Terutoshi Hishita

CCAAT/enhancer-binding protein β expressed by bone marrow mesenchymal stromal cells regulates early B-cell lymphopoiesis.

The transcription factor CCAAT/enhancer‐binding protein β (C/EBPβ) regulates the differentiation of a variety of cell types. Here, the role of C/EBPβ expressed by bone marrow mesenchymal stromal cells (BMMSCs) in B‐cell lymphopoiesis was examined. The size of the precursor B‐cell population in bone marrow was reduced in C/EBPβ‐knockout (KO) mice. When bone marrow cells from C/EBPβ‐KO mice were transplanted into lethally irradiated wild‐type (WT) mice, which provide a normal bone marrow microenvironment, the size of the precursor B‐cell population was restored to a level equivalent to that generated by WT bone marrow cells. In coculture experiments, BMMSCs from C/EBPβ‐KO mice did not support the differentiation of WT c‐Kit+ Sca‐1+ Lineage− hematopoietic stem cells (KSL cells) into precursor B cells, whereas BMMSCs from WT mice did. The impaired differentiation of KSL cells correlated with the reduced production of CXCL12/stromal cell‐derived factor‐1 by the cocultured C/EBPβ‐deficient BMMSCs. The ability of C/EBPβ‐deficient BMMSCs to undergo osteogenic and adipogenic differentiation was also defective. The survival of leukemic precursor B cells was poorer when they were cocultured with C/EBPβ‐deficient BMMSCs than when they were cocultured with WT BMMSCs. These results indicate that C/EBPβ expressed by BMMSCs plays a crucial role in early B‐cell lymphopoiesis. Stem Cells 2014;32:730–740

Parathyroid Hormone Enhances Hematopoietic Expansion Via Upregulation of Cadherin-11 in Bone Marrow Mesenchymal Stromal Cells

Parathyroid hormone (PTH) stimulates hematopoiesis in mouse models. The involvement of osteoblasts in this process has been well investigated; however, the effects of PTH on human hematopoiesis and bone marrow mesenchymal stromal cells (BM‐MSCs) are unclear. Here, we show that BM‐MSCs contribute to the hematopoiesis‐stimulating effects of PTH via upregulation of cadherin‐11 (CDH11). When culture‐expanded human BM‐MSCs were stimulated with PTH, their ability to expand cocultured CD34+ hematopoietic progenitor cells (HPCs) was enhanced. Furthermore, when PTH‐treated BM‐MSCs were subcutaneously implanted into NOD/SCID mice, the induction of hematopoietic cells was enhanced. Culture‐expanded human BM‐MSCs expressed CDH11, and the level of CDH11 expression increased following PTH stimulation. Depletion of CDH11 expression in BM‐MSCs using small interfering RNA abolished the enhancement of HPC expansion by PTH‐treated BM‐MSCs. In lethally irradiated mice that underwent BM transplantation, CDH11 expression in BM‐MSCs was higher and survival was better in PTH‐treated mice than in control mice. The number of hematopoietic cells in BM and the number of red blood cells in peripheral blood were higher in PTH‐treated mice than in control mice. Our results demonstrate that PTH stimulates hematopoiesis through promoting the upregulation of CDH11 expression in BM‐MSCs, at least in part. PTH treatment may be an effective strategy to enhance the ability of BM‐MSCs to support hematopoiesis. Stem Cells 2014;32:2245–2255

Leukemic and meningeal relapse of CD5+ intravascular large B-cell lymphoma with down-modulation of CD20 after rituximab therapy

Although CD20- relapses of B-cell lymphoma following rituximab therapy have increasingly been reported recently, coexistence of both the original and selected clones on relapse in a single patient have not been described. We experienced such a case with rare CD5+ intravascular lymphomatosis (IVL). A 46-year-old woman was admitted because of IVL complicated with cauda equina syndrome and pulmonary infarction. Complete remission was successfully achieved with multidrug chemotherapy in combination with rituximab. However, the disease recurred after 8 months with leukemic progression and meningeal involvement. The phenotype of the abnormal lymphocytes in the peripheral blood was fundamentally the same (CD20+CD5+CD10-CD19+CD23-sIgλ+) as that of the cells in the cerebrospinal fluid (CSF). However, CD20 expression was decreased remarkably compared with that in the CSF and that in the bone marrow before therapy. The targeting of CD20 molecules on the tumor cell surface by rituximab may have provided a selective pressure on lymphoma cells. The escape phenomenon of the lymphoma cells from rituximab was observed by simultaneously comparing the CD20 expression of cells in the peripheral blood and in a site of sanctuary from rituximab, the CSF.

Effects of the Tyrosine Kinase Inhibitor Imatinib Mesylate on a Bcr-Abl-Positive Cell Line: Suppression of Autonomous Cell Growth but No Effect on Decreased Adhesive Property and Morphological Changes

Expression of the Bcr-Abl oncoprotein alters various aspects of hematopoietic cells. We investigated the effects of a Bcr-Abl tyrosine kinase inhibitor, imatinib mesylate, on the proliferation, adhesive properties, and morphology of a Bcr-Abl-transferred cell line, TF-1 Bcr-Abl, in comparison with parental TF-1. First, the factor-independent growth of TF-1 Bcr-Abl was inhibited in the presence of imatinib mesylate, but this inhibition was overcome by addition of exogenous granulocyte-macrophage colony-stimulating factor. Imatinib mesylate remarkably reduced tyrosine phosphorylation of Bcr-Abl, Cbl, and Crkl in a time-dependent manner, and their complex formation also was affected. Imatinib mesylate inhibited activation of Stat5 rather than the MEK-ERK1/2 pathway. TF-1 Bcr-Abl cells exhibited a round shape, unlike TF-1, and the adhesive property to fibronectin was much lower than that of TF-1. Although the Bcr-Abl oncoprotein may be involved negatively in cell adhesion, the decreased adhesion and altered morphology of TF-1 Bcr-Abl cells were minimally affected by imatinib mesylate and seemed independent of Bcr-Abl kinase activity. The present data indicated that the Bcr-Abl-specific kinase inhibitor cannot control Bcr-Abl-induced cell alterations other than autonomous growth.Int J Hematol. 2003;78:233-240.

