[Show abstract][Hide abstract] ABSTRACT: T-cell prolymphocytic leukemia (T-PLL), a rare type of peripheral T-cell leukemia, is characterized by marked splenomegaly with rapidly progressive lymphocytosis and a poor prognosis. Nine kinds of ABL1 chimeric genes have been identified in various kinds of hematological malignancies, such as chronic myeloid leukemia and B- or T- lymphoblastic leukemia. However, there have been no reports describing T-PLL cases with ABL1 rearrangements. We herein report a case of T-PLL with a novel SEPT9-ABL1 fusion gene which induced strong resistance to tyrosine kinase inhibitors such as imatinib and dasatinib.
[Show abstract][Hide abstract] ABSTRACT: We analyzed the function of a SEPT9-ABL1 fusion identified in a case of T-prolymphocytic leukemia with tyrosine kinase inhibitor (TKI) resistance. Five isoforms with different N-termini, including SEPT9a-ABL1, SEPT9b-ABL1, SEPT9d-ABL1, SEPT9e-ABL1 and SEPT9f-ABL1, were detected in the leukemic cells. All isoforms except SEPT9d-ABL1 are localized in the cytoplasm, undergo autophosphorylation and phosphorylate the downstream targets, STAT-5 and Crkl, and provided IL-3-independence and in vivo invasiveness to 32D cells. Additionally, these SEPT9-ABL1 isoforms were resistant to TKIs in vitro and in vivo, in comparison to BCR-ABL1. These findings demonstrated that SEPT9-ABL1 had oncogenic activity and conferred resistance to TKIs.
[Show abstract][Hide abstract] ABSTRACT: We examined the antileukemic effects of high concentrations of L-ascorbic acid (high AA) on human leukemic cells. In vitro, high AA markedly induced apoptosis in various leukemic cell lines by generating hydrogen peroxide (H2O2) but not in normal hematopoietic stem/progenitor cells. High AA significantly repressed leukemic cell proliferation as well as neoangiogenesis in immunodeficient mice. We then noted that in leukemic cells, HIF-1α transcription was strongly suppressed by high AA and correlated with the transcription of VEGF. Our data indicate that exposure to high AA markedly increased the intracellular AA content of leukemic cells and inhibited the nuclear translocation of NF-κB, which mediates expression of HIF-1α. We next generated K562 cells that overexpressed HIF-1α (K562-HIF1α cells) and assessed the mechanistic relationship between inhibition of HIF-1α transcription and the antileukemic effect of high AA. The ability of high AA to induce apoptosis was significantly lower in K562-HIF1α cells than in K562 cells in vitro. We found that expression of HIF-1α-regulated antiapoptotic proteins of the Bcl-2 family, such as Mcl-1, Bcl-xL, and Bcl-2, was significantly suppressed by high AA in K562 cells, but was sustained at higher levels in K562-HIF1α cells, regardless of high AA exposure. Moreover, repression of cell proliferation and neoangiogenesis by high AA was completely abrogated in mice receiving transplants of K562-HIF1α cells. These results indicate that, along with H2O2 generation, downregulation of HIF-1α transcription plays a crucial role in growth inhibition of human leukemic cells by high AA.
PLoS ONE 01/2013; 8(4):e62717. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Stem cells of highly regenerative organs including blood are susceptible to endogenous DNA damage caused by both intrinsic and extrinsic stress. Response mechanisms to such stress equipped in hematopoietic stem cells (HSCs) are crucial in sustaining hematopoietic homeostasis but remain largely unknown. In this study, we demonstrate that serial transplantation of human HSCs into immunodeficient mice triggers replication stress that induces incremental elevation of intracellular reactive oxygen species (ROS) levels and the accumulation of persistent DNA damage within the human HSCs. This accumulation of DNA damage is also detected in HSCs of clinical HSC transplant patients and elderly individuals. A forced increase of intracellular levels of ROS by treatment with a glutathione synthetase inhibitor aggravates the extent of DNA damage, resulting in the functional impairment of HSCs in vivo. The oxidative DNA damage activates the expression of cell-cycle inhibitors in a HSC specific manner, leading to the premature senescence among HSCs, and ultimately to the loss of stem cell function. Importantly, treatment with an antioxidant can antagonize the oxidative DNA damage and eventual HSC dysfunction. The study reveals that ROS play a causative role for DNA damage and the regulation of ROS have a major influence on human HSC aging.
