Akira Tanimura

NAD-dependent histone deacetylase, SIRT1, plays essential roles in the maintenance of hematopoietic stem cells.

Sir2 has been shown to be essential for transcriptional silencing and longevity provided by calorie restriction in Saccharomyces cerevisiae and Caenorhabditis elegans. In this study, we investigated the role for its mammalian homologue, SIRT1, in hematopoietic cells. SIRT1 inhibitor, nicotinamide (NA), promoted and its activator, resveratrol, inhibited the differentiation of murine bone marrow c-Kit(high)Sca-1(+)Lineage(-) (KSL) cells during the culture system ex vivo. To further clarify the roles of SIRT1 in hematopoietic cells, we isolated KSL cells from fetal liver of SIRT1 knockout (KO) mice and cultured them for 5days, because SIRT1 KO mice die shortly after the delivery. In agreement with the results from the experiments using NA and resveratrol, KSL cells isolated from SIRT1 KO mice more apparently differentiated and lost the KSL phenotype than those from wild-type (WT) mice. Furthermore, in each of colony assay, replating assay, or serial transplantation assay, SIRT1 KO KSL cells lost earlier the characteristics of stem cells than WT KSL cells. In addition, we found that SIRT1 maintains prematurity of hematopoietic cells through ROS elimination, FOXO activation, and p53 inhibition. These results suggest that SIRT1 suppresses differentiation of hematopoietic stem/progenitor cells and contributes to the maintenance of stem cell pool.

Fulminant septicemia of Bacillus cereus resistant to carbapenem in a patient with biphenotypic acute leukemia

We report a case of fulminant septicemia with Bacillus cereus resistant to carbapenem. A 33-year-old man was suffering from febrile neutropenia (FN) on day 15 after the start of remission-induction therapy for biphenotypic acute leukemia under gut decontamination with polymyxin B and nystatin. Meropenem, a carbapenem, was administered according to the guideline for FN. Two days later (on day 17), he complained of severe abdominal pain, lost consciousness, went into sudden cardiopulmonary arrest, and died. Autopsy showed multiple spots of hemorrhage and necrosis caused by bacterial plaque in the brain, lungs, and liver. B. cereus was isolated from a blood sample obtained in the morning on day 17 and it was after his death that the isolated B. cereus was revealed to be resistant to carbapenem. B. cereus obtained from blood samples has been reported to be usually sensitive to carbapenem and also to vancomycin, new quinolones, and clindamycin. If B. cereus resistant to carbapem increases, our method of gut decontamination with polymyxin B and nystatin may have to be changed to one containing a new quinolone for the prevention of septicemia. Careful watching to determine whether B. cereus resistant to carbapem increases may be also important for empiric therapy, because carbapenem is often selected as the initial therapy for FN in patients with severe neutropenia.

The Clathrin Assembly Protein PICALM Is Required for Erythroid Maturation and Transferrin Internalization in Mice

Phosphatidylinositol binding clathrin assembly protein (PICALM), also known as clathrin assembly lymphoid myeloid leukemia protein (CALM), was originally isolated as part of the fusion gene CALM/AF10, which results from the chromosomal translocation t(10;11)(p13;q14). CALM is sufficient to drive clathrin assembly in vitro on lipid monolayers and regulates clathrin-coated budding and the size and shape of the vesicles at the plasma membrane. However, the physiological role of CALM has yet to be elucidated. Here, the role of CALM in vivo was investigated using CALM-deficient mice. CALM-deficient mice exhibited retarded growth in utero and were dwarfed throughout their shortened life-spans. Moreover, CALM-deficient mice suffered from severe anemia, and the maturation and iron content in erythroid precursors were severely impaired. CALM-deficient erythroid cells and embryonic fibroblasts exhibited impaired clathrin-mediated endocytosis of transferrin. These results indicate that CALM is required for erythroid maturation and transferrin internalization in mice.

PICOT is a molecule which binds to anamorsin.

Anamorsin (AM) (also called CIAPIN-1) is a cell-death-defying factor. AM deficient mice die during late gestation; AM deficient embryos are anemic and very small compared to wild type (WT) embryos. It is thought that AM plays crucial roles in hematopoiesis and embryogenesis. To clarify the mechanisms of AM functions, we performed the yeast-two-hybrid assay to identify AM-interacting molecules; we found that PICOT (PKCθ interacting cousin of thioredoxin) preferentially bound to AM. We also showed that the N-terminal regions of both AM and PICOT were essential for their bindings and the inhibition of interaction of both molecules might lead to the cell growth retardation. Both PICOT and the yeast homolog of AM are known to be iron-sulfur proteins. The phenotype of PICOT deficient mice is very similar to that of anamorsin deficient mice; both mice are embryonic lethal. These data suggest that AM and PICOT might play cooperatively essential roles in embryogenesis as iron-sulfur cluster proteins.

