[Show abstract][Hide abstract] ABSTRACT: Multiple myeloma (MM) is a clonal plasma cell disorder affecting the immune system with various systemic symptoms. MM remains incurable even with high dose chemotherapy using conventional drugs, thus necessitating development of novel therapeutic strategies. Gossypol (Gos) is a natural polyphenolic compound extracted from cotton plants, and has been shown to possess anti-neoplastic activity against various tumors. Recent studies have shown that Gos is an inhibitor for Bcl-2 or Bcl-XL acting as BH3 mimetics that interfere interaction between pro-apoptotic BH3-only proteins and Bcl-2/Bcl-XL. Since most of the patients with MM overexpress Bcl-2 protein, we considered Gos might be a promising therapeutic agent for MM. We herein show that Gos efficiently induced apoptosis and inhibited proliferation of the OPM2 MM cell line, in a dose- and time-dependent manner. Gos induced activation of caspase-3 and cytochrome c release from mitochondria, showing mitochondrial dysfunction pathway is operational during apoptosis. Further investigation revealed that phosphorylation of Bcl-2 at serine-70 was attenuated by Gos treatment, while protein levels were not affected. In addition, Mcl-1 was downregulated by Gos. Interestingly, phosphorylation of JAK2, STAT3, ERK1/2 and p38MAPK was inhibited by Gos-treatment, indicating that Gos globally suppressed interleukin-6 (IL-6) signals. Moreover, JAK2 inhibition mimicked the effect of Gos in OPM2 cells including Bcl-2 dephosphorylation and Mcl-1 downregulation. These results demonstrated that Gos induces apoptosis in MM cells not only through displacing BH3-only proteins from Bcl-2, but also through inhibiting IL-6 signaling, which leads to Bcl-2 dephosphorylation and Mcl-1 downregulation.
International Journal of Oncology 09/2014; · 2.77 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Familial platelet disorder (FPD) with predisposition to acute myelogenous leukaemia (AML) is characterized by platelet defects with a propensity for the development of haematological malignancies. Its molecular pathogenesis is poorly understood, except for the role of germline RUNX1 mutations. Here we show that CDC25C mutations are frequently found in FPD/AML patients (53%). Mutated CDC25C disrupts the G2/M checkpoint and promotes cell cycle progression even in the presence of DNA damage, suggesting a critical role for CDC25C in malignant transformation in FPD/AML. The predicted hierarchical architecture shows that CDC25C mutations define a founding pre-leukaemic clone, followed by stepwise acquisition of subclonal mutations that contribute to leukaemia progression. In three of seven individuals with CDC25C mutations, GATA2 is the target of subsequent mutation. Thus, CDC25C is a novel gene target identified in haematological malignancies. CDC25C is also useful as a clinical biomarker that predicts progression of FPD/AML in the early stage.
[Show abstract][Hide abstract] ABSTRACT: DNA methylation is one of the critical epigenetic modifications regulating various cellular processes such as differentiation or proliferation, and its dysregulation leads to disordered stem cell function or cellular transformation. Ten-eleven translocation (TET) family genes, initially found as a chromosomal translocation partner in leukemia, turned out to be a key enzyme for DNA demethylation. They hydroxylate 5-methylcytosine (5-mC) to 5-hydroxymethylcytosine (5-hmC), which is then converted to unmodified cytosine through multiple mechanisms. On the other hand, somatic mutations of TET2 gene were reported in a variety of human hematological malignancies such as leukemia, myelodysplastic syndrome or malignant lymphoma, suggesting a critical role for TET2 in hematopoiesis. Importance of TET-mediated cytosine demethylation pathway is also underscored by a recurrent mutation of isocytrate dehydrogenase (IDH) 1 and IDH2 in hematological malignancies, whose mutation inhibits TET function through a novel oncometabolite, 2-hydroxyglutarate (2-HG). Studies using mouse models revealed that TET2 is critical for the function of hematopoietic stem cells (HSCs), and disruption of TET2 results in the expansion of multipotent as well as myeloid progenitors, leading to the accumulation of premalignant clones. In addition to cytosine demethylation, TET proteins are involved in chromatin modifications and other cellular processes through the interaction with O-linked β-N-acetylglucosamine transferase (OGT). In summary, TET2 is a critical regulator for HSC homeostasis whose functional impairment leads to hematological malignancies. Future studies will uncover the whole picture of epigenetic and signaling networks wired with TET2, which helps developing ways to intervene dysregulated cellular pathways by TET2 mutations.This article is protected by copyright. All rights reserved.
