Language：English   Publisher：JCI insight  

Human T lymphotropic virus type 1-assoicated (HTLV-1-associated) myelopathy/tropical spastic paraparesis (HAM/TSP) is a neuroinflammatory disease caused by the persistent proliferation of HTLV-1-infected T cells. Here, we performed a T cell receptor (TCR) repertoire analysis focused on HTLV-1-infected cells to identify and track the infected T cell clones that are preserved in patients with HAM/TSP and migrate to the CNS. TCRβ repertoire analysis revealed higher clonal expansion in HTLV-1-infected cells compared with noninfected cells from patients with HAM/TSP and asymptomatic carriers (ACs). TCR clonality in HTLV-1-infected cells was similar in patients with HAM/TSP and ACs. Longitudinal analysis showed that the TCR repertoire signature in HTLV-1-infected cells remained stable, and highly expanded infected clones were preserved within each patient with HAM/TSP over years. Expanded HTLV-1-infected clones revealed different distributions between cerebrospinal fluid (CSF) and peripheral blood and were enriched in the CSF of patients with HAM/TSP. Cluster analysis showed similarity in TCRβ sequences in HTLV-1-infected cells, suggesting that they proliferate after common antigen stimulation. Our results indicate that exploring TCR repertoires of HTLV-1-infected cells can elucidate individual clonal dynamics and identify potential pathogenic clones expanded in the CNS.

DOI： 10.1172/jci.insight.167422

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

DOI： 10.1016/j.jdermsci.2023.02.001

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Language：English   Publisher：エルゼビア・ジャパン(株)  

Language：English   Publishing type：Research paper (scientific journal)  

BACKGROUND/AIM: Acute myeloid leukemia (AML) with high expression of the oncogenic transcription factor ecotropic viral integration site-1 (EVI1) (EVI1high AML) is refractory, and there is an urgent need to develop treatment for EVI1high AML. We previously showed that calcitonin receptor-like receptor (CRLR)/receptor activity modifying protein 1 (RAMP1) is highly expressed in EVI1high AML and participates in calcitonin gene-related peptide (CGRP)-induced stress hematopoiesis. This study examined whether MK0974 (a CGRP antagonist) acts as a therapeutic agent in CRLR/RAMP1high AML cell lines. MATERIALS AND METHODS: An in vitro experimental system was used to determine the effect of MK0974 on EVI1high AML cell lines. The expression of CRLR and RAMP1-3 in EVI1high and EVI1low AML lines was evaluated by reverse-transcription polymerase chain reaction (RT-PCR). Next, MK0974 was added to the AML cell lines, and cell proliferation, cell cycle and apoptosis assays were carried out using flow cytometry (FCM). Proteins were evaluated using western blot analysis. We also generated AML cell lines with CRLR knockdown and evaluated whether the effect of MK0974 was reduced. RESULTS: Apoptosis was induced by adding MK0974 to the EVI1high AML cell line. In the EVI1high AML cell line, the addition of MK0974 attenuated the phosphorylation of ERK and p38. These effects were also attenuated by CRLR knockdown. CONCLUSION: MK0974, a CGRP receptor antagonist, inhibits the CRLR/RAMP1 complex and induces apoptosis, making it a potential therapeutic agent for CRLR/RAMP1high AML.

DOI： 10.21873/anticanres.15979

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

DOI： 10.1016/j.exphem.2022.04.004

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Both natural viral infections and therapeutic interventions using viral vectors pose significant risks of malignant transformation. Monitoring for clonal expansion of infected cells is important for detecting cancer. Here we developed a novel method of tracking clonality via the detection of transgene integration sites. RAISING (Rapid Amplification of Integration Sites without Interference by Genomic DNA contamination) is a sensitive, inexpensive alternative to established methods. Its compatibility with Sanger sequencing combined with our CLOVA (Clonality Value) software is critical for those without access to expensive high throughput sequencing. We analyzed samples from 688 individuals infected with the retrovirus HTLV-1, which causes adult T-cell leukemia/lymphoma (ATL) to model our method. We defined a clonality value identifying ATL patients with 100% sensitivity and 94.8% specificity, and our longitudinal analysis also demonstrates the usefulness of ATL risk assessment. Future studies will confirm the broad applicability of our technology, especially in the emerging gene therapy sector.

DOI： 10.1038/s42003-022-03467-w

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Language：Japanese   Publisher：日本家畜臨床学会  

牛伝染性リンパ腫(enzootic bovine leucosis:EBL)は、国内において増加傾向にあり、清浄国を除く世界中で問題になっている。EBLの原因は、牛白血病ウイルス(bovine leukemia virus:BLV)であり、病理学的特徴は、多形型B細胞性リンパ腫である。本症例は宮崎県内のと畜場に搬入され、解体時に乳房に数個のソフトボール大の腫瘤を認めたため、一時保留処分となった。その後、食肉衛生検査所における病理組織学的検査および抗BLV抗体検査の結果、EBLと診断され全部廃棄処分になった。乳房の腫瘤の由来は肉眼的には不明であったが、病理組織学的検査において、腫瘤組織中に腫瘍細胞の浸潤とリンパ節の髄洞に相当する部分を認めたことから、乳房リンパ節が腫瘍細胞の浸潤によって腫大したものと思われた。また乳房腫瘤内にCD20、IgM抗体陽性のB細胞が浸潤し、核分裂像が散見された。EBLの診断において、本例のように臨床所見だけでは判断ができないため、血液検査、ウイルスの抗体検査・PCR検査、剖検所見、病理組織学的所見の全ての総合的な診断が、極めて重要であることを再確認した。(著者抄録)

Language：English   Publishing type：Research paper (scientific journal)  

BACKGROUND/AIM: Pancreatic ductal adenocarcinoma (PDAC) is one of the most common cancers worldwide, with a poor prognosis. Owing to the difficulty of early diagnosis, the aim of this study was to isolate biomarkers from extracellular vesicles (EVs) that can lead to early diagnosis. MATERIALS AND METHODS: EVs in the culture supernatant were isolated from a pancreatic cancer cell line (PK-1) and expanded by using two-dimensional gel electrophoresis, and protein identification from each spot was performed by using matrix-assisted laser desorption ionization mass spectrometry. The identified proteins were classified and compared with previously reported results for EVs from murine pancreatic cancer PAN02 cells, and their expression specificity was examined using PDAC cell lines and patient-derived PDAC tissues. In addition, the significance of selected biomarker(s) was examined based on the changes in biomarkers in the blood EVs of PDAC patients after surgery. RESULTS: We found that the ITGA6A splice variant was predominantly expressed in several pancreatic cancer cell lines and blood EVs from patients with PDAC, whereas the ITGA6B splice variant was predominantly expressed in EVs from the blood of normal volunteers. In the expression pattern of ITGA6 in EVs from blood samples of two PDAC patients before and after resection surgery, the expression of ITGA6A in EVs significantly decreased after surgery and increased several months before clinical recurrence. Furthermore, the increased expression of ITGA6A in EVs occurred much earlier than that of CA19-9. CONCLUSION: Determination of ITGA6A expression in blood EVs in PDAC patients could be a useful blood marker for the early diagnosis of PDAC recurrence.

DOI： 10.21873/anticanres.15653

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Language：English   Publisher：Veterinary Medicine International  

Recent research has focused on the receptor tyrosine kinase (RTK) KIT which is involved in the pathogenesis of canine mast cell tumors (MCT). However, the role of other RTKs in this neoplasm remains unclear. The present study aimed to determine the frequency of FLT3 mutations and to evaluate the mutational status and clinicopathological relevance of canine MCT patients. There were a total of 20 cases that were cytologically and histopathological diagnosed as canine MCTs; genomic polymerase chain reaction (PCR) and Sanger sequencing were used to identify mutations. For the juxtamembrane (JM) domain, the FLT3 14/15 primer pair was used to investigate exon 14/15 loci. Based on genomic PCR amplification of exon 14/15 and 20 of the FLT3 gene and Sanger sequencing of 20 cases of canine MCTs, the overall frequency of FLT3 mutation in canine MCTs was 75%. The majority of FLT3 mutations (70%) were internal tandem duplications (ITD) of the JM domain, while one case arose from deletion mutations of the tyrosine kinase domain (TKD). However, double mutations were not observed in this study. Furthermore, there is also clinicopathological relevance to MCT dogs carrying FLT3-ITD mutations, showing a tendency toward leukocytosis due to neutrophilia, and resembling human acute myeloid leukemia (AML) with FLT3-ITD mutations. A subset of MCTs with FLT3-ITD mutations, showing an enhanced signal of phosphorylated ERK1/2 identified by immunoblotting, suggests that an activating mutation may be driven by a distinct signal of the ERK pathway. Our results indicate that FLT3-ITD mutation is an oncogenic driver of canine MCTs, and that it shares some clinicopathologic features with human AML. These findings may offer new opportunities for further studies on canine mast cell tumorigenesis and a novel therapeutic target for canine MCT cases harboring FLT3-ITD mutations.

