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Yuko Ozaki,
Hirotaka Matsui, Hiroya Asou,
Akiko Nagamachi,
Daisuke Aki,
Hiroaki Honda,
Shin'ichiro Yasunaga,
Yoshihiro Takihara,
Tadashi Yamamoto,
Shunsuke Izumi,
Miho Ohsugi,
Toshiya Inaba
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ABSTRACT: During prometaphase, dense microtubule nucleation sites at centrosomes form robust spindles that align chromosomes promptly. Failure of centrosome maturation leaves chromosomes scattered, as seen routinely in cancer cells, including myelodysplastic syndrome (MDS). We previously reported that the Miki (LOC253012) gene is frequently deleted in MDS patients, and that low levels of Miki are associated with abnormal mitosis. Here we demonstrate that Miki localizes to the Golgi apparatus and is poly(ADP-ribosyl)ated by tankyrase-1 during late G2 and prophase. PARsylated Miki then translocates to mitotic centrosomes and anchors CG-NAP, a large scaffold protein of the γ-tubulin ring complex. Due to impairment of microtubule aster formation, cells in which tankyrase-1, Miki, or CG-NAP expression is downregulated all show prometaphase disturbances, including scattered and lagging chromosomes. Our data suggest that PARsylation of Miki by tankyrase-1 is a key initial event promoting prometaphase.
Molecular cell 08/2012; 47(5):694-706. · 14.61 Impact Factor
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ABSTRACT: The dynactin complex is required for activation of the dynein motor complex, which plays a critical role in various cell functions including mitosis. During metaphase, the dynein-dynactin complex removes spindle checkpoint proteins from kinetochores to facilitate the transition to anaphase. Three components (p150(Glued), dynamitin, and p24) compose a key portion of the dynactin complex, termed the projecting arm. To investigate the roles of the dynactin complex in mitosis, we used RNA interference to down-regulate p24 and p150(Glued) in human cells. In response to p24 down-regulation, we observed cells with delayed metaphase in which chromosomes frequently align abnormally to resemble a "figure eight," resulting in cell death. We attribute the figure eight chromosome alignment to impaired metaphasic centrosomes that lack spindle tension. Like p24, RNA interference of p150(Glued) also induces prometaphase and metaphase delays; however, most of these cells eventually enter anaphase and complete mitosis. Our findings suggest that although both p24 and p150(Glued) components of the dynactin complex contribute to mitotic progression, p24 also appears to play a role in metaphase centrosome integrity, helping to ensure the transition to anaphase.
Journal of Biological Chemistry 02/2011; 286(7):5589-98. · 4.77 Impact Factor
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ABSTRACT: A 28-year-old man with marked eosinophilia is described. FIP1L1/PDGFRA mRNA showed multiple alternatively-spliced fusion transcripts. Sequencing analysis showed that the deduced DNA breakpoints were intron 10 in the FIP1L1 gene and exon 12 in the PDGFRA gene. Then, a diagnosis of chronic eosinophilic leukemia (CEL) was made. Whereas the response to the treatments with prednisolone and hydroxyurea were unsatisfactory, treatment with imatinib showed a rapid decrease of eosinophils. The hemoglobin level also dropped and bone marrow examination showed pure red cell aplasia. Continued administration of very low dose imatinib (100 mg every 5 days) led to and maintained complete molecular remission, with good tolerability.