[Show abstract][Hide abstract] ABSTRACT: A 62-year-old man with refractory leukemia transformed from myelodysplastic syndrome was placed on hydroxyurea (hydroxycarbamide) at a daily dose of 500 mg. Because of insufficient cytoreductive efficacy, the dose was increased to 1,500 mg five days later. Eight days after the initiation of hydroxyurea, the patient started complaining of chills, fever, and vomiting. Serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were markedly elevated to 5,098 and 3,880 IU/l from 44 and 59 IU/l in one day, respectively. Tests for hepatitis viruses were all negative. With the discontinuation of hydroxyurea, AST and ALT returned to their former levels within two weeks. A drug-induced lymphocyte stimulation test for hydroxyurea was positive with a stimulating index of 2.0. Hepatic dysfunction has been recognized as one of the side effects of hydroxyurea. However, there have been only a limited number of reports demonstrating drug allergy to have a role in hepatic dysfunction accompanied by fever and gastrointestinal symptoms. The findings of our case strongly suggest that all presentations could be explained by drug allergy. Physicians should be mindful of the potential for acute and severe hepatic dysfunction due to allergic reaction against hydroxyurea.
[Rinshō ketsueki] The Japanese journal of clinical hematology 01/2014; 55(1):125-9.
[Show abstract][Hide abstract] ABSTRACT: Oncogenic transformation requires unlimited self-renewal. Currently it remains unclear whether a normal capacity for self-renewal is required for acquiring an aberrant self-renewal capacity. Our results in a new conditional transgenic mouse showed that a Mixed Lineage Leukemia (MLL) fusion oncogene, MLL-ENL, at an endogenous-like expression level led to leukemic transformation selectively in a restricted subpopulation of hematopoietic stem cells (HSCs) through upregulation of Promyelocytic leukemia zinc finger (Plzf). Interestingly, forced expression of Plzf itself immortalized HSCs and myeloid progenitors in vitro without upregulation of Hoxa9/Meis1, which are well-known targets of MLL fusion proteins, while its mutant lacking the BTB/POZ domain did not. In contrast, depletion of Plzf suppressed the MLL-fusion-induced leukemic transformation of HSCs in vitro and in vivo. Gene expression analyses of human clinical samples showed that a subtype of PLZF-high MLL-rearranged myeloid leukemia cells was closely associated with the gene expression signature of HSCs. These findings suggested that MLL fusion protein enhances the self-renewal potential of normal HSCs to develop leukemia, in part through a Plzf-driven self-renewal program.
[Show abstract][Hide abstract] ABSTRACT: Roundabout (Robo) family proteins are immunoglobulin-type surface receptors critical for cellular migration and pathway finding of neuronal axons. We have previously shown that Robo4 was specifically expressed in hematopoietic stem and progenitor cells and its high expression correlated with long-term repopulating (LTR) capacity. To reveal the physiological role of Robo4 in hematopoiesis, we examined the effects of Robo4 disruption on the function of hematopoietic stem cells (HSCs) and progenitors. In Robo4-deficient mice, basic hematological parameters including complete blood cell count and differentiation profile were not affected. In contrast to the previous report, HSC/hematopoietic progenitor (HPC) frequencies in the bone marrow (BM) were perfectly normal in Robo4(-/-) mice. Moreover, Robo4(-/-) HSCs were equally competitive as wild-type HSCs in transplantation assays and had normal long-term repopulating (LTR) capacity. Of note, the initial engraftment at 4-weeks after transplantation was slightly impaired by Robo4 ablation, suggesting a marginal defect in BM homing of Robo4(-/-) HSCs. In fact, homing efficiencies of HSCs/HPCs to the BM was significantly impaired in Robo4-deficient mice. On the other hand, granulocyte-colony stimulating factor-induced peripheral mobilization of HSCs was also impaired by Robo4 disruption. Lastly, marrow recovery from myelosuppressive stress was equally efficient in WT- and Robo4-mutant mice. These results clearly indicate that Robo4 plays a role in HSC trafficking such as BM homing and peripheral mobilization, but is not essential in the LTR and self-renewal capacity of HSCs.
PLoS ONE 11/2012; 7(11):e50849. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Hematopoietic progenitors have been shown to retain plasticity and switch lineages by appropriate stimuli. However, mature blood cells hardly showed such differentiation plasticity. In this paper, we tried to reprogram mature B cells into erythroid lineage by expressing various hematopoietic transcription factors. Among various factors, GATA-1, SCL together with CCAAT/enhancer binding protein (C/EBP) α turned out to be a minimal set of factors that efficiently reprogrammed terminally differentiated mature B cells into erythroid lineage, as evidenced by colony forming assays and erythroid-specific gene expressions. This study sets an avenue to generate autologous erythrocytes from peripheral B cells.