DOI： 10.1155/2022/9509900

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Language：English  

Adult T-cell leukemia/lymphoma (ATLL) originates from human T-cell leukemia virus type 1 (HTLV-1) infection due to the activation of the nuclear factor-κB (NF-κB) signaling pathway to maintain proliferation and survival. An important mechanism of the activated NF-κB signaling pathway in ATLL is the activation of the macroautophagy (herafter referred to as autophagy in the remainder of this manuscript)-lysosomal degradation of p47 (NSFL1C), a negative regulator of the NF-κB pathway. Therefore, we considered the use of chloroquine (CQ) or hydroxychloroquine (HCQ) (CQ/HCQ) as an autophagy inhibitor to treat ATLL; these drugs were originally approved by the FDA as antimalarial drugs and have recently been used to treat autoimmune diseases, such as systemic lupus erythematosus (SLE). In this paper, we determined the therapeutic efficacy of CQ/HCQ, as NF-κB inhibitors, in ATLL mediated by blockade of p47 degradation. Administration of CQ/HCQ to ATLL cell lines and primary ATLL cells induced cell growth inhibition in a dose-dependent manner, and the majority of cells underwent apoptosis after CQ administration. As to the molecular mechanism, autophagy was inhibited in CQ-treated ATLL cells, and activation of the NF-κB pathway was suppressed with the restoration of the p47 level. When the antitumor effect of CQ/HCQ was examined using immunodeficient mice transplanted with ATLL cell lines, CQ/HCQ significantly suppressed tumor growth and improved the survival rate in the ATLL xenograft mouse model. Importantly, HCQ selectively induced ATLL cell death in the ATLL xenograft mouse model at the dose used to treat SLE. Taken together, our results suggest that the inhibition of autophagy by CQ/HCQ may become a novel and effective strategy for the treatment of ATLL.

Language：English   Publishing type：Research paper (scientific journal)  

Adult T-cell leukemia/leukemia (ATLL) is an aggressive peripheral T-cell malignancy, caused by infection with the human T-cell leukemia virus type 1 (HTLV-1). We have recently shown that cell adhesion molecule 1 (CADM1), a member of the immunoglobulin superfamily, is specifically and consistently overexpressed in ATLL cells, and functions as a novel cell surface marker. In this study, we first show that a soluble form of CADM1 (sCADM1) is secreted from ATLL cells by mainly alternative splicing. After developing the Alpha linked immunosorbent assay (AlphaLISA) for sCADM1, we showed that plasma sCADM1 concentrations gradually increased during disease progression from indolent to aggressive ATLL. Although other known biomarkers of tumor burden such as soluble interleukin-2 receptor α (sIL-2Rα) also increased with sCADM1 during ATLL progression, multivariate statistical analysis of biomarkers revealed that only plasma sCADM1 was selected as a specific biomarker for aggressive ATLL, suggesting that plasma sCADM1 may be a potential risk factor for aggressive ATLL. In addition, plasma sCADM1 is a useful marker for monitoring response to chemotherapy as well as for predicting relapse of ATLL. Furthermore, the change in sCADM1 concentration between indolent and aggressive type ATLL was more prominent than the change in the percentage of CD4+CADM1+ ATLL cells. As plasma sCADM1 values fell within normal ranges in HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) patients with higher levels of serum sIL-2Rα, a measurement of sCADM1 may become a useful tool to discriminate between ATLL and other inflammatory diseases, including HAM/TSP.

DOI： 10.3324/haematol.2019.234096

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Language：English   Publishing type：Research paper (scientific journal)  

N-myc downstream-regulated gene 2 (NDRG2) is a candidate tumor suppressor in various cancers, including adult T-cell leukemia/lymphoma (ATLL). NDRG2, as a stress-responsive protein, is induced by several stress-related signaling pathways and NDRG2 negatively regulates various signal transduction pathways. Although it has not been found to function alone, NDRG2 binds serine/threonine protein phosphatase 2A (PP2A), generating a complex that is involved in the regulation of various target proteins. The main function of NDRG2 is to maintain cell homeostasis by suppressing stress-induced signal transduction; however, in cancer, genomic deletions and/or promoter methylation may inhibit the expression of NDRG2, resulting in enhanced tumor development via overactivated signal transduction pathways. A wide variety of tumors develop in Ndrg2-deficient mice, including T-cell lymphoma, liver, lung and other tumors, the characteristics of which are similar to those in Pten-deficient mice. In particular, PTEN is a target molecule of the NDRG2/PP2A complex, which enhances PTEN phosphatase activity by dephosphorylating residues in the PTEN C-terminal region. In ATLL cells, loss of NDRG2 expression leads to the failed recruitment of PP2A to PTEN, resulting in the inactivation of PTEN phosphatase with phosphorylation, ultimately leading to the activation of PI3K/AKT. Thus, NDRG2, as a PP2A adaptor, regulates the global phosphorylation of important signaling molecules. Moreover, the downregulation of NDRG2 expression via long-term stress-induced methylation is directly correlated with the development of ATLL and other cancers. Thus, NDRG2 might be important for the development of stress-induced leukemia and other cancers and has become an important target for novel molecular therapies.

DOI： 10.1111/cas.14716

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Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1111/bjh.17214

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Public Library of Science  

Adult T-cell leukemia/lymphoma (ATLL) originates from human T-cell leukemia virus type 1 (HTLV-1) infection due to the activation of the nuclear factor-κB (NF-κB) signaling pathway to maintain proliferation and survival. An important mechanism of the activated NF-κB signaling pathway in ATLL is the activation of the macroautophagy (herafter referred to as autophagy in the remainder of this manuscript)-lysosomal degradation of p47 (NSFL1C), a negative regulator of the NF-κB pathway. Therefore, we considered the use of chloroquine (CQ) or hydroxychloroquine (HCQ) (CQ/HCQ) as an autophagy inhibitor to treat ATLL
these drugs were originally approved by the FDA as antimalarial drugs and have recently been used to treat autoimmune diseases, such as systemic lupus erythematosus (SLE). In this paper, we determined the therapeutic efficacy of CQ/HCQ, as NF-κB inhibitors, in ATLL mediated by blockade of p47 degradation. Administration of CQ/HCQ to ATLL cell lines and primary ATLL cells induced cell growth inhibition in a dose-dependent manner, and the majority of cells underwent apoptosis after CQ administration. As to the molecular mechanism, autophagy was inhibited in CQ-treated ATLL cells, and activation of the NF-κB pathway was suppressed with the restoration of the p47 level. When the antitumor effect of CQ/ HCQ was examined using immunodeficient mice transplanted with ATLL cell lines, CQ/ HCQ significantly suppressed tumor growth and improved the survival rate in the ATLL xenograft mouse model. Importantly, HCQ selectively induced ATLL cell death in the ATLL xenograft mouse model at the dose used to treat SLE. Taken together, our results suggest that the inhibition of autophagy by CQ/HCQ may become a novel and effective strategy for the treatment of ATLL.