Internal Medicine 01/2010; 49(12):1195-200. · 0.94 Impact Factor
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ABSTRACT: Monosomy 7 and interstitial deletions in the long arm of chromosome 7 (-7/7q-) is a common nonrandom chromosomal abnormality found frequently in myeloid disorders including acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), and juvenile myelomonocytic leukemia (JMML). Using a short probe-based microarray comparative genomic hybridization (mCGH) technology, we identified a common microdeletion cluster in 7q21.3 subband, which is adjacent to 'hot deletion region' thus far identified by conventional methods. This common microdeletion cluster contains three poorly characterized genes; Samd9, Samd9L, and a putative gene LOC253012, which we named Miki. Gene copy number assessment of three genes by real-time PCR revealed heterozygous deletion of these three genes in adult patients with AML and MDS at high frequency, in addition to JMML patients. Miki locates to mitotic spindles and centrosomes and downregulation of Miki by RNA interference induced abnormalities in mitosis and nuclear morphology, similar to myelodysplasia. In addition, a recent report indicated Samd9 as a tumor suppressor. These findings indicate the usefulness of the short probe-based CGH to detect microdeletions. The three genes located to 7q21.3 would be candidates for myeloid tumor-suppressor genes on 7q.
Biochemical and Biophysical Research Communications 05/2009; 383(2):245-51. · 2.48 Impact Factor
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ABSTRACT: Clinical, cytogenetic, and molecular genetic studies were performed to clarify the pathophysiology of Japanese B cell chronic lymphocytic leukaemia (B-CLL), since the incidence of B-CLL in Japan is significantly lower than in western countries. The clinical and laboratory features of 55 Japanese patients with B-CLL in this study did not differ from those of Americans or Europeans with B-CLL. In the chromosome analyses, suitable metaphases with good band quality were obtained from 48 patients (87.2%), of whom 22 patients (45.8%) showed clonal chromosome aberrations and 14 (29.2%) had non-clonal aberrations. Trisomy 12 and abnormalities of 14q and 13q were found in four (18.2%), two (9.1%) and six patients (27.2%). respectively. There were no particular chromosome abnormalities or specific breakpoints in Japanese B-CLL. However, complex karyotype was found in higher incidence than in western countries. In the Southern blot analyses, rearranged band patterns were observed in the major breakpoint region (mbr) of the bcl-2 gene in one case, in the 5′-breakpoint region (5′-bcl-2) in two, and bcl-3 in one. Of the two patients with 5′-bcl-2 rearrangements, one had a normal karyotype and the other had t(2:18)(p12:q21). The incidence of rearrangements of the bcl-1, bcl-2 and bcl-3 genes in Japanese B-CLL was similar to that in western countries. These findings suggest that the biological characteristics of B-CLL in Japan are almost the same as those in western countries, although the incidence of B-CLL in Japan is quite different: this may be related to racial differences, which seem to be an important factor in the development of B-CLL.
British Journal of Haematology 03/2008; 85(3):492 - 497. · 4.94 Impact Factor
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ABSTRACT: Three cases of acute leukaemia with t(4;12) (qll-12;pl3) karyotypic abnormalities were analysed. They had the following common clinical and biological characteristics: (1) dysplasia of three haemopoietic lineages; (2) absent or low myeloperoxidase activity; and (3) retention of platelets in the peripheral blood and megakaryocytes in the bone marrow. There were increased numbers of basophils in the bone marrow and peripheral blood in two of the cases. In all, the blast cells displayed the unique immunophenotype CD7+CD13+CD34+HLA-DR+. The blasts analysed in one case expressed c-kit on the membrane surface. These findings suggest that the t(4:12) (qll-12:pl3) abnormality is associated with a particular type of acute leukaemia, one in which the morphology and immunophenotype suggest that the translocation may have occurred at an early stage of haemopoiesis.
British Journal of Haematology 03/2008; 90(4):850 - 854. · 4.94 Impact Factor
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ABSTRACT: Previous gene-targeting studies indicated that Bim, a BH3-only death activator, regulates total blood cell number. Cytokines contribute to this process by negatively regulating steady-state levels of Bim mRNA. Here we present a molecular mechanism for cytokine-mediated posttranscriptional regulation of Bim mRNA by heat-shock cognate protein 70 (Hsc70), which binds to AU-rich elements (AREs) in the 3'-untranslated region of specific mRNAs and enhances their stability. The RNA binding potential of Hsc70 is regulated by cochaperones including Bag-4 (also SODD), CHIP, Hip, and Hsp40. Cytokines regulate the expression or function of these cochaperones by activating Ras pathways. Thus, exposure of cells to cytokines ultimately leads to destabilization of Bim mRNA and promotion of cell survival. This unanticipated role of a chaperone/cochaperone complex in mRNA stability appears to be critical for hematopoiesis and leukemogenesis.