[Show abstract][Hide abstract] ABSTRACT: Somatic mutation of ten-eleven translocation 2 (TET2) gene is frequently found in human myeloid malignancies. Recent reports showed that loss of Tet2 led to pleiotropic hematopoietic abnormalities including increased competitive repopulating capacity of bone marrow (BM) HSCs and myeloid transformation. However, precise impact of Tet2 loss on the function of fetal liver (FL) HSCs has not been examined. Here we show that disruption of Tet2 results in the expansion of Lin(-)Sca-1(+)c-Kit(+) (LSK) cells in FL. Furthermore, Tet2 loss led to enhanced self-renewal and long-term repopulating capacity of FL-HSCs in in vivo serial transplantation assay. Disruption of Tet2 in FL also led to altered differentiation of mature blood cells, expansion of common myeloid progenitors and increased resistance for hematopoietic progenitor cells (HPCs) to differentiation stimuli in vitro. These results demonstrate that Tet2 plays a critical role in homeostasis of HSCs and HPCs not only in the BM, but also in FL.
[Show abstract][Hide abstract] ABSTRACT: A 61-year-old female was diagnosed with multiple myeloma (MM) in 2001. After treatment with chemotherapy containing alkylating agents and thalidomide, she underwent autologous stem cell transplantation in 2003, with high-dose melphalan as a conditioning regimen. Thalidomide was also given after the transplant from July 2003 to November 2005 for residual disease and she remained in partial remission. In October 2008, she developed pancytopenia. Bone marrow examination confirmed the diagnosis of acute B lymphoblastic leukemia (B-ALL). We performed IgH gene rearrangement studies on genomic DNA which revealed the MM, and ALL seemed to be derived from different clones. The development of MM and ALL in the same patient is a very rare event. Further accumulation of the cases to understand the mechanism underlying this event is warranted.
[Rinshō ketsueki] The Japanese journal of clinical hematology 02/2012; 53(2):219-23.
[Show abstract][Hide abstract] ABSTRACT: Differentiation of hematopoietic cells is a sequential process of cell fate decision originating from hematopoietic stem cells (HSCs), allowing multi- or oligopotent progenitors to commit to certain lineages. HSCs are cells that are able to self-renew and repopulate the marrow for the long term. They first differentiate into multipotent progenitors (MPPs), which give rise to common lymphoid progenitors (CLPs) and common myeloid progenitors (CMPs). CMPs then differentiate into granulocyte monocyte progenitors (GMPs) and megakaryocyte erythroid progenitors (MEPs), which are the precursors of granulocytes/monocytes and erythrocytes/megakaryocytes, respectively. Lineage specification at differentiation branch points is dictated by the activation of lineage-specific transcription factors such as C/EBPα, PU.1, and GATA-1. The role of these transcription factors is generally instructive, and the expression of a single factor can often determine cell fate. Differentiation was long regarded as an irreversible process, and it was believed that somatic cells would not change their fate once they were differentiated. This paradigm was first challenged by the finding that ectopic cytokine signals could change the fate of differentiation, probably through modulating internal transcription networks. Subsequently, we and others showed that virtually all progenitors, including CLPs, CMPs, GMPs, and MEPs, still retain differentiation plasticity, and they can be converted into lineages other than their own by ectopic activation of only a single lineage-specific transcription factor. These findings established a novel paradigm for cellular differentiation and opened up an avenue for artificially manipulating cell fate for clinical use.
The Keio Journal of Medicine 06/2011; 60(2):47-55.
[Show abstract][Hide abstract] ABSTRACT: Various key cell cycle components, especially G0/G1 regulators, have effects not only on cell proliferation but also on cell differentiation. Cdh1, one of the co-activators that maintain anaphase-promoting complex/cyclosome activity, plays a crucial role in the mitotic phase, but has recently been identified as a G0/G1 regulator, suggesting that the role of Cdh1 in cell differentiation. Here, we generated Cdh1 conditional gene-trap mice to examine Cdh1 functions in adult tissues by overcoming the embryonic lethality of Cdh1 homozygous gene-trap mice. We focused on the hematopoietic system and found that Cdh1-deficient mice exhibited a general decrease in mature lineage progenitor cells and a significant increase in short-term hematopoietic stem cells. This phenomenon became conspicuous by irradiation shortly after Cdh1 downregulation, suggesting that Cdh1 regulates the pool sizes of the hematopoietic stem cells and mature lineage progenitor cells by protecting cells from genotoxic stress. We also found that the irradiation-induced G2/M checkpoint was defective in Cdh1-deficient BM cells, causing the loss of stem/progenitor cells. This is the first report revealing Cdh1 function in adult hematopoiesis and showing a role of Cdh1 in a G2/M checkpoint regulation in vivo.