DOI： 10.1371/journal.pone.0256320

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Language：English   Publishing type：Research paper (scientific journal)  

Adult T-cell leukemia/lymphoma (ATLL) is a highly invasive and refractory T-cell malignancy, with poor prognosis. We previously identified that cell adhesion molecule 1 (CADM1) is overexpressed consistently in ATLL cells, and that CADM1 expression increases the adhesion capacity of ATLL cells to endothelial cells and promotes the organ invasion of ATLL cells in a xenograft mouse model. In this study, we first show that newly developed several anti-human CADM1 antibodies, which were complete human IgG antibodies generated by phage display method, specifically recognize CADM1 on ATLL cells. Although most of the CADM1 antibodies did not have a direct cytotoxic effect against CADM1-positive ATLL cells, clone 089-084 exhibited weak but significant antibody-dependent cell-mediated cytotoxic activity. Moreover, clone 103-189 effectively inhibits the interaction between endothelial cells and CADM1-positive ATLL cells. Furthermore, in mice bearing intra-splenic transplantation of EL4 mouse lymphoma cells expressing CADM1, the treatment of 103-189 significantly suppressed the organ invasion of CADM1-positive EL4 cells, resulting in improved survival time of mice. Therefore, since the anti-CADM1 antibody may be useful for the suppression of organ invasion in ATLL patients, combination use of the anti-CADM1 antibody with chemotherapy drugs could be beneficial for the efficient elimination of ATLL cells.

DOI： 10.1007/s12185-020-02939-1

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DOI： 10.1016/j.bbrc.2020.05.147

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Language：English   Publishing type：Research paper (scientific journal)  

Approximately 10-20 million of Human T-cell leukemia virus type-1 (HTLV-1)-infected carriers have been previously reported, and approximately 5% of these carriers develop adult T-cell leukemia/lymphoma (ATL) with a characteristic poor prognosis. In Japan, Southern blotting has long been routinely performed for detection of clonally expanded ATL cells in vivo, and as a confirmatory diagnostic test for ATL. However, alternative methods to Southern blotting, such as sensitive, quantitative, and rapid analytical methods, are currently required in clinical practice. In this study, we developed a high-throughput method called rapid amplification of integration site (RAIS) that could amplify HTLV-1-integrated fragments within 4 h and detect the integration sites in > 0.16% of infected cells. Furthermore, we established a novel quantification method for HTLV-1 clonality using Sanger sequencing with RAIS products, and the validity of the quantification method was confirmed by comparing it with next-generation sequencing in terms of the clonality. Thus, we believe that RAIS has a high potential for use as an alternative routine molecular confirmatory test for the clonality analysis of HTLV-1-infected cells.

DOI： 10.1007/s12185-020-02935-5

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N-myc downstream-regulated gene 2 (NDRG2) as a tumor suppressor is frequently downregulated in human T-lymphotropic retrovirus (HTLV-1)-infected adult T-cell leukemia (ATL) and variety of cancers, and negatively regulates PI3K signaling pathways through dephosphorylation of PTEN with protein phosphatase 2A (PP2A). We recently identified that protein arginine methyltransferase 5 (PRMT5) is one of novel NDRG2 binding proteins and the knockdown of PRMT5 induces cell apoptosis with degradation of several signaling molecules. To investigate how the apoptosis is induced by the knockdown PRMT5 expression, heat shock protein 90 alpha (HSP90A) was identified as a binding protein for NDRG2 or PRMT5 by immunoprecipitation-mass analysis. NDRG2/PP2A complex inhibited arginine methyltransferase activity of PRMT5 through dephosphorylation at Serine 335 (S335); however, in NDRG2low ATL-related cells, highly phosphorylated PRMT5 at S335 was mainly localized in cytoplasm with binding to HSP90A, resulting in enhancing arginine-methylation at the middle domain (R345 and R386). Since knockdown of PRMT5 expression or forced expression of HSP90A with alanine replacement of R345 or R386 induced apoptosis with the degradation of client proteins in NDRG2low ATL-related and other cancer cells, we here identified that the novel arginine methylations of HSP90A are essential for maintenance of its function in NDRG2low ATL and other cancer cells.

DOI： 10.1016/j.bbamcr.2019.118615

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Language：English   Publisher：Cancer Genomics and Proteomics  

Background: Since bromodomain-containing protein 4 (BRD4) facilitates the transcription of genes important for neoplastic cells in a cancer-type specific manner, BRD4-regulated molecules may also include therapeutic targets for mantle cell lymphoma (MCL), a treatment-refractory subtype of malignant lymphoma. Materials and Methods: In order to uncover direct BRD4-regulated targets in MCL, we performed integrated analysis using the pathway database and the results of both gene-expression profiling and chromatin immunoprecipitation with parallel sequencing for BRD4. Results: Treatment with BRD4 inhibitor I-BET151 exerted a dose-dependent inhibitory effect on cell proliferation in MCL cell lines. BRD4 was found to directly regulate series of genes involved in the B-cell receptor (BCR) signaling pathway, including B-cell linker (BLNK), paired box 5 (PAX5), and IKAROS family zinc finger 3 (IKZF3), and several oncogenes, such as MYB. Indeed, the combinatory inhibition of BCR pathway and IKZF showed an additive antitumor effect. Conclusion: Concomitant targeting multiple BRD4-regulated molecules may constitute a rational therapeutic strategy for MCL.

DOI： 10.21873/cgp.20169

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Language：English   Publishing type：Research paper (scientific journal)  

N-myc downstream-regulated gene 2 (NDRG2) is a candidate tumor suppressor that is frequently downregulated in adult T-cell leukemia/lymphoma (ATLL) and functions to negatively regulate several cellular signaling pathways as PP2A recruiter. To clarify the molecular mechanisms of suppression of NDRG2 expression, we initially determined the expression pattern of NDRG2 in various types of T-cells and ATLL cells. NDRG2 expression was significantly upregulated in HTLV-1/Tax-immortalized T-cells, which was mediated by NF-κB activation through Tax expression. On the other hand, NDRG2 expression was suppressed in HTLV-1-infected cell lines and various types of ATLL cells, which was dependent on the DNA methylation of the NDRG2 promoter. We found that the expression of enhancer of zeste homolog 2 (EZH2), a member of the polycomb family, is increased in ATLL, and that EZH2 directly binds to the NDRG2 promoter and induces DNA methylation of the NDRG2 promoter. Since the expression of EZH2 were anti-parallelly regulated with the NDRG2 expression, EZH2 might be one of the most important regulators of the downregulation of NDRG2, contributing to enhanced activation of signaling pathways during ATLL development.

DOI： 10.1016/j.bbadis.2019.07.001

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Cell adhesion molecule 1 (CADM1), a member of the immunoglobulin superfamily, is identified as a novel cell surface marker for human T-cell leukemia virus (HTLV-1)-infected T cells. Adult T-cell leukemia/lymphoma (ATLL) is developed in HTLV-1-infected T-cells after a long infection period. To examine the mechanism of CADM1 overexpression in ATLL, we first identified that CADM1 is transcriptionally up-regulated by a transcriptional enhancer element through NF-κB signaling pathway. In HTLV-1-infected T-cells, CADM1 expression is dependent on HTLV-1/Tax through activation of canonical and non-canonical NF-κB; however, in ATLL cells with frequent loss of Tax expression, the activation of canonical NF-κB only enhances the CADM1 expression. Along with active mutations in signaling molecules under T-cell recepor (TCR) signaling, degradation of p47, a negative regulator of NF-κB, was essential for activation of canonical NF-κB through stabilization of NEMO (NF-κB essential modulator). The mechanism of p47 degradation is primarily dependent on activation of lysosomal-autophagy and the autophagy is activated in most of the HTLV-infected and ATLL cells, suggesting that the p47 degradation may be a first key molecular event during HTLV-1 infection to T-cells as a connector of two important signaling pathways, NF-κB and autophagy.

DOI： 10.1038/s41598-019-39424-7

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We had previously reported that in addition to p53 inactivation, overexpression of the DNA sensor protein-absent in melanoma 2 (AIM2)-contributes to tumorigenesis of oral squamous cell carcinoma (OSCC). Given that AIM2 is highly expressed in the OSCC tumors from patients with metastasis, we investigated whether AIM2 expression contributes to the progression of OSCC metastasis. In in vitro assays using OSCC cell lines, the high migration and invasion capacity of OSCC cells were dependent on the increased expression of AIM2, resulting in enhanced epithelial-mesenchymal transition (EMT), with EMT-related gene expression. Moreover, the in vivo short-term metastasis assay using orthotopic implantation into immunodeficient mice demonstrated that OSCC cells with high levels of AIM2 expression exhibited enhanced tumor growth in the tongue, resulting in decreased survival of the mice. Further, the cells overexpressing AIM2 dominantly invaded into the tumor lymphatic vessels, unlike OSCC cells with low AIM2 expression. Thus, the high expression of AIM2 in OSCC enhances progression of tumor growth.