Molecular Cell 02/2007; 25(1):99-112. · 14.18 Impact Factor
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ABSTRACT: PC-SPES is an eight herbal mixture which has been shown to be active against prostate cancer cells in vitro as well as in patients. In this study, we discovered that it has anti-leukemia activity. HL-60, NB4, U937 and THP-1 human acute myeloid leukemia cells were cultured in the presence of various concentrations of PC-SPES (0.06-0.5 micro l/ml) for 4 days, and cell numbers were counted by Trypan blue exclusion. PC-SPES inhibited proliferation of these cells with an ED50 of 0.17, 0.09, 0.18, 0.32 micro l/ml, respectively. In clonogenic assay, PC-SPES inhibited growth of HL-60 cells (ED50, 0.043 micro l/ml). On the other hand, PC-SPES (0.1 micro l/ml) stimulated growth of normal myeloid committed stem cells (CFU-GM) by 1.4-fold of control (p=0.03). Anti-leukemia effects also occurred against freshly isolated leukemia cells from acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) patients. Interestingly, when PC-SPES was combined with ATRA, the antiproliferative effect was markedly enhanced. For example, PC-SPES (0.125 micro l/ml) or ATRA (10(-8) mol/l) inhibited growth of HL-60 cells after 4 days of culture, by approximately 40 and 30%, respectively; simultaneous treatment with both, suppressed growth by 80%. In addition, PC-SPES induced differentiation of HL-60 and NB4 cells, as measured by expression of CD11b and reduction of NBT. ATRA synergistically enhanced this activity. For example, either PC-SPES (0.5 micro l/ml) or ATRA (10(-8) mol/l) induced 23 and 18% of HL-60 cells, respectively to express CD11b on day 2 of culture; and when both were combined, 60% of HL-60 cells were stimulated to express CD11b antigen. Furthermore, PC-SPES (0.5 micro l/ml) produced apoptosis of HL-60 and NB4 cells, as measured by TUNEL assay, with 17% of HL-60 cells and 52% of NB4 cells becoming apoptotic on their third day of culture. Importantly, PC-SPES stimulated expression of the novel myeloid specific transcription factor C/EBPepsilon in HL-60 and NB4 cells. Taken together, PC-SPES inhibits growth and induces differentiation and apoptosis of myeloid leukemia cells, and enhances the antiproliferative and prodifferentiative effects of ATRA on these cells. PC-SPES might be useful with ATRA for treatment of patients with acute promyelocytic leukemia (APL), and it could have a role in other types of cancers including MDS.
International Journal of Oncology 11/2003; 23(4):1203-11. · 2.40 Impact Factor
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ABSTRACT: A myeloid leukemia cell line designated Kasumi-6 was established from the bone marrow cells of an individual with acute myeloid leukemia, subtype M2. Both the original leukemic cells and the Kasumi-6 cell line harbor a hemizygous point mutation in the gene encoding the CCAAT/enhancer binding protein alpha (C/EBPalpha), a critical myeloid transcriptional factor. The C to G transition at nucleotide 1063 of the C/EBPalpha gene results in amino acid transition R305P in the fork or hinge region between the DNA-binding basic region and the leucine zipper dimerization domain of the C/EBPalpha protein. The Kasumi-6 cells expressed both the p42 and p30 isoforms of the C/EBPalpha protein endogenously, but electrophoretic mobility shift assays demonstrated an absence of C/EBPalpha binding to its respective site. Exogenous expression of the mutant form of C/EBPalpha demonstrated that it was unable to bind DNA and activate transcription from a G-CSF receptor-luciferase reporter construct. Furthermore, coexpression of the wild-type and mutant forms revealed that the mutant form repressed reporter gene activation by the wild type in a dose-responsive manner. This was concomitant with a dose-responsive decrease in wild-type protein binding to the G-CSF receptor C/EBP site. The data suggest that the R305P alteration confers a dominant-negative property on the mutant C/EBPalpha protein whereby the mutant polypeptide heterodimerizes with the wild-type polypeptide and prevents it from binding to DNA, thus blocking transcriptional activation. The Kasumi-6 cell line can serve as a model to study the cellular and molecular biology of the non-t(8;21) M2 type of myeloid leukemia and can elucidate the role of mutated C/EBPalpha in leukemogenesis.
