Hiroya Asou

Haploinsufficiency of SAMD9L, an endosome fusion facilitator, causes myeloid malignancies in mice mimicking human diseases with monosomy 7.

Monosomy 7 and interstitial deletion of 7q (-7/7q-) are well-recognized nonrandom chromosomal abnormalities frequently found among patients with myelodysplastic syndromes (MDSs) and myeloid leukemias. We previously identified candidate myeloid tumor suppressor genes (SAMD9, SAMD9-like = SAMD9L, and Miki) in the 7q21.3 subband. We established SAMD9L-deficient mice and found that SAMD9L(+/-) mice as well as SAMD9L(-/-) mice develop myeloid diseases resembling human diseases associated with -7/7q-. SAMD9L-deficient hematopoietic stem cells showed enhanced colony formation potential and in vivo reconstitution ability. SAMD9L localizes in early endosomes. SAMD9L-deficient cells showed delays in homotypic endosome fusion, resulting in persistence of ligand-bound cytokine receptors. These findings suggest that haploinsufficiency of SAMD9L and/or SAMD9 gene(s) contributes to myeloid transformation.

Poly-ADP Ribosylation of Miki by tankyrase-1 Promotes Centrosome Maturation

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.

Resveratrol, a natural product derived from grapes, is a new inducer of differentiation in human myeloid leukemias.

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 μM, 4 days) alone inhibited the growth in liquid culture of each of the 6 cell lines. Resveratrol (10 μM) enhanced the expression of adhesion molecules (CD11a, CD11b, CD18, CD54) in each of the cell lines except for Kasumi-1. Moreover, resveratrol (25 μM, 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 μM) 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 μM, 3 days), the binding activity of nuclear factor-κB (NFκB) protein was suppressed. Eight of 19 samples of fresh acute leukemia cells reduced NBT after exposure to resveratrol (20 μM, 4 days). Taken together, these findings show that resveratrol inhibits proliferation and induces differentiation of myeloid leukemia cells.

Japanese B cell chronic lymphocytic leukaemia: a cytogenetic and molecular biological study

Summary. 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.

Hidden monosomy 7 in acute myeloid leukemia and myelodysplastic syndrome detected by interphase fluorescence in situ hybridization

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.

Establishment of the acute myeloid leukemia cell line Kasumi‐6 from a patient with a dominant‐negative mutation in the DNA‐binding region of the C/EBPα gene

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/EBPα), a critical myeloid transcriptional factor. The C to G transition at nucleotide 1063 of the C/EBPα 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/EBPα protein. The Kasumi‐6 cells expressed both the p42 and p30 isoforms of the C/EBPα protein endogenously, but electrophoretic mobility shift assays demonstrated an absence of C/EBPα binding to its respective site. Exogenous expression of the mutant form of C/EBPα 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/EBPα 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/EBPα in leukemogenesis.

Establishment of an Undifferentiated Leukemia Cell Line (Kasumi-3) with t(3;7)(q27;q22) and Activation of the EVI1 Gene

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.

Antiproliferative and proapoptotic activity of GUT-70 mediated through potent inhibition of Hsp90 in mantle cell lymphoma

Background:Mantle cell lymphoma (MCL) is an aggressive B-cell lymphoma with poor prognosis, requiring novel anticancer strategies.Methods:Mantle cell lymphoma cell lines with known p53 status were treated with GUT-70, a tricyclic coumarin derived from Calophyllum brasiliense, and the biological and biochemical consequences of GUT-70 were studied.Results:GUT-70 markedly reduced cell proliferation/viability through G1 cell cycle arrest and increased apoptosis, with greater sensitivity in mutant (mt)-p53-expressing MCL cells than in wild-type (wt)-p53-bearing cells. Mechanistically, GUT-70 showed binding affinity to heat-shock protein 90 (Hsp90) and ubiquitin-dependent proteasomal degradation of Hsp90 client proteins, including cyclin D1, Raf-1, Akt, and mt-p53. Depletion of constitutively overexpressed cyclin D1 by GUT-70 was accompanied by p27 accumulation and decreased Rb phosphorylation. GUT-70 induced mitochondrial apoptosis with Noxa upregulation and Mcl-1 downregulation in mt-p53 cells, but Mcl-1 accumulation in wt-p53 cells. Noxa and Mcl-1 were coimmunoprecipitated, and activated BAK. Treatment with a combination of GUT-70 and bortezomib or doxorubicin had synergistic antiproliferative effects in MCL cells that were independent of p53 status.Conclusion:GUT-70 has pronounced antiproliferative effects in MCL with mt-p53, a known negative prognostic factor for MCL, through Hsp90 inhibition. These findings suggest that GUT-70 has potential utility for the treatment of MCL.

A specific chromosome abnormality of t(4;12)(qll‐12;pl3) in CD7+ acute leukaemia

Summary. 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.

Establishment of a myeloid leukaemia cell line (Kasumi-4) with t(9;22;11)(q34;q11;q13), inv(3)(q21q26) and the EVI1 gene activation from a patient with chronic myelogenous leukaemia in blast crisis

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.