DOI： 10.1016/j.bbrc.2018.12.066

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Ecotropic viral integration site-1 (EVI1) has a critical role in normal and malignant hematopoiesis. Since we previously identified high expression of calcitonin receptor like receptor (CRLR) in acute myeloid leukemia (AML) with high EVI1 expression, we here characterized the function of CRLR in hematopoiesis. Since higher expression of CRLR and receptor activity modifying protein 1 (RAMP1) was identified in immature hematopoietic bone marrow (BM) cells, we focused on calcitonin gene-related peptide (CGRP), a specific ligand for the CRLR/RAMP1 complex. To elucidate the role of CGRP in hematopoiesis, Ramp1-deficient (Ramp1-/-) mice were used. The steady-state hematopoiesis was almost maintained in Ramp1-/- mice; however, the BM repopulation capacity of Ramp1-/- mice was significantly decreased, and the transplanted Ramp1-/- BM mononuclear cells had low proliferation capacity with enhanced reactive oxygen species (ROS) production and cell apoptosis. Thus, CGRP is important for maintaining hematopoiesis during temporal exposures with proliferative stress. Moreover, continuous CGRP exposure to mice for two weeks induced a reduction in the number of BM immature hematopoietic cells along with differentiated myeloid cells. Since CGRP is known to be increased under inflammatory conditions to regulate immune responses, hematopoietic exhaustion by continuous CGRP secretion under chronic inflammatory conditions is probably one of the important mechanisms of anti-inflammatory responses.

DOI： 10.1038/s41598-018-36796-0

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Language：English   Publishing type：Research paper (scientific journal)  

G protein-coupled receptor 56 (GPR56) is highly expressed in acute myeloid leukemia (AML) cells with high EVI1 expression (EVI1high AML). Because GPR56 is a transcriptional target of EVI1 and silencing of GPR56 expression induces apoptosis, we developed a novel drug to suppress GPR56 expression in EVI1high AML cells. For this purpose, we generated pyrrole-imidazole (PI) polyamides specific to GPR56 (PIP/56-1 or PIP/56-2) as nuclease-resistant novel compounds that interfere with the binding of EVI1 to the GPR56 promoter in a sequence-specific manner. Treatment of EVI1high AML cell lines (UCSD/AML1 and Kasumi-3) with PIP/56-1 or PIP/56-2 effectively suppressed GPR56 expression by inhibiting binding of EVI1 to its promoter, leading to suppression of cell growth with increased rates of apoptosis. Moreover, intravenous administration of PIP/56-1 into immunodeficient Balb/c-RJ mice subcutaneously transplanted with UCSD/AML1 cells significantly inhibited tumor growth and extended survival. Furthermore, organ infiltration by leukemia cells in immunodeficient Balb/c-RJ mice, which were intravenously transplanted using UCSD/AML1 cells, was successfully inhibited by PIP/56-1 treatment with no apparent effects on murine hematopoietic cells. In addition, PIP treatment did not inhibit colony formation of human CD34+ progenitor cells. Thus, PI polyamide targeting of GPR56 using our compound is promising, useful, and safe for the treatment of EVI1high AML.

DOI： 10.1038/s41598-018-32205-8

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Language：English   Publisher：(一社)日本癌学会  

Web of Science

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Language：Japanese   Publisher：Japanese Biochemical Society  

DOI： 10.14952/seikagaku.2017.890877

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Language：English   Publisher：(一社)日本癌学会  

Language：English   Publishing type：Research paper (scientific journal)  

Human T-cell leukemia virus type 1 (HTLV-1) is a causative agent of adult T-cell leukemia-lymphoma (ATLL). The HTLV-1-encoded protein Tax plays important roles in the proliferation of HTLV-1-infected T-cells by affecting cellular proteins. In this study, we showed that Tax transcriptionally and post-transcriptionally downregulates the expression of the tumor suppressor gene B-cell leukemia/lymphoma 11B (BCL11B), which encodes a lymphoid-related transcription factor. BCL11B expression was downregulated in HTLV-1-infected T-cell lines at the mRNA and protein levels, and forced expression of BCL11B suppressed the proliferation of these cells. The proteasomal inhibitor MG132 increased BCL11B expression in HTLV-1-infected cell lines, and colocalization of Tax with BCL11B was detected in the cytoplasm of HTLV-1-infected T-cells following MG132 treatment. shRNA knock-down of Tax expression also increased the expression of BCL11B in HTLV-1-infected cells. Moreover, we found that Tax physically binds to BCL11B protein and induces the polyubiquitination of BCL11B and proteasome-dependent degradation of BCL11B. Thus, inactivation of BCL11B by Tax protein may play an important role in the Tax-mediated leukemogenesis.

DOI： 10.1016/j.bbrc.2017.06.172

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Loss of the tumor suppressor NDRG2 has been implicated in the development of oral squamous cell carcinoma (OSCC), acting by modulating PI3K/AKT-mediated dephosphorylation of PTEN at S380/S382/T383 (STT). Here, we show that the majority of OSCC tumors with lymph node metastasis, a major prognostic factor, exhibit high levels of phosphorylated AKT-S473 and PTEN-STT and low levels of NDRG2 expression. In Ndrg2-deficient mice, which develop a wide range of tumors, we developed a model of OSCC by treatment with the tobacco surrogate 4-nitroquinoline-1-oxide (4-NQO). In this model, both the number and size of OSCC tumors were increased significantly by Ndrg2 deficiency, which also increased invasion of cervical lymph nodes. 4-NQO treatment of human OSCC cell lines exhibiting low NDRG2 expression induced epithelial-mesenchymal transition via activation of NF-κB signaling. Conversely, ectopic expression of NDRG2 reversed the EMT phenotype and inhibited NF-κB signaling via suppression of PTEN-STT and AKT-S473 phosphorylation. Our results show how NDRG2 expression serves as a critical determinant of the invasive and metastatic capacity of OSCC. Cancer Res; 77(9); 2363-74. ©2017 AACR.

DOI： 10.1158/0008-5472.CAN-16-2114

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Language：English   Publishing type：Research paper (scientific journal)  

Iron is an essential nutrient for normal cell growth, and reprogramming of iron metabolism is essential to tumor cell survival and progression. HTLV-1-associated adult T-cell leukemia/lymphoma (ATLL) has no effective therapy and high levels of cell surface transferrin receptor 1 (TFR1) expression have been reported in ATLL by us and other groups. In this study, to develop a novel molecular-targeted therapy against TFR1 to modulate iron metabolism, we initially determined the expression pattern of several iron-related genes along with TFR1 and found that ATLL cells presented characteristic of an iron-deficiency state such as high expression of iron-regulatory protein 2 (IRP2) and low expression of its E3 ubiquitin-ligase, FBXL5. Therefore, we developed human IgG monoclonal antibodies to human TFR1 using a phage display method (ICOS method) to block the incorporation of the transferrin (TF)-iron complex into ATLL cells for inhibiting cell growth. One of the mAbs, JST-TFR09, presented its greater affinity to TFR1 on ATLL cells in flow cytometry (FCM) analysis than those of commercially available anti-TFR1 antibodies and identified high expression of TFR1 in most of the acute-type ATLL cells. Moreover, JST-TFR09 could interfere with binding between TFR1 and TF, which resulted in effective blockade of TFR1 internalization and induction of cell apoptosis by the treatment of ATLL cells with JST-TFR09. JST-TFR09 showed dual activities through direct cell cytotoxicity and antibody-dependent cellular cytotoxicity (ADCC), and the treatment of JST-TFR09 significantly suppressed cell growth of ATLL cells with induction of apoptosis in in vitro and in vivo experiments. Thus, JST-TFR09 described here may become a promising therapeutic antibody for the treatment of ATLL.