Genes Chromosomes and Cancer 03/2003; 36(2):167-74. · 3.31 Impact Factor
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ABSTRACT: A natural product, resveratrol (3,4,40-trihydroxy-trans-stilbene), a phytoalexin found in grapes and other food products, is known as a cancer chemopreventive agent. We studied the in vitro biological activity of this compound by examining its effect on proliferation and differentiation in myeloid leukemia cell lines (HL-60, NB4, U937,THP-1, ML-1, Kasumi-1) and fresh samples from 17 patients with acute myeloid leukemia. Resveratrol (20 microM, 4 days) alone inhibited the growth in liquid culture of each of the 6 cell lines. Resveratrol (10 microM) enhanced the expression of adhesion molecules (CD11a, CD11b, CD18, CD54) in each of the cell lines except for Kasumi-1. Moreover, resveratrol (25 microM, 4 days) induced 37% of U937 cells to produce superoxide as measured by the ability to reduce nitroblue tetrazolium (NBT). The combination of resveratrol (10 microM) and all-trans-retinoic acid (ATRA) (50 nM, 4 days) induced 95% of the NB4 cells to become NBT-positive, whereas <1% and 12% of the cells became positive for NBT after a similar exposure to either resveratrol or ATRA alone, respectively. In U937 cells exposed to resveratrol (25 microM, 3 days), the binding activity of nuclear factor-kappaB (NFkappaB) protein was suppressed. Eight of 19 samples of fresh acute leukemia cells reduced NBT after exposure to resveratrol (20 microM, 4 days). Taken together, these findings show that resveratrol inhibits proliferation and induces differentiation of myeloid leukemia cells.
International Journal of Hematology 06/2002; 75(5):528-33. · 1.27 Impact Factor
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Adrian F Gombart,
Wolf-K Hofmann,
Seiji Kawano,
Seisho Takeuchi,
Utz Krug,
Scott H Kwok,
Renee J Larsen, Hiroya Asou,
Carl W Miller,
Dieter Hoelzer,
H Phillip Koeffler
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ABSTRACT: The CCAAT/enhancer binding protein alpha (C/EBPalpha) protein is essential for proper lung and liver function and granulocytic and adipose tissue differentation. It was hypothesized that abnormalties in C/EBPalpha function contribute to the development of malignancies in a variety of tissues. To test this, genomic DNA from 408 patient samples and 5 cell lines representing 11 different cancers was screened for mutations in the C/EBPalpha gene. Two silent polymorphisms termed P1 and P2 were present at frequencies of 13.5% and 2.2%, respectively. Of the 12 mutations detected in 10 patients, silent changes were identified in one nonsmall cell lung cancer, one prostate cancer, and one acute myelogenous leukemia (AML) subtype M4. The 9 remaining mutations were detected in 1 of 92 (1.1%) myelodysplastic syndrome (MDS) samples and 6 of 78 (7.7%) AML (AML-M2 and AML-M4) samples. Some mutations truncated the predicted protein with loss of the DNA-binding (basic region) and dimerization (leucine zipper [ZIP]) domains by either deletions or nonsense codons. Also, inframe deletions or insertions in the fork region located between the leucine zipper and basic region, or within the leucine zipper, disrupted the alpha-helical phase of the bZIP domain. The inframe deletion and insertion mutations abrogated the transcriptional activation function of C/EBPalpha on the granulocyte colony-stimulating factor receptor promoter. These mutants localized properly to the nucleus, but were unable to bind to the C/EBP site in the promoter and did not possess dominant-negative activity. The mutations in the MDS patient and one AML-M2 patient were biallelic, indicating a loss of C/EBPalpha function. These results suggest that mutation of C/EBPalpha is involved in specific subtypes of AML and in MDS, but may occur rarely in other types of leukemias or nonhematologic malignancies.