[Show abstract][Hide abstract] ABSTRACT: Regulating transition of hematopoietic stem cells (HSCs) between quiescent and cycling states is critical for maintaining homeostasis of blood cell production. The cycling states of HSCs are regulated by the extracellular factors such as cytokines and extracellular matrix; however, the molecular circuitry for such regulation remains elusive. Here we show that tissue inhibitor of metalloproteinase-3 (TIMP-3), an endogenous regulator of metalloproteinases, stimulates HSC proliferation by recruiting quiescent HSCs into the cell cycle. Myelosuppression induced TIMP-3 in the bone marrow before hematopoietic recovery. Interestingly, TIMP-3 enhanced proliferation of HSCs and promoted expansion of multipotent progenitors, which was achieved by stimulating cell-cycle entry of quiescent HSCs without compensating their long-term repopulating activity. Surprisingly, this effect did not require metalloproteinase inhibitory activity of TIMP-3 and was possibly mediated through a direct inhibition of angiopoietin-1 signaling, a critical mediator for HSC quiescence. Furthermore, bone marrow recovery from myelosuppression was accelerated by over-expression of TIMP-3, and in turn, impaired in TIMP-3-deficient animals. These results suggest that TIMP-3 may act as a molecular cue in response to myelosuppression for recruiting dormant HSCs into active cell cycle and may be clinically useful for facilitating hematopoietic recovery after chemotherapy or ex vivo expansion of HSCs.
[Show abstract][Hide abstract] ABSTRACT: Two types of mutations of a transcription factor CCAAT-enhancer binding protein α (C/EBPα) are found in leukemic cells of 5%-14% of acute myeloid leukemia (AML) patients: N-terminal mutations expressing dominant negative p30 and C-terminal mutations in the basic leucine zipper domain. Our results showed that a mutation of C/EBPα in one allele was observed in AML after myelodysplastic syndrome, while the 2 alleles are mutated in de novo AML. Unlike an N-terminal frame-shift mutant (C/EBPα-N(m))-transduced cells, a C-terminal mutant (C/EBPα-C(m))-transduced cells alone induced AML with leukopenia in mice 4-12 months after bone marrow transplantation. Coexpression of both mutants induced AML with marked leukocytosis with shorter latencies. Interestingly, C/EBPα-C(m) collaborated with an Flt3-activating mutant Flt3-ITD in inducing AML. Moreover, C/EBPα-C(m) strongly blocked myeloid differentiation of 32Dcl3 cells, suggesting its class II mutation-like role in leukemogenesis. Although C/EBPα-C(m) failed to inhibit transcriptional activity of wild-type C/EBPα, it suppressed the synergistic effect between C/EBPα and PU.1. On the other hand, C/EBPα-N(m) inhibited C/EBPα activation in the absence of PU.1, despite low expression levels of p30 protein generated by C/EBPα-N(m). Thus, 2 types of C/EBPα mutations are implicated in leukemo-genesis, involving different and cooperating molecular mechanisms.
[Show abstract][Hide abstract] ABSTRACT: Wnt signaling has been implicated in the self-renewal of hematopoietic stem cells (HSCs). Secreted frizzled-related proteins (SFRPs) are a family of soluble proteins containing a region homologous to a receptor for Wnt, Frizzled, and are thought to act as endogenous modulators for Wnt signaling. This study examined the role of SFRPs in HSC regulation. Among the four family members, SFRP-1 and SFRP-2 are specifically induced in the bone marrow in response to myelosuppression, and immunostaining revealed that both proteins were expressed in osteoblasts. Interestingly, SFRP-1 reduced the number of multipotent progenitors in in vitro culture of CD34(-)KSL cells, while SFRP-2 did not. Furthermore, SFRP-1 compromised the long-term repopulating activity of HSCs, whereas SFRP-2 did not affect or even enhanced it in the same setting. These results indicate that although both SFRP-1 and SFRP-2 act as inhibitors for Wnt signaling in vitro, they differentially affect the homeostasis of HSCs.
