Takeyuki Sato

Establishment of a human leukaemic cell line (CMK) with megakaryocytic characteristics from a Down's syndrome patient with acute megakaryoblastic leukaemia

Summary. A new megakaryoblastic cell line (CMK), which also exhibits erythroid and myeloid markers, was established from a Downs syndrome patient suffering from acute megakaryoblastic leukaemia. The CMK cells were found to be positive in reactions with anti‐platelet antibodies (anti‐glycoproteins IIb/IIIa and Ib, and Plt‐1). Platelet peroxidase (PPO) reactivity was found to be associated with the nuclear envelope and the endoplasmic reticulum but not with the Golgi apparatus. Some cells possessed cytoplasmic granules with the characteristics of α‐granules and demarcation membranes. Karyotyping revealed near‐tetraploidy (modal chromosome number of 95; ranging 87–98) and a translocation der(17)t(11;17), also found in the original leukaemic cells, confirming that the cells were derived from the patients malignant blasts. The CMK cells were also found to be positive in reaction with anti‐glycophorin A antibody, as well as with anti‐myeloid antibodies (MY4, MY7 and MY9). Treatment of CMK cells with phorbol ester 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA) greatly enhanced the reactivity with anti‐platelet antibodies, increased the number of cells in which cytoplasm was dissociated into numerous segments and suppressed the reactivity with anti‐glycophorin A. The proliferation of CMK cells was stimulated by interleukin‐3 (IL‐3) and granulocyte‐macrophage colony stimulation factor (GM‐CSF). This cell line should be a useful tool for analysing the basis of the afferent association between megakaryoblastic leukaemia and Downs syndrome, as well as for further study of megakaryocytic differentiation.

Six Months of Maintenance Chemotherapy After Intensified Treatment for Acute Lymphoblastic Leukemia of Childhood

PURPOSE We postulated that intensification of chemotherapy immediately after remission induction might reduce the leukemic cell burden sufficiently to allow an abbreviated period of antimetabolite therapy. PATIENTS AND METHODS Three hundred forty-seven children (ages 1 to 15 years) with previously untreated acute lymphoblastic leukemia (ALL) were enrolled onto the Tokyo L92-13 study, which excluded patients with mature B-cell ALL and patients less than 1 year old. One hundred twenty-four patients were classified as standard risk, 122 as high risk, and 101 as extremely high risk, according to age, peripheral-blood leukocyte count, selected genetic abnormalities, and immunophenotype. All subjects received four drugs for remission induction, followed by a risk-directed multidrug intensification phase and therapy for presymptomatic leukemia in the CNS. Maintenance chemotherapy with oral mercaptopurine and methotrexate was administered for 6 months, with all treatment stopped by 1 year after diagnosis. RESULTS The mean (+/- SD) event-free survival (EFS) and overall survival rates for all patients were 59.5% +/- 3.4% and 81.5% +/- 2.2%, respectively, at 5. 5 years after diagnosis. EFS rates by risk category were similar (60. 2% +/- 6.0% for standard risk, 57.7% +/- 5.6% for high risk, and 62. 5% +/- 5.7% for extremely high risk), whereas overall survival rates differed significantly (91.2% +/- 2.7%, 80.0% +/- 4.1%, and 72.1% +/- 4.5%, respectively, P <.0001 by the log-rank test). There were 107 relapses. Eighty-five (79.4%) of these 107 patients achieved second complete remissions, with subsequent EFS rates of 61.5% +/- 7. 9% (standard risk), 42.6% +/- 8.1% (high risk), and 9.6% +/- 6.4% (extremely high risk). Of the five risk factors analyzed, only the response to prednisolone monotherapy among extremely high-risk patients proved important. CONCLUSION Early treatment intensification did not compensate for a truncated phase of maintenance chemotherapy in children with standard- or high-risk ALL. However, 6 months of antimetabolite treatment seemed adequate for extremely high-risk patients who were good responders to prednisolone and received intensified chemotherapy that included high-dose cytarabine early in the clinical course.