Blood 03/2002; 99(4):1332-40. · 9.90 Impact Factor
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ABSTRACT: Induction of differentiation and apoptosis in cancer cells through ligands of nuclear hormone receptors (NHRs) is a novel
and promising approach to cancer therapy. All-trans-retinoic acid (ATRA), an RA receptor-specific NHR ligand, is now used for selective cancers. The NHR, peroxisome proliferator-activated
receptor γ (PPARγ) is expressed in breast cancer cells. Activation of PPARγ through a synthetic ligand, troglitazone (TGZ),
and other PPARγ-activators cause inhibition of proliferation and lipid accumulation in cultured breast cancer cells. TGZ (10−5 M, 4 days) reversibly inhibits clonal growth of MCF7 breast cancer cells and the combination of TGZ (10−5 M) and ATRA (10−6 M, 4 days) synergistically and irreversibly inhibits growth and induces apoptosis of MCF7 cells, associated with a dramatic
decrease of their bcl-2 protein levels. Similar effects are noted with in vitro cultured breast cancer tissues from patients, but not with normal breast epithelial cells. The observed apoptosis mediated
by TGZ and ATRA may be related to the striking down-regulation of bcl-2, because forced over-expression of bcl-2 in MCF7 cells
cultured with TGZ and ATRA blocks their cell death. TGZ significantly inhibits MCF7 tumor growth in triple immunodeficient
mice. Combined administration of TGZ and ATRA causes prominent apoptosis and fibrosis of these tumors without toxic effects
on the mice. Taken together, this combination may provide a novel, nontoxic and selective therapy for human breast cancers.
Proceedings of the National Academy of Sciences 07/1998; 95(15):8806-8811. · 9.68 Impact Factor
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ABSTRACT: Fifty patients [25 acute myeloid leukemia (AML) and 25 myelodysplastic syndrome (MDS)], without monosomy 7 according to conventional cytogenetics, were re-examined by fluorescence in situ hybridization (FISH). Eleven (44.0%) patients with AML and nine (36.0%) with MDS showed hidden monosomy 7. Two samples who had both monosomy 7 and iso chromosome 17 were analyzed by dual color FISH to identify their clonal origin, and showed that these two abnormalities can occur together or independently. Only one of 16 MDS patients without monosomy 7 transformed into AML whereas four of eight MDS patients with the hidden monosomy 7 transformed into AML, suggesting patients with this abnormality are more likely to undergo transformation to AML.
Leukemia Research 10/1996; · 2.92 Impact Factor
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ABSTRACT: A novel human leukaemia cell line (Kasumi-4) was established from the peripheral blood of a 6-year-old girl suffering from chronic myelogenous leukaemia (CML) in blast crisis. The Kasumi-4 cells had the following characteristic features: undifferentiated blasts which were positive for CD34, CD33 and CD13 surface markers, but negative for myeloperoxidase platelet peroxidase, CD36, CD41 and CD42; chromosome abnormalities of t(9;22;11) (q34;q11;q13), inv(3)(q21q26); and elevated expression of EVI1 gene which is located at chromosome band 3q26. Megakaryocytic maturation was not observed in the liquid culture following the addition of TPA, IL-3, IL-6 or GM-CSF. b2-a2 type of BCR-ABL chimaeric messenger RNA was detected by RT-PCR analysis. This is the first leukaemia cell line with a three-way translocation containing the Ph chromosome and the second cell line with an inv(3)(q21q26). This cell line appears to be useful for studying the mechanisms of leukaemogenesis involving these chromosomal abnormalities and related oncogenes.