DOI： 10.1016/j.bbrc.2017.02.039

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Language：English   Publishing type：Research paper (scientific journal)  

The presence of a BCR-ABL1 fusion gene is necessary for the pathogenesis of chronic myeloid leukemia (CML) through t(9;22)(q34;q11) translocation. Imatinib, an ABL tyrosine kinase inhibitor, is dramatically effective in CML patients; however, 30% of CML patients will need further treatment due to progression of CML to blast crisis (BC). Aberrant high expression of ecotropic viral integration site 1 (EVI1) is frequently observed in CML during myeloid-BC as a potent driver with a CML stem cell signature; however, the precise molecular mechanism of EVI1 transcriptional regulation during CML progression is poorly defined. Here, we demonstrate the transcriptional activity of EVI1 is dependent on activation of lymphoid enhancer-binding factor 1 (LEF1)/β-catenin complex by BCR-ABL with loss of p53 function during CML-BC. The activation of β-catenin is partly dependent on BCR-ABL expression through enhanced GSK3β phosphorylation, and EVI1 expression is directly enhanced by the LEF1/β-catenin complex bound to the EVI1 promoter region. Moreover, the loss of p53 expression is inversely correlated with high expression of EVI1 in CML leukemia cells with an aggressive phase of CML, and a portion of the activation mechanism of EVI1 expression is dependent on β-catenin activation through GSK3β phosphorylation by loss of p53. Therefore, we found that the EVI1 activation in CML-BC is dependent on LEF1/β-catenin activation by BCR-ABL expression with loss of p53 function, representing a novel selective therapeutic approach targeting myeloid blast crisis progression.

DOI： 10.1016/j.bbrc.2016.11.146

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Language：English   Publishing type：Research paper (scientific journal)  

N-myc downstream-regulated gene 2 (NDRG2) is one of the important stress-inducible genes and plays a critical role in negatively regulating PI3K/AKT signaling during hypoxia and inflammation. Through recruitment of PP2A phosphatase, NDRG2 maintains the dephosphorylated status of PTEN to suppress excessive PI3K/AKT signaling, and loss of NDRG2 expression is frequently seen in various types of cancer with enhanced activation of PI3K/AKT signaling. Because NDRG2 is highly expressed in the nervous system, we investigated whether NDRG2 plays a functional role in the nervous system using Ndrg2-deficient mice. Ndrg2-deficient mice do not display any gross abnormalities in the nervous system, but they have a diminished behavioral response associated with anxiety. Ndrg2-deficient mice exhibited decreased immobility and increased head-dipping and rearing behavior in two behavioral models, indicating an improvement of emotional anxiety-like behavior. Moreover, treatment of wild-type mice with the antidepressant drug imipramine reduced the expression of Ndrg2 in the frontal cortex, which was due to the degradation of HIF-1α through reduced expression of HSP90 protein. Furthermore, we found that the down-regulation of Ndrg2 in Ndrg2-deficient mice and imipramine treatment improved mood behavior with enhanced phosphorylation of GSK3β through activation of PI3K/AKT signaling, suggesting that the expression level of NDRG2 has a causal influence on mood-related phenotypes. Collectively, these results suggest that NDRG2 may be a potential target for mood disorders such as depression and anxiety.

DOI： 10.1016/j.cellsig.2015.07.012

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Language：English   Publisher：ELSEVIER SCIENCE INC  

DOI： 10.1016/j.exphem.2015.06.204

Web of Science

Language：English   Publishing type：Research paper (scientific journal)  

The activation of nuclear factor kappa B (NF-κB) signaling has a central role in the development of adult T-cell leukemia/lymphoma (ATL) and many other cancers. However, the activation mechanism of the NF-κB pathways remains poorly understood. Recently, we reported that N-myc downstream-regulated gene 2 (NDRG2) is a negative regulator of the phosphoinositide 3-kinase (PI3K)/AKT pathway by promoting the active dephosphorylated form of PTEN at its C-terminus via the recruitment of PP2A. Additionally, the down-regulation of NDRG2 expression promotes the inactive phosphorylated form of PTEN, which results in constitutively active PI3K/AKT signaling in various cancer cell types. Here, we investigated the involvement of NDRG2 in modulating NF-κB signaling. The forced expression of NDRG2 in ATL cells down-regulates not only the canonical pathway by inhibiting AKT signaling but also the non-canonical pathway by inducing NF-κB-inducing kinase (NIK) dephosphorylation via the recruitment of PP2A. Therefore, NDRG2 works as a PP2A recruiter to suppress not only PI3K/AKT signaling but also NF-κB signaling, which is particularly important in host defenses or immune responses to Human T-cell leukemia virus type 1 (HTLV-1) infection. Furthermore, the loss of NDRG2 expression might play an important role in the progression of tumor development after HTLV-1 infection.

DOI： 10.1038/srep12841

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Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1182/blood-2014-08-594093

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Language：English   Publishing type：Research paper (scientific journal)  

Oral squamous cell carcinoma (OSCC) is the sixth most common cancer worldwide. Up to 20% of oral dysplasia cases have been suggested to undergo malignant transformation to OSCC; however, there are no methods to predict OSCC development. In this study, to identify the genes associated with oral dysplasia progression, we performed genomic copy number analyses of genomic DNA samples isolated from primary oral dysplasia and OSCC via the microdissection method and found elevated expression of transferrin receptor C (TfR1/TFRC) with genomic amplification in oral dysplasia and OSCC. The expression rate of TFRC in OSCC was significantly higher than that in dysplasia, suggesting that OSCC disease progression might be related to TFRC expression. Additionally, we investigated the in vitro and in vivo impacts of a newly established anti-human TFRC monoclonal antibody, which was isolated from a human cDNA library using the phage-display method, on cell proliferation and survival. The anti-TFRC antibody blocked the interaction between transferrin and TFRC and consequently inhibited iron uptake, leading to the iron deprivation-mediated suppression of cell growth and induction of apoptosis. Moreover, we demonstrated that the anti-TFRC antibody efficiently inhibited tumor growth in a murine xenograft OSCC model. Therefore, we suggest our developed complete human anti-human TFRC antibody as a useful, novel treatment for oral dysplasia and OSCC.

DOI： 10.1002/cam4.267

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Language：English   Publishing type：Research paper (scientific journal)  

Constitutive phosphatidylinositol 3-kinase (PI3K)-AKT activation has a causal role in adult T-cell leukaemia-lymphoma (ATLL) and other cancers. ATLL cells do not harbour genetic alterations in PTEN and PI3KCA but express high levels of PTEN that is highly phosphorylated at its C-terminal tail. Here we report a mechanism for the N-myc downstream-regulated gene 2 (NDRG2)-dependent regulation of PTEN phosphatase activity via the dephosphorylation of PTEN at the Ser380, Thr382 and Thr383 cluster within the C-terminal tail. We show that NDRG2 is a PTEN-binding protein that recruits protein phosphatase 2A (PP2A) to PTEN. The expression of NDRG2 is frequently downregulated in ATLL, resulting in enhanced phosphorylation of PTEN at the Ser380/Thr382/Thr383 cluster and enhanced activation of the PI3K-AKT pathway. Given the high incidence of T-cell lymphoma and other cancers in NDRG2-deficient mice, PI3K-AKT activation via enhanced PTEN phosphorylation may be critical for the development of cancer.

DOI： 10.1038/ncomms4393

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Language：English   Publishing type：Research paper (scientific journal)  

Acute myeloid leukemia with high ecotropic viral integration site-1 expression (EVI1(high) AML) is classified as a refractory type of leukemia with a poor prognosis. To provide new insights into the prevention and treatment of this disease, we identified the high expression of EVI1-regulated G protein-coupled receptor 56 (GPR56), and the association of high cell adhesion and antiapoptotic activities in EVI1(high) AML cells. Knockdown of GPR56 expression decreased the cellular adhesion ability through inactivation of RhoA signaling, resulting in a reduction of cellular growth rates and enhanced apoptosis. Moreover, in Gpr56(-/-) mice, the number of hematopoietic stem cells (HSCs) was significantly decreased in the bone marrow (BM) and, conversely, was increased in the spleen, liver and peripheral blood. The number of Gpr56(-/-) HSC progenitors in the G0/G1-phase was significantly reduced and was associated with impaired cellular adhesion. Finally, the loss of GPR56 function resulted in a reduction of the in vivo repopulating ability of the HSCs. In conclusion, GPR56 may represent an important GPCR for the maintenance of HSCs by acting as a co-ordinator of interactions with the BM osteosteal niche; furthermore, this receptor has the potential to become a novel molecular target in EVI1(high) leukemia.

