2 mRNA transcripts. Amplification of the cdc2 gene was found in three cases. Cdc2 mRNA was over‐expressed in these three cases, suggesting that gene amplification is a direct cause of mRNA over‐expression in a subset of cases. Cell proliferative capacity was well correlated with the amount of cdc2 mRNA transcripts, i.e. 3H‐thymidine incorporation was highest in cases with cdc2 mRNA over‐expression and was significantly higher in cdc2‐positive cases than in cdc2‐negative cases. These results suggest that over‐expression of CDC2, which is due to the gene amplification in some cases, might play a role in altered growth of leukaemic cells.