British Journal of Haematology 03/1996; 93(1):68 - 74. · 4.94 Impact Factor
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ABSTRACT: A novel human leukemia cell line (Kasumi-3) was established from the blast cells of a 57-year-old man suffering from myeloperoxidase-negative acute leukemia. The cell line had five distinctive features, as follows. 1) Flow cytometric analyses showed cell surface expression of CD7, CD4, CD13, CD33, CD34, HLA-DR and c-Kit. This phenotype is compatible with that of acute myelocytic leukemia cells with the M0 subtype in the French-American-British classification. 2) Kasumi-3 cells carried chromosomal abnormalities of t(3;7)(q27:q22), del(5)(q15), del(9)(q32), and add(12)(pll). The breakpoint of 3q27 was located near the EVI1 gene, and a high level of expression of the EVI1 gene was observed. 4) Kasumi-3 cells treated with TPA showed maturation to monocytic lineage. 5) Treatment with either interleukin (IL)-2, IL-3, IL-4, granulocyte-macrophage colony-stimulating or stem cell factor induced the proliferation of Kasnmi-3 cells. Thus, the Kasumi-3 cell line shows the characteristic features of undifferentiated leukemia. It should, therefore, be useful both for studying the biological characteristics of acute myelogenous leukemia M0 subtype and for investigating the role of the EVI1 gene in leukemogenesis.
Cancer Science 02/1996; 87(3):269 - 274. · 3.33 Impact Factor
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ABSTRACT: Serial cytogenetic analysis revealed karyotypically unrelated clones in four patients with acute myeloblastic leukaemia (AML) in remission. At diagnosis, three patients had t(8;21)(q22;q22) and one had an inv(16)(p13q22). After 18-22 months in remission, different clones emerged in each patient with myelodysplastic features of the bone marrow cells. The emergence of clones with abnormalities of chromosome 7 in remission seems to be an unfavourable factor for prognosis.
British Journal of Haematology 02/1995; 89(3):531 - 534. · 4.94 Impact Factor
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Hiroya Asou,
Hirotaka Matsui,
Yuko Ozaki,
Akiko Nagamachi,
Megumi Nakamura,
Daisuke Aki,
Toshiya Inaba,
ヒロヤ アソウ,
Hiroya Asō,
ヒロタカ マツイ, [......],
メグミ ナカムラ,
ダイスケ アキ,
トシヤ イナバ,
博也 麻生,
啓隆 松井,
佑子 尾崎,
安希子 長町,
めぐみ 中村,
大輔 安藝,
俊哉 稲葉
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ABSTRACT: Monosomy 7 and interstitial deletions in the long arm of chromosome 7 (-7/7q-) is a common non-random chromosomal abnormality found frequently in rnyeloid disorders including acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), and juvenile myelomonocytic leukemia (JMML). Using a short probe-based microarray comparative genomic hybridization (mCGH) technology, we identified a common microdeletion Cluster in 7q21.3 subband, which is adjacent to 'hot deletion region' thus far identified by conventional methods. This common microdeletion cluster contains three poorly characterized genes; Samd9, Samd9L, and a putative gene LOC253012, which we named Miki. Gene copy number assessment of three genes by real-time PCR revealed heterozygous deletion of these three genes in adult patients with AML and MDS at high frequency, in addition to JMML patients. Miki locates to mitotic spindles and centrosomes and downregulation of Miki by RNA interference induced abnormalities in mitosis and nuclear morphology, similar to myelodysplasia. In addition, a recent report indicated Samd9 as a tumor suppressor. These findings indicate the usefulness of the short probe-based CGH to detect microdeletions. The three genes located to 7q21.3 would be candidates for myeloid tumor-suppressor genes on 7q.