DOI： 10.1038/leu.2013.75

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Language：English  

Cell adhesion molecule 1 (CADM1/TSLC1) was recently identified as a novel cell surface marker for adult T-cell leukemia/lymphoma (ATLL). In this study, we developed various antibodies as diagnostic tools to identify CADM1-positive ATLL leukemia cells. In flow cytometric analysis, the percentages of CD4(+)CADM1(+) double-positive cells correlated well with both the percentages of CD4(+)CD25(+) cells and with abnormal lymphocytes in the peripheral blood of patients with various types of ATLL. Moreover, the degree of CD4(+)CADM1(+) cells over 1% significantly correlated with the copy number of the human T-lymphotropic virus type 1 (HTLV-1) provirus in the peripheral blood of HTLV-1 carriers and ATLL patients. We also identified a soluble form of CADM1 in the peripheral blood of ATLL patients, and the expression levels of this form were correlated with the levels of soluble interleukin 2 receptor alpha. Moreover, lymphomas derived from ATLL were strongly and specifically stained with a CADM1 antibody. Thus, detection of CD4(+)CADM1(+) cells in the peripheral blood, measurement of serum levels of soluble CADM1 and immunohistochemical detection of CADM1 in lymphomas would be a useful set of markers for disease progression in ATLL and may aid in both the early diagnosis and measurement of treatment efficacy for ATLL.

DOI： 10.1038/leu.2011.379

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Language：English   Publishing type：Research paper (scientific journal)  

The development of oral squamous cell carcinoma (OSCC) is a multistep process that requires the accumulation of genetic alterations. To identify genes responsible for OSCC development, we performed high-density single nucleotide polymorphism array analysis and genome-wide gene expression profiling on OSCC tumors. These analyses indicated that the absent in melanoma 2 (AIM2) gene and the interferon-inducible gene 16 (IFI16) mapped to the hematopoietic interferon-inducible nuclear proteins. The 200-amino-acid repeat gene cluster in the amplified region of chromosome 1q23 is overexpressed in OSCC. Both AIM2 and IFI16 are cytoplasmic double-stranded DNA sensors for innate immunity and act as tumor suppressors in several human cancers. Knockdown of AIM2 or IFI16 in OSCC cells results in the suppression of cell growth and apoptosis, accompanied by the downregulation of nuclear factor kappa-light-chain-enhancer of activated B cells activation. Because all OSCC cell lines have reduced p53 activity, wild-type p53 was introduced in p53-deficient OSCC cells. The expression of wild-type p53 suppressed cell growth and induced apoptosis via suppression of nuclear factor kappa-light-chain-enhancer of activated B cells activity. Finally, the co-expression of AIM2 and IFI16 significantly enhanced cell growth in p53-deficient cells; in contrast, the expression of AIM2 and/or IFI16 in cells bearing wild-type p53 suppressed cell growth. Moreover, AIM2 and IFI16 synergistically enhanced nuclear factor kappa-light-chain-enhancer of activated B cells signaling in p53-deficient cells. Thus, expression of AIM2 and IFI16 may have oncogenic activities in the OSCC cells that have inactivated the p53 system.

DOI： 10.1111/j.1349-7006.2012.02211.x

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Language：English   Publishing type：Research paper (scientific journal)  

CADM1/TSLC1 (Cell adhesion molecule 1/Tumor suppressor in lung cancer 1) is a cell adhesion molecule that was originally identified as a tumor suppressor in lung cancer. CADM1/TSLC1 expression is reduced in a variety of cancers via promoter methylation, and this reduction is associated with poor prognosis and enhanced metastatic potential. In contrast, we observed that CADM1/TSLC1 is highly and ectopically expressed in all primary adult T-cell leukemia/lymphoma (ATLL) cells and in most human T-cell leukemia virus type (HTLV)-1-infected T-cell and ATLL cell lines. No expression, however, was detected in CD4⁺ T cells or in several other non-HTLV-1-infected leukemia cells. Moreover, we identified that high CADM1/TSLC1 expression plays an important role in enhanced cell-cell adhesion to the vascular endothelium, tumor growth and the ability of ATLL cells to infiltrate organs. We developed various antibodies as diagnostic tools to identify CADM1⁺ ATLL cells. Using flow cytometry, we determined that CADM1/TSLC1 is present on the surface of ATLL cells. The percentage of CD4⁺CADM1⁺ cells in the peripheral blood of HTLV-1 carriers and ATLL patients was highly correlated with the DNA copy number of HTLV-1 in lymphocytes. In particular, we identified the soluble form of CADM1/TSLC1 in the peripheral blood of HTLV-1 carriers and ATLL patients. Therefore, measurements of soluble CADM1/TSLC1 serum levels and the detection of CD4⁺CADM1⁺ cells in the blood, when combined with standard diagnostic methods, would be useful for identifying and monitoring disease progression in HTLV-1 carriers. Such tests would provide increased accuracy and may aid in early diagnosis and in determining the effects of ATLL treatments.

PubMed

Language：English   Publisher：The Japanese Society for Lymphoreticular Tissue Research  

<I>CADM1/TSLC1 (Cell adhesion molecule 1/Tumor suppressor in lung cancer 1)</I> is a cell adhesion molecule that was originally identified as a tumor suppressor in lung cancer. <I>CADM1/TSLC1</I> expression is reduced in a variety of cancers via promoter methylation, and this reduction is associated with poor prognosis and enhanced metastatic potential. In contrast, we observed that <I>CADM1/TSLC1</I> is highly and ectopically expressed in all primary adult T-cell leukemia/lymphoma (ATLL) cells and in most human T-cell leukemia virus type (HTLV)-1-infected T-cell and ATLL cell lines. No expression, however, was detected in CD4<SUP>+</SUP> T cells or in several other non-HTLV-1-infected leukemia cells. Moreover, we identified that high CADM1/TSLC1 expression plays an important role in enhanced cell-cell adhesion to the vascular endothelium, tumor growth and the ability of ATLL cells to infiltrate organs. We developed various antibodies as diagnostic tools to identify CADM1<SUP>+</SUP> ATLL cells. Using flow cytometry, we determined that CADM1/TSLC1 is present on the surface of ATLL cells. The percentage of CD4<SUP>+</SUP>CADM1<SUP>+</SUP> cells in the peripheral blood of HTLV-1 carriers and ATLL patients was highly correlated with the DNA copy number of HTLV-1 in lymphocytes. In particular, we identified the soluble form of CADM1/TSLC1 in the peripheral blood of HTLV-1 carriers and ATLL patients. Therefore, measurements of soluble CADM1/TSLC1 serum levels and the detection of CD4<SUP>+</SUP>CADM1<SUP>+</SUP> cells in the blood, when combined with standard diagnostic methods, would be useful for identifying and monitoring disease progression in HTLV-1 carriers. Such tests would provide increased accuracy and may aid in early diagnosis and in determining the effects of ATLL treatments. [<I>J Clin Exp Hematopathol 52(1) : 17-22, 2012</I>]

DOI： 10.3960/jslrt.52.17

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Language：Japanese   Publisher：Oncogene  

MEL1 (MDS1/EVI1-like gene 1/PRDM16), which was identified as a gene near the chromosomal breakpoint in t(1;3)(p36;q21)-positive human acute myeloid leukemia cells, belongs to the PRDI-BF1-RIZ1 homologous (PR) domain (PRDM) family of transcription repressors. The short form of MEL1 (MEL1S), which lacks the PR-domain at the N-terminus, is the main form expressed in t(1;3)(p36;q21)-positive acute myeloid leukemia cells. The overexpression of MEL1S blocks granulocyte colony-stimulating factor (G-CSF)-induced myeloid differentiation in interleukin-3-dependent murine myeloid L-G3 cells. In this study, we show that treatment with the histone deacetylase inhibitor trichostatin A abolished the blockade of myeloid differentiation in L-G3 cells overexpressing MEL1S. The expression of MEL1S containing mutated CtBP-interacting motif (CIM) in L-G3 cells still blocked the myeloid differentiation induced by G-CSF. We found that the small ubiquitin-related modifier (SUMO) motif (SM) at lysine 568 (VKAE) adjacent to the CIM was necessary to obtain the maximum transcriptional repressor activity of MEL1S. L-G3 cells expressing MEL1S, and bearing mutated CIM and SM differentiated into granulocytes in response to G-CSF; this indicated that both the SUMO modification at lysine 568 and CtBP binding were required for MEL1S-mediated transcriptional repression and blockade of differentiation, which might be relevant for the process of leukemogenesis. © 2011 Macmillan Publishers Limited All rights reserved.