Allelic loss of chromosome 13q14.3 in human oral cancer: Correlation with lymph node metastasis

To evaluate the role of chromosome 13 deletions in oral squamous cell carcinoma (SCC) progression and to define the precise localization of putative tumor suppressor genes, we studied tumors from 34 unrelated patients with oral SCC by the polymerase chain reaction (PCR)‐loss of heterozygosity (LOH) assay, using 18 different polymorphic loci. Chromosome 13q allelic losses (LOH) were observed in 67.6% at 1 or more loci. These results enabled the identification of a putative minimal region of deletion mapped at 13q14.3. The commonly deleted region is located close, but telomeric to the RB1 locus. We also examined the same samples for inactivation of the RB1 gene by immunohistochemical analysis of paraffin‐embedded samples, but no significant variation in RB protein expression was detected. In addition, we also performed PCR‐single‐strand conformational polymorphism (SSCP) analysis to detect any mutation of the RB1 gene using 52 primer pairs, which covers all exons of this gene. We found no mutations of the RB1 gene in our samples. Interestingly, we found significant correlation between LOH of 13q14.3 and lymph node metastasis. Our results indicate that LOH of 13q is a common event in oncogenesis and/or progression of oral SCC, and also suggest the existence of a new suppressor gene near D13S273‐D13S176 loci which may play a role in these events. Int. J. Cancer (Pred. Oncol.) 79:312–317, 1998.

Localization of a tumour-suppressor gene associated with human oral cancer on 7q31.1.

To search for the existence of a tumour‐suppressor gene (TSG) associated with oral squamous cell carcinoma (SCC), PCR analysis of microsatellite polymorphisms corresponding to 14 loci which map to chromosome 7q21.3‐qter was performed to screen 35 patients with oral SCC for loss of heterozygosity (LOH). LOH was observed in at least one of the loci in 19 of 34 (55.9%) informative cases. Among the loci tested, frequent LOH was restricted at D7S522 on chromosome 7q31.1, which was measured within 1 cM. Furthermore, we detected microsatellite instability (MI) in 11 of 35 (31.4%) cases tested. Our observations indicate that alterations of chromosome 7q are associated with oral SCC tumorigenesis and that 7q31.1 might harbour at least one putative TSG. Int. J. Cancer 75:671–674, 1998.© 1998 Wiley‐Liss, Inc.

Enhancement by interferon of natural cytotoxic activities of lymphocytes from human cord blood and peripheral blood of aged persons

Abstract Effects of interferon on natural (or “spontaneous”) cytotoxicity of lymphocytes from cord blood and peripheral blood of aged persons were tested in a chromium-release assay against RSb target cells. These natural cytotoxic activities were enhanced by leukocyte and fibroblast interferon as shown in adult lymphocytes.

Chimeric MLL products with a Ras binding cytoplasmic protein AF6 involved in t(6;11) (q27;q23) leukemia localize in the nucleus

In infantile leukemias and therapy-related leukemias, the MLL gene is frequently found to be disrupted and fused to various translocation partner genes, such as AF4/FEL, LTG9/AF9 and LTG19/ENL as a result of 11q23 translocations. We previously showed that the N-terminal portion common to various chimeric MLL products, as well as to MLL-LTG9 and MLL-LTG19, localizes in the nuclei, and therefore suggested that it might play an important role in leukemogenesis. In the present study, MLL-AF6 chimeric products found in the t(6;11) (q27;q23) translocation were analysed since AF6, a Ras-binding protein, exhibits a different subcellular localization from that of LTG9/AF9 and LTG19/ENL. Immunofluorescence staining data and cell fractionation analyses demonstrated that MLL-AF6 chimeric products localize in the nuclei despite the fact that AF6 itself localizes in the cytoplasm, confirming the importance of the nuclear localization of chimeric MLL products. The region in the N-terminal portion of MLL responsible for this nuclear localization was examined and found to be a region containing AT-hook motifs.