Clathrin Assembly Protein CALM Plays a Critical Role in KIT Signaling by Regulating Its Cellular Transport from Early to Late Endosomes in Hematopoietic Cells

CALM is implicated in the formation of clathrin-coated vesicles, which mediate endocytosis and intracellular trafficking of growth factor receptors and nutrients. We previously found that CALM-deficient mice suffer from severe anemia due to the impaired clathrin-mediated endocytosis of transferrin receptor in immature erythroblast. However, CALM has been supposed to regulate the growth and survival of hematopoietic stem/progenitor cells. So, in this study, we focused on the function of CALM in these cells. We here show that the number of Linage−Sca-1+KIT+ (LSK) cells decreased in the fetal liver of CALM −/− mice. Also, colony forming activity was impaired in CALM−/− LSK cells. In addition, SCF, FLT3, and TPO-dependent growth was severely impaired in CALM−/− LSK cells, while they can normally proliferate in response to IL-3 and IL-6. We also examined the intracellular trafficking of KIT using CALM −/− murine embryonic fibroblasts (MEFs) engineered to express KIT. At first, we confirmed that endocytosis of SCF-bound KIT was not impaired in CALM −/− MEFs by the internalization assay. However, SCF-induced KIT trafficking from early to late endosome was severely impaired in CALM −/− MEFs. As a result, although intracellular KIT disappeared 30 min after SCF stimulation in wild-type (WT) MEFs, it was retained in CALM −/− MEFs. Furthermore, SCF-induced phosphorylation of cytosolic KIT was enhanced and prolonged in CALM −/− MEFs compared with that in WT MEFs, leading to the excessive activation of Akt. Similar hyperactivation of Akt was observed in CALM −/− KIT+ cells. These results indicate that CALM is essential for the intracellular trafficking of KIT and its normal functions. Also, our data demonstrate that KIT located in the early endosome can activate downstream molecules as a signaling endosome. Because KIT activation is involved in the pathogenesis of some malignancies, the manipulation of CALM function would be an attractive therapeutic strategy.

BCR-ABL but Not JAK2 V617F Inhibits Erythropoiesis through the Ras Signal by Inducing p21CIP1/WAF1

BCR-ABL is a causative tyrosine kinase (TK) of chronic myelogenous leukemia (CML). In CML patients, although myeloid cells are remarkably proliferating, erythroid cells are rather decreased and anemia is commonly observed. This phenotype is quite different from that observed in polycythemia vera (PV) caused by JAK2 V617F, whereas both oncogenic TKs activate common downstream molecules at the level of hematopoietic stem cells (HSCs). To clarify this mechanism, we investigated the effects of BCR-ABL and JAK2 V617F on erythropoiesis. Enforced expression of BCR-ABL but not of JAK2 V617F in murine LSK (Lineage−Sca-1hiCD117hi) cells inhibited the development of erythroid cells. Among several signaling molecules downstream of BCR-ABL, an active mutant of N-Ras (N-RasE12) but not of STAT5 or phosphatidylinositol 3-kinase (PI3-K) inhibited erythropoiesis, while N-RasE12 enhanced the development of myeloid cells. BCR-ABL activated Ras signal more intensely than JAK2 V617F, and inhibition of Ras by manumycin A, a farnesyltransferase inhibitor, ameliorated erythroid colony formation of CML cells. As for the mechanisms of Ras-induced suppression of erythropoiesis, we found that GATA-1, an erythroid-specific transcription factor, blocked Ras-mediated mitogenic signaling at the level of MEK through the direct interaction. Furthermore, enforced expression of N-RasE12 in LSK cells derived from p53-, p16INK4a/p19ARF-, and p21CIP1/WAF1-null/wild-type mice revealed that suppressed erythroid cell growth by N-RasE12 was restored only by p21CIP1/WAF1 deficiency, indicating that a cyclin-dependent kinase (CDK) inhibitor, p21CIP1/WAF1, plays crucial roles in Ras-induced suppression of erythropoiesis. These data would, at least partly, explain why respective oncogenic TKs cause different disease phenotypes.