MALT lymphoma of the thymus with Sjögren’s syndrome: biphasic changes in serological abnormalities over a 4-year period following thymectomy

Thymic mucosa-associated lymphoid tissue (MALT) lymphoma shows distinct immunological characteristics, such as the expression of the IgA isotype, the frequent presence of immunoglobulin abnormalities, and a strong association with autoimmune disease, especially Sjögren’s syndrome (SjS). We report a case of thymic MALT lymphoma, who exhibited biphasic changes in her clinical characteristics during the 4-year observation period after thymectomy. A 71-year-old woman was admitted because of suspected SjS. A diagnosis of primary thymic MALT lymphoma was made, and SjS was confirmed. Serological abnormalities such as polyclonal hypergammaglobulinemia, IgA M protein, and elevated levels of rheumatoid factor were noted. These abnormalities improved rapidly after the thymectomy, but did not completely disappear. Interestingly, the remaining abnormalities, which can be ascribed to the proliferation of B cells throughout the body under the influence of SjS, have been improving slowly but steadily during the 4-year observation period. It is suspected that the removal of the tumor by thymectomy has more or less normalized the immunological environment and alleviated the SjS disease activity.

Sustained activation of MEK1-ERK1/2 pathway in membrane skeleton occurs dependently on cell adhesion in megakaryocytic differentiation

A human megakaryoblastic cell line, CMK, was treated with 12-o-tetradecanoylphorbol-13-acetate (TPA) for differentiation-induction. We examined TPA-induced activation of the MEK1-ERK1/2 pathway in the 100,000g Triton X-insoluble fraction of CMK cells as the membrane skeleton and researched the relation of the MEK1-ERK1/2 activation with integrin expression. We found that this activation was divided into two phases: the first activation occurred transiently in the membrane skeleton fraction of the suspended cell status and diminished after 1h; and the second sustained activation was maintained by cell adhesion. TPA-treated CMK cells revealed increased expression of integrins alphaIIb and beta3 only when the cell adhesion persisted, regardless of the difference of culture substratum. Sustained activation of the MEK1-ERK1/2 pathway is generated in the membrane skeleton by continuous cell adhesion and seems to be essential to TPA-induced megakaryocytic differentiation of CMK cells.

Bortezomib interferes with adhesion of B cell precursor acute lymphoblastic leukemia cells through SPARC up-regulation in human bone marrow mesenchymal stromal/stem cells

The poor prognosis of adults with B cell precursor acute lymphoblastic leukemia (BCP-ALL) is attributed to leukemia cells that are protected by the bone marrow (BM) microenvironment. In the present study, we explored the pharmacological targeting of mesenchymal stromal/stem cells in BM (BM-MSCs) to eliminate chemoresistant BCP-ALL cells. Human BCP-ALL cells (NALM-6 cells) that adhered to human BM-MSCs (NALM-6/Ad) were highly resistant to multiple anti-cancer drugs, and exhibited pro-survival characteristics, such as an enhanced Akt/Bcl-2 pathway and increased populations in the G0 and G2/S/M cell cycle stages. Bortezomib, a proteasome inhibitor, interfered with adhesion between BM-MSCs and NALM-6 cells and up-regulated the matricellular protein SPARC (secreted protein acidic and rich in cysteine) in BM-MSCs, thereby reducing the NALM-6/Ad population. Inhibition of SPARC expression in BM-MSCs using a small interfering RNA enhanced adhesion of NALM-6 cells. Conversely, recombinant SPARC protein interfered with adhesion of NALM-6 cells. These results suggest that SPARC disrupts adhesion between BM-MSCs and NALM-6 cells. Co-treatment with bortezomib and doxorubicin prolonged the survival of BCP-ALL xenograft mice, with a significant reduction of leukemia cells in BM. Our findings demonstrate that bortezomib contributes to the elimination of BCP-ALL cells through disruption of their adhesion to BM-MSCs, and offer a novel therapeutic strategy for BCP-ALL through targeting of BM-MSCs.

Ca2+Mg2+-dependent endonuclease in marrow CD34 positive and erythroid cells in myelodysplasia

Endonucleases capable of producing internucleosomal DNA cleavage are one of the key enzymes in apoptosis. We examined endonuclease activities contained in nuclei of CD34+ and erythroid cells in the bone marrow (BM) from 12 patients with the myelodysplastic syndromes. The levels of Mg(2+)-dependent and acidic endonucleases showed little changes as compared with those from normal BM. By contrast, the level of Ca2+/Mg(2+)-dependent endonuclease was appreciably higher in MDS erythroid cells than normal counterparts, although the activity varied markedly in CD34+ and erythroid cells. Our results suggested that Ca2+/Mg(2+)-dependent endonuclease is related to ineffective erythropoiesis in MDS.