DOI： 10.1038/onc.2011.132

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Ecotropic viral integration site 1 (EVI1) is an oncogenic transcription factor in human acute myeloid leukemia (AML) with chromosomal alterations at 3q26. Because a high expression of EVI1 protein in AML cells predicts resistance to chemotherapy with a poor outcome, we have searched for molecular targets that will treat these patients with AML. In this study, we determined that CD52, which is mainly expressed on lymphocytes, is highly expressed in most cases of AML with a high EVI1 expression (EVI1(High)). CAMPATH-1H, a humanized monoclonal antibody against CD52, has been used to prevent graft-versus-host disease and treat CD52-positive lymphoproliferative disorders. Here, we investigated the antitumor effect of CAMPATH-1H on EVI1(High) AML cells. CAMPATH-1H significantly inhibited cell growth and induced apoptosis in CD52-positive EVI1(High) leukemia cells. Furthermore, CAMPATH-1H induced complement-dependent cytotoxicity and antibody-dependent cellular cytotoxicity against CD52-positive EVI1(High) leukemia cells. After an intravenous injection of CAMPATH-1H into NOD/Shi-scid/IL-2Rγ;null mice with subcutaneous engraftment of EVI1(High) leukemia cells, tumor growth rates were significantly reduced, and the mice survived longer than those in the phosphate-buffered saline-injected control group. Thus, CAMPATH-1H is a potential therapeutic antibody for the treatment of patients with EVI1(High) leukemia.

DOI： 10.1038/leu.2011.36

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Language：Japanese   Publisher：(一社)日本血液学会-東京事務局  

Language：Japanese   Publisher：Oncogene  

Zinc-finger E-box binding homeobox 1 (ZEB1) is a candidate tumor-suppressor gene in adult T-cell leukemia/lymphoma (ATLL). ZEB1 binds phosphorylated Smad2/3 to enhance transforming growth factor-Β1 (TGF-Β1) signaling. In addition to downregulation of ZEB1 mRNA, we found overexpression of inhibitory Smad, Smad7, in resistance of ATLL cells to growth suppression by TGF-Β1. A protein complex of Smad7 and histone deacetylase constantly bound to the promoter region of TGF-Β1 responsive genes with the Smad-responsive element (SRE) to inhibit TGF-Β1 signaling; however, ectopic expression of ZEB1 reactivated TGF-Β1 signaling by binding to Smad7 and recruiting the Smad3/p300 histone acetyltransferase complex to the promoter after TGF-Β1 stimulation in ATLL. Conversely, because ZEB1 mRNA was detected in the late stages of T-cell development, we used CTLL2 cells with ZEB1 expression, a murine peripheral T-cell lymphoma, and found that a complex of Smad3, Smad7 and ZEB1 was bound to the SRE of the p21 CDKN1A promoter after the induction of Smad7 by TGF-Β1 treatment. Because the duration of TGF-Β1-induced transcriptional activation of PAI-1 and p21 was shortened in shZEB1-expressing CTLL2 cells, ZEB1 may have a role in enhancing TGF-Β1 signaling by binding not only to Smad3 but also to Smad7 in the nucleus. Altogether, these results suggest that both ZEB1 downregulation and Smad7 overexpression contribute to resistance to TGF-Β1-mediated growth suppression in ATLL. © 2010 Macmillan Publishers Limited All rights reserved.

DOI： 10.1038/onc.2010.172

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Language：English   Publishing type：Research paper (scientific journal)  

Human T-lymphotropic virus 1 (HTLV-1) causes an aggressive malignancy of T lymphocytes called adult T-cell leukemia/lymphoma (ATLL), and expression of HTLV-1 Tax influences cell survival, proliferation, and genomic stability in the infected T lymphocytes. Cyclin-dependent kinase inhibitor 1A (CDKN1A/p21(waf1/Cip1)) is upregulated by Tax, without perturbation of cell cycle control. During an analysis of the gene expression profiles of ATLL cells, we found very low expression of CDKN1A in ATLL-derived cell lines and ATLL cells from patient samples, and epigenetic abnormalities including promoter methylation are one of the mechanisms for the low CDKN1A expression in ATLL cells. Three HTLV-1-infected cell lines showed high levels of expression of both CDKN1A and Tax, but expression of CDKN1A was detected in only two of six ATLL-derived cell lines. In both the HTLV-1-infected and ATLL cell lines, we found that activated Akt phosphorylates CDKN1A at threonine 145 (T145), leading to cytoplasmic localization of CDKNIA. In HTLV-1-infected cell lines, cytoplasmic CDKN1A did not inhibit the cell cycle after UV irradiation; however, following treatment with LY294002, a PI3K inhibitor, CDKN1A was dephosphorylated and relocalized to the nucleus, resulting in suppression of the cell cycle. In the ATLL cell lines, treatment with LY294002 did not inhibit the cell cycle but induced apoptosis with the cytoplasmic localization. Therefore, the low CDKN1A expression in ATLL cells may be a key player in ATLL leukemogenesis, and the abnormal genomic methylation may influence the expression of not only HTLV-1 Tax but also CDKN1A during long-term development of ATLL from the HTLV-1-infected T lymphocytes.

DOI： 10.1128/JVI.00073-10

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Language：English   Publishing type：Research paper (scientific journal)  

Oral cancer is one of the most common cancers worldwide, and squamous-cell carcinoma (OSCC) is the most common phenotype of oral cancer. Although patients with OSCC have poor survival rates and a high incidence of metastasis, the molecular mechanisms of OSCC development have not yet been elucidated. This study investigated whether N-myc downstream-regulated gene 2 (NDRG2) contributes to the carcinogenesis of OSCC, as NDRG2 is reported to be a candidate tumor-suppressor gene in a wide variety of cancers. The down-regulation of NDRG2 mRNA, which was dependent on promoter methylation, was seen in the majority of OSCC cases and in several cases of precancerous leukoplakia with dysplasia. Induction of NDRG2 expression in an HSC-3/OSCC cell line significantly inhibited cell proliferation and decreased colony formation ability on soft agar. The majority of OSCC cell lines showed an activation of PI3K/Akt signaling, and enforced expression of NDRG2 in HSC-3 cells decreased the level of phosphorylated Akt at Serine 473 (p-Akt). Immunohistochemical p-Akt staining was detected in 56.5% of the OSCC tumors, and 80.4% of the tumors were negative for NDRG2 staining. Moreover, positive p-Akt staining was inversely correlated with decreased NDRG2 expression in OSCC tumors with moderate to poor differentiation (p<0.005). Therefore, NDRG2 is a candidate tumor-suppressor gene for OSCC development and probably contributes to the tumorigenesis of OSCC partly via the modulation of Akt signaling.

DOI： 10.1016/j.bbrc.2009.12.156

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Language：English   Publishing type：Research paper (scientific journal)  

Adult T-cell leukemia/lymphoma (ATLL) is a malignant tumor caused by latent human T-lymphotropic virus 1 (HTLV-1) infection. We previously identified a common breakpoint cluster region at 10p11.2 in acute-type ATLL by spectral karyotyping. Single nucleotide polymorphism array comparative genomic hybridization analysis of the breakpoint region in three ATLL-related cell lines and four patient samples revealed that the chromosomal breakpoints are localized within the enhancer of polycomb 1 (EPC1) gene locus in an ATLL-derived cell line (SO4) and in one patient with acute-type ATLL. EPC1 is a human homologue of the E(Pc) enhancer of polycomb gene of Drosophila. Inappropriate expression of the polycomb group gene family has been linked to the loss of normal gene silencing pathways, which can contribute to the loss of cell identity and malignant transformation in many kinds of cancers. In the case of the SO4 cell line, which carried a der(10)t(2;10)(p23;p11.2) translocation, EPC1 was fused with the additional sex combs-like 2 (ASXL2) gene at 2p23.3 (EPC1/ASXL2). In the case with an acute-type ATLL, who carried a der(10)del(10)(p11.2)del(10)(q22q24) translocation, a putative truncated EPC1 gene (EPC1tr) was identified. Overexpression of EPC1/ASXL2 enhanced cell growth in T-leukemia cells, and a GAL4-EPC1/ASXL2 fusion protein showed high transcriptional activity. Although a GAL4-EPC1tr fusion protein did not activate transcription, overexpression of EPC1tr accelerated cell growth in leukemia cells, suggesting that the EPC1 structural abnormalities in the SO4 cell line and in the patient with acute-type ATLL may contribute to leukemogenesis.