Biochemical and Biophysical Research Communications 09/2009; 390(1):65-70. · 2.28 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: It is now conceivable that leukemogenesis requires two types of mutations, class I and class II mutations. We previously established a mouse bone marrow-derived HF6, an IL-3-dependent cell line, that was immortalized by a class II mutation MLL/SEPT6 and can be fully transformed by class I mutations such as FLT3 mutants. To understand the molecular mechanism of leukemogenesis, particularly progression of myelodysplastic syndrome (MDS) to acute leukemia, we made cDNA libraries from the samples of patients and screened them by expression-cloning to detect class I mutations that render HF6 cells factor-independent. We identified RasGRP4, an activator of Ras, as a candidate for class I mutation from three of six patients (MDS/MPD = 1, MDS-RA = 1, MDS/AML = 2, CMMoL/AML = 1 and AML-M2 = 1). To investigate the potential roles of RasGRP4 in leukemogenesis, we tested its in vivo effect in a mouse bone marrow transplantation (BMT) model. C57BL/6J mice transplanted with RasGRP4-transduced primary bone marrow cells died of T cell leukemia, myeloid leukemia, or myeloid leukemia with T cell leukemia. To further examine if the combination of class I and class II mutations accelerated leukemic transformation, we performed a mouse BMT model in which both AML1 mutant (S291fsX300) and RasGRP4 were transduced into bone marrow cells. The double transduction led to early onset of T cell leukemia but not of AML in the transplanted mice when compared to transduction of RasGRP4 alone. Thus, we have identified RasGRP4 as a gene potentially involved in leukemogenesis and suggest that RasGRP4 cooperates with AML1 mutations in T cell leukemogenesis as a class I mutation.
International journal of hematology 05/2009; 89(4):470-81. · 1.17 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: STAT5 is a critical mediator of a variety of cytokine signaling whose transcriptional activity is regulated by associating with various proteins. During a search for STAT5-interacting proteins, we identified SHD1, a mammalian homologue of yeast gene Sac3, as a potential interacter. SHD1 was localized in the nucleus, and induced by cytokines that activate STAT5, such as erythropoietin, interleukin-2 (IL-2), or IL-3. SHD1 interacted specifically with STAT5A and STAT5B, and interestingly, it specifically repressed STAT5-dependent transcription in vitro without affecting the stability or phosphorylation of STAT5 protein. Gene disruption study revealed that T, B, or bone marrow cells from mice lacking SHD1 were hyperresponsive to T-cell-receptor engagement, or stimulation with various STAT5-activating cytokines. These results suggest that SHD1 is a novel cytokine-inducible negative feedback regulator of STAT5.
[Show abstract][Hide abstract] ABSTRACT: Roundabout (Robo) family proteins are immunoglobulin-type cell surface receptors that are expressed predominantly in the nervous system. The fourth member of this family, Robo4, is distinct from the other family members in that it is expressed specifically in endothelial cells. In this study, we examined the expression of Robo4 in hematopoietic stem cells (HSCs) and its possible role in HSC regulation. Robo4 mRNA was specifically expressed in murine HSCs and the immature progenitor cell fraction but not in lineage-positive cells or differentiated progenitors. Moreover, flow cytometry showed a correlation between higher expression of Robo4 and immature phenotypes of hematopoietic cells. Robo4(high) hematopoietic stem/progenitor cells presented higher clonogenic activity or long-term repopulating activity by colony assays or transplantation assays, respectively. A ligand for Robo4, Slit2, is specifically expressed in bone marrow stromal cells, and its expression was induced in osteoblasts in response to myelosuppressive stress. Interestingly, overexpression of Robo4 or Slit2 in HSCs resulted in their decreased residence in the c-Kit(+)Sca-1(+)Lineage(-)-side population fraction. These results indicate that Robo4 is expressed in HSCs, and Robo4/Slit2 signaling may play a role in HSC homeostasis in the bone marrow niche.
[Show abstract][Hide abstract] ABSTRACT: Previous studies using loss-of-function mutants revealed that CCAAT/enhancer-binding protein alpha (C/EBPalpha) and PU.1 are potential regulators for hematopoietic stem cells (HSCs). To gain further insight into the HSC regulation by C/EBPalpha or PU.1, we used transgenic mice expressing conditional forms of these transcription factors to examine whether their activation alone is sufficient for modulating HSC functions. The activation of C/EBPalpha or PU.1 in HSCs in vitro or in vivo led to their suppression of growth, decreased mixed colony formation, and impaired competitive repopulating activities because of their defective self-renewal. These effects were more prominently observed when C/EBPalpha was activated, and the differentiation capacity to megakaryocytic lineage was selectively impaired upon C/EBPalpha activation. Unexpectedly, the expression of Bmi-1 and HoxB4, well-known regulators for self-renewal of HSCs, was not affected by the activation of C/EBPalpha or PU.1, suggesting that they regulate HSC function through an as yet unknown mechanism. Our data suggest that the activation of C/EBPalpha or PU.1 is sufficient to repress stem cell capacities in HSCs, and their fine-tuned regulation is critical for HSC homeostasis.