Donor Cell-Derived Chronic Myeloproliferative Disease with t(7;11)(p15;p15) after Cord Blood Transplantation in a Patient with Philadelphia Chromosome-Positive Acute Lymphoblastic Leukemia

We report a case of donor cell-derived chronic myeloproliferative disease with t(7;11)(p15;p15) occurring after cord blood transplantation (CBT). A 41-year-old man developed precursor B-cell acute lymphoblastic leukemia with a karyotype of 46, XY, t(9;22)(q34;q11) and inv(9)(p11;q13), for which he received CBT from a sex-mismatched donor at the first complete remission of the leukemia. Five months after CBT, gradual neutrophilia of unknown origin developed following the myeloid reconstitution after CBT. Karyotyping of bone marrow cells at 9 months after CBT showed 46,XX, t(7;11)(p15;p15) in 17/20 dividing cells, but neither Philadelphia chromosome (Ph) nor inv(9)(p11;q13) was present. This is the first report of chronic myeloproliferative disease with t(7;11)(p15;p15) that developed in donor cells after CBT. The donor was well-developed and healthy, at least at the time of follow-up, half a year after the birth.

A cell-death-defying factor, anamorsin mediates cell growth through inactivation of PKC and p38MAPK

Anamorsin (AM) plays crucial roles in hematopoiesis and embryogenesis. AM deficient (AM KO) mice die during late gestation; AM KO embryos are anemic and very small compared to wild type (WT) embryos. To determine which signaling pathways AM utilizes for these functions, we used murine embryonic fibroblast (MEF) cells generated from E-14.5 AM KO or WT embryos. Proliferation of AM KO MEF cells was markedly retarded, and PKCθ, PKCδ, and p38MAPK were more highly phosphorylated in AM KO MEF cells. Expression of cyclinD1, the target molecule of p38MAPK, was down-regulated in AM KO MEF cells. p38MAPK inhibitor as well as PKC inhibitor restored expression of cyclinD1 and cell growth in AM KO MEF cells. These data suggest that PKCθ, PKCδ, and p38MAPK activation lead to cell cycle retardation in AM KO MEF cells, and that AM may negatively regulate novel PKCs and p38MAPK in MEF cells.

Clinical role of FDG PET/CT for methotrexate-related malignant lymphoma.

Methotrexate-related malignant lymphoma (MTX-RML) is a type of therapy-related lymphoma, and it often occurs in patients with rheumatoid arthritis. The most distinctive characteristic of MTX-RML is a quick response to withdrawal of MTX. However, because there is a risk of recurrence without a distinctive indicator of disease, close follow-up is needed. We present F-18 2-fluoro-2-deoxyglucose (FDG) postitron emission tomography (PET) or computed tomography (CT) images of MTX-RML along with the characteristic clinical presentation of MTX-RML. FDG PET/CT has the advantage of being able to detect malignant lymphoma in patients who have undergone MTX treatment. After withdrawal of MTX, FDG uptake decreases along with a reduction in the volume of lesions. Although recurrent lesion develops independent to the initial FDG PET/CT findings, FDG PET/CT is useful for early detection of unexpected recurrent lesions. FDG PET/CT allows for the assessment of malignant lymphoma and recurrent lesions in patients who received MTX therapy, which is crucial for the management of MTX-RML.

ESAM is a novel human hematopoietic stem cell marker associated with a subset of human leukemias.

Reliable markers are essential to increase our understanding of the biological features of human hematopoietic stem cells and to facilitate the application of hematopoietic stem cells in the field of transplantation and regenerative medicine. We previously identified endothelial cell-selective adhesion molecule (ESAM) as a novel functional marker of hematopoietic stem cells in mice. Here, we found that ESAM can also be used to purify human hematopoietic stem cells from all the currently available sources (adult bone marrow, mobilized peripheral blood, and cord blood). Multipotent colony-forming units and long-term hematopoietic-reconstituting cells in immunodeficient mice were found exclusively in the ESAM(High) fraction of CD34(+)CD38(-) cells. The CD34(+)CD38(-) fraction of cord blood and collagenase-treated bone marrow contained cells exhibiting extremely high expression of ESAM; these cells are likely to be related to the endothelial lineage. Leukemia cell lines of erythroid and megakaryocyte origin, but not those of myeloid or lymphoid descent, were ESAM positive. However, high ESAM expression was observed in some primary acute myeloid leukemia cells. Furthermore, KG-1a myeloid leukemia cells switched from ESAM negative to ESAM positive with repeated leukemia reconstitution in vivo. Thus, ESAM is a useful marker for studying both human hematopoietic stem cells and leukemia cells.