DOI： 10.1002/gcc.20681

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Language：English   Publishing type：Research paper (scientific journal)  

Intracytoplasmic sperm injection (ICSI) is a technique that has been successfully used for assisting reproduction in mammals. However, this method is still not reliable in nonmammalian species, including teleosts. We succeeded in producing medaka individuals by ICSI with a rate of 13.4% (28 hatched embryos out of 209 eggs fertilized by ICSI), the best value reported so far in teleosts, including zebrafish and Nile tilapia. Although the technique was based on that developed for mammalian eggs, some critical modifications were made to adjust it to the medaka egg, which has a thick and hard envelope (the chorion) and a single sperm entry site (the micropyle). Medaka ICSI was performed by injecting a demembranated spermatozoon into an egg cytoplasm through the micropyle 10-15 sec after egg activation induced by a piezo-actuated vibration, the site and timing of sperm penetration being consistent with those in normal fertilization in medaka. To increase the efficiency of ICSI in medaka, we found that the fertilization by ICSI should precisely mimic the fertilization by insemination with intact sperm, both spatially and temporally. The success rate of ICSI was highly variable in batches of eggs (ranging from 0% to 56%), suggesting that the conditions of eggs are important factors in stabilizing the production of individuals by ICSI. The success in medaka ICSI provides a basis for future research to understand the basic mechanisms in gamete biology of teleosts as well as for development of new technology that can yield valuable applications in fisheries science.

DOI： 10.1095/biolreprod.108.069880

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Language：English   Publishing type：Research paper (scientific journal)  

Adult T-cell leukemia (ATL) is associated with human T-cell leukemia virus type 1 infection. The tumor suppressor lung cancer 1 (TSLC1) gene was previously identified as a novel cell surface marker for ATL, and this study demonstrated the involvement of TSLC1 expression in tumor growth and organ infiltration of ATL cells. In experiments using NOD/SCID/gamma c(null) mice, both leukemia cell lines and primary ATL cells with high TSLC1 expression caused more tumor formation and aggressive infiltration of various organs of mice. Our results suggest that TSLC1 expression in ATL cells plays an important role in the growth and organ infiltration of ATL cells.

DOI： 10.1128/JVI.01149-08

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Language：English   Publishing type：Research paper (scientific journal)  

Adult T-cell leukemia/lymphoma (ATLL) is caused by latent human T-lymphotropic virus-1 (HTLV-1) infection. To clarify the molecular mechanism underlying leukemogenesis after viral infection, we precisely mapped 605 chromosomal breakpoints in 61 ATLL cases by spectral karyotyping and identified frequent chromosomal breakpoints in 10p11, 14q11, and 14q32. Single nucleotide polymorphism (SNP) array-comparative genomic hybridization (CGH), genetic, and expression analyses of the genes mapped within a common breakpoint cluster region in 10p11.2 revealed that in ATLL cells, transcription factor 8 (TCF8) was frequently disrupted by several mechanisms, including mainly epigenetic dysregulation. TCF8 mutant mice frequently developed invasive CD4(+) T-cell lymphomas in the thymus or in ascitic fluid in vivo. Down-regulation of TCF8 expression in ATLL cells in vitro was associated with resistance to transforming growth factor beta1 (TGF-beta1), a well-known characteristic of ATLL cells, suggesting that escape from TGF-beta1-mediated growth inhibition is important in the pathogenesis of ATLL. These findings indicate that TCF8 has a tumor suppressor role in ATLL.

DOI： 10.1182/blood-2008-01-131185

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Language：English   Publishing type：Research paper (scientific journal)  

An intronic hexanucleotide UGCAUG has been shown to play a critical role in the regulation of tissue-specific alternative splicing of pre-mRNAs in a wide range of tissues. Vertebrate Fox-1 has been shown to bind to this element, in a highly sequence-specific manner, through its RNA recognition motif (RRM). In mammals, there are at least two Fox-1-related genes, ataxin-2 binding protein 1 (A2BP1)/Fox-1 and Fxh/Rbm9, which encode an identical RRM. Here, we demonstrate that both mouse Fxh and A2BP1 transcripts undergo tissue-specific alternative splicing, generating protein isoforms specific to brain and muscle. These tissue-specific isoforms are characterized for their abilities to regulate neural cell-specific alternative splicing of a cassette exon, N30, in the non-muscle myosin heavy chain II-B pre-mRNA, previously shown to be regulated through an intronic distal downstream enhancer (IDDE). All Fxh and A2BP1 isoforms with the RRM are capable of binding to the IDDE in vitro through the UGCAUG elements. Each isoform, however, shows quantitative differences in splicing activity and nuclear distribution in transfected cells. All Fxh isoforms and a brain isoform of A2BP1 show a predominant nuclear localization. Brain isoforms of both Fxh and A2BP1 promote N30 splicing much more efficiently than do the muscle-specific isoforms. Skeletal muscles express additional isoforms that lack a part of the RRM. These isoforms are incapable of activating neural cell-specific splicing and, moreover, can inhibit UGCAUG-dependent N30 splicing. These findings suggest that tissue-specific isoforms of Fxh and A2BP1 play an important role in determining tissue specificity of UGCAUG-mediated alternative splicing.

DOI： 10.1093/nar/gki338

PubMed

Language：English   Publishing type：Research paper (scientific journal)  

Protein synthesis of cyclin B by translational activation of the dormant mRNA stored in oocytes is required for normal progression of maturation. In this study, we investigated the involvement of Xenopus Pumilio (XPum), a cyclin B1 mRNA-binding protein, in the mRNA-specific translational activation. XPum exhibits high homology to mammalian counterparts, with amino acid identity close to 90%, even if the conserved RNA-binding domain is excluded. XPum is bound to cytoplasmic polyadenylation element (CPE)-binding protein (CPEB) through the RNA-binding domain but not to its phosphorylated form in mature oocytes. In addition to the CPE, the XPum-binding sequence of cyclin B1 mRNA acts as a cis-element for translational repression. Injection of anti-XPum antibody accelerated oocyte maturation and synthesis of cyclin B1, and, conversely, over-expression of XPum retarded oocyte maturation and translation of cyclin B1 mRNA, which was accompanied by inhibition of poly(A) tail elongation. The injection of antibody and the over-expression of XPum, however, had no effect on translation of Mos mRNA, which also contains the CPE. These findings provide the first evidence that XPum is a translational repressor specific to cyclin B1 in vertebrates. We propose that in cooperation with the CPEB-maskin complex, the master regulator common to the CPE-containing mRNAs, XPum acts as a specific regulator that determines the timing of translational activation of cyclin B1 mRNA by its release from phosphorylated CPEB during oocyte maturation.

DOI： 10.1016/s0925-4773(03)00160-6

PubMed

Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

Translational activation of dormant cyclin B1 mRNA stored in oocytes is a prerequisite for the initiation or promotion of oocyte maturation in many vertebrates. Using a monoclonal antibody against the domain highly homologous to that of Drosophila Pumilio, we have shown for the first time in any vertebrate that a homolog of Pumilio is expressed in Xenopus oocytes, This 137-kDa protein binds to the region including the sequence UGUA at nucleotides 1335-1338 in the 3'-untranslated region of cyclin B1 mRNA, which is close to but does not overlap the cytoplasmic polyadenylation elements (CPEs). Physical in vitro association of Xenopus Pumilio with a Xenopus homolog of Nanos (Xcat-2) was demonstrated by a protein pull-down assay. The results of immunoprecipitation experiments showed in vivo interaction between Xenopus Pumilio and CPE-binding protein (CPEB), a key regulator of translational repression and activation of mRNAs stared in oocytes, This evidence provides a new insight into the mechanism of translational regulation through the 3'-end of mRNA during oocyte maturation. These results also suggest the generality of the function of Pumilio as a translational regulator of dormant mRNAs in both invertebrates and vertebrates.

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