Interleukin 6, a possible autocrine growth and differentiation factor for the human megakaryocytic cell line, CMK

Summary. CMK is a human cell line derived from a megakaryoblastic leukaemia. It has characteristics of the megakaryocytic lineage, such as the presence of platelet peroxidase, membrane glycoproteins (GP)Ib and GPIIb/IIIa, α‐granules, and demarcation membranes. The cell line proliferates autonomously in serum‐containing medium. Here we report that the cell line expresses the gene for IL‐6 and releases small quantities of the cytokine into the medium. Addition of exogenous IL‐6 to cultures seeded into medium was found to promote growth of the cells. Conversely, addition of a neutralizing anti‐IL‐6 antibody inhibited cell growth. These data support the notion that autocrine IL‐6 is one of the factors accounting for autonomous growth of the cell line.

Ultrastructural and ultracytochemical differences between transient myeloproliferative disorder and megakaryoblastic leukaemia in Down's syndrome

Ultrastructural and ultracytochemical studies were performed on blast cells from 12 Downs syndrome neonates with transient myeloproliferative disorder (TMD) and 13 Downs syndrome patients with megakaryoblastic leukaemia (MKL), in order to clarify the cytological characteristics of these cells. Average platelet peroxidase‐positivity in blast cells of TMD patients was similar to that found in cases of MKL. Blast cells from subjects with TMD contained a number of different granules, namely, alpha granules, those that were myeloperoxidase (MPO)‐positive, electron‐lucent or basophil‐like, and those containing membrane components or ferritin particles. On the other hand, granules found in the blast cells of MKL patients with Downs syndrome included the electron‐lucent variety, those with membrane components and a few that were basophil‐like, but not alpha and MPO‐positive granules nor those containing ferritin particles. A demarcation membrane system was observed in blasts from the TMD group, but not in the MKL group.

Antithymocyte globulin and cyclosporine for treatment of 44 children with hepatitis associated aplastic anemia.

We analyzed the outcomes of 44 children with hepatitis associated aplastic anemia (HAA) who received immunosuppressive therapy (IST) with antithymocyte globulin (ATG) and cyclosporine (CsA). Fourteen (31.8%) patients achieved complete response and 17 (38.6%) achieved partial response, for an overall response rate of 70.4% after 6 months. Seven non-responders received bone marrow transplantation from an HLA-matched unrelated donor and 6 out of 7 are alive. The probability of overall survival at 10 years was 88.3±4.9%, which supports the role of IST with ATG and CsA as treatment of choice for children with HAA without an HLA identical sibling donor.

The neuroprotective effect of a novel calmodulin antagonist, 3-[2-[4-(3-chloro-2-methylphenyl)-1-piperazinyl]ethyl]-5,6-dimethoxy-1-(4-imidazolylmethyl)-1H-indazole dihydrochloride 3.5 hydrate, in transient forebrain ischemia.

A novel calmodulin (CaM) antagonist DY-9760e, (3-[2-[4-(3-chloro-2-methylphenyl)-1-piperazinyl]ethyl]-5,6-dimethoxy-1-(4-imidazolylmethyl)-1H-indazole dihydrochloride 3.5 hydrate), with an apparent neuroprotective effect in vivo, potently inhibits CaM-dependent nitric oxide synthase in situ. In the present study, we determined whether DY-9760e inhibits nitric oxide (NO) production and protein nitration by peroxynitrite (ONOO(-)) formation in the hippocampal CA1 region of gerbils after transient forebrain ischemia. In freely moving gerbils, NO production after 10-minute forebrain ischemia was monitored consecutively with in vivo brain microdialysis. Pretreatment with DY-9760e (50 mg/kg i.p.) significantly decreased the increased levels of NO(x)(-) (NO metabolites, NO(2)(-) plus NO(3)(-)) immediately after, 24 h after cerebral ischemia-reperfusion to the control levels of sham-operated animals. Western blot and immunohistochemical analyses using an anti-nitrotyrosine antibody as a marker of ONOO(-) formation indicated a marked increase in nitrotyrosine immunoreactivity in the pyramidal neurons of the CA1 region 2 h after reperfusion, and DY-9760e significantly inhibited increased nitrotyrosine immunoreactivity. Coincident with the inhibition of the NO production and protein tyrosine nitration, pretreatment with DY-9760e rescued the delayed neuronal death in the hippocampal CA1 region. These results suggest that the inhibitory effects of DY-9760e on the NO-ONOO(-) pathway partly account for its neuroprotective effects in cerebral ischemia.