[Show abstract][Hide abstract] ABSTRACT: Cultured epidermal autografts and cultured skin substitute are vulnerable to infection. Human beta defensin (HBD)-3 is an antimicrobial peptide that exhibits a wide-spectrum antimicrobial activity against gram-positive/negative bacteria and fungi. This study determined whether normal human keratinocytes (NHKs) and human dermal fibroblasts (HDFs) transfected with the HBD-3 gene secrete HBD-3 peptide with an antimicrobial activity. An adenovirus vector with an HBD-3 cDNA inserted downstream of the CMV promoter (ADhBD3) was created. The HBD-3 gene was introduced into NHKs and HDFs via ADhBD3 infection. HBD-3 gene expression in each type of transfected cells was evaluated by RT-PCR. The presence of HBD-3 peptide in the culture supernatants of each type of transfected cells was evaluated by Western blotting. The antimicrobial activities of the culture supernatants of each type of transfected cells against several bacterial strains were also measured. Both NHKs and HDFs infected with ADhBD3 expressed the HBD-3 gene and secreted HBD-3 peptide into culture supernatants. These supernatants exhibited a strong bacteriocidal activity against a Staphylococcus aureus reference strain and methicillin-resistant S. aureus (MRSA). NHKs and HDFs transfected with the HBD-3 gene secrete HBD-3 peptide with an antimicrobial activity against S. aureus and MRSA.
Burns: journal of the International Society for Burn Injuries 09/2010; 37(1):109-16. · 1.95 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Hematopoiesis is a dynamic and strictly regulated process orchestrated by self-renewing hematopoietic stem cells (HSCs) and the supporting microenvironment. However, the exact mechanisms by which individual human HSCs sustain hematopoietic homeostasis remain to be clarified. To understand how the long-term repopulating cell (LTRC) activity of individual human HSCs and the hematopoietic hierarchy are maintained in the bone marrow (BM) microenvironment, we traced the repopulating dynamics of individual human HSC clones using viral integration site analysis. Our study presents several lines of evidence regarding the in vivo dynamics of human hematopoiesis. First, human LTRCs existed in a rare population of CD34(+)CD38(-) cells that localized to the stem cell niches and maintained their stem cell activities while being in a quiescent state. Second, clonally distinct LTRCs controlled hematopoietic homeostasis and created a stem cell pool hierarchy by asymmetric self-renewal division that produced lineage-restricted short-term repopulating cells and long-lasting LTRCs. Third, we demonstrated that quiescent LTRC clones expanded remarkably to reconstitute the hematopoiesis of the secondary recipient. Finally, we further demonstrated that human mesenchymal stem cells differentiated into key components of the niche and maintained LTRC activity by closely interacting with quiescent human LTRCs, resulting in more LTRCs. Taken together, this study provides a novel insight into repopulation dynamics, turnover, hierarchical structure, and the cell cycle status of human HSCs in the recipient BM microenvironment.
[Show abstract][Hide abstract] ABSTRACT: Hematopoietic stem cells (HSCs) consist of heterogenous subpopulations, one of which is CD34(-) HSCs. Recent development of successful engraftment by intra-bone marrow transplantation revealed severe combined immunodeficiency (scid) mouse-repopulating cell (SRC) activity in human CD34(-) cord blood (CB) cells. On the other hand, CD34(-) cells from bone marrow (BM) cells remain relatively undefined. Here, we investigated pre-SRC populations in human BM CD34(-) cells and the effect of the niche-related factor, angiopoietin-1, on them.
Two populations in BM CD34(-) cells (namely M cells and S cells) were purified by flow cytometry. Then, they were cocultured with six growth factors on the hematopoietic-supportive mouse BM stromal cell line, HESS-5 or AHESS-5 that were engineered to produce human angiopoietin-1, because we detected Tie2 expression on M cells and S cells. Cultured cells were assessed for their in vitro and in vivo hematopietic activities.
After 7 days in coculture, AHESS-5 was stronger more effective than HESS-5 in converting M and S cells to CD34(+) cells (M cells: 67.4% vs 17.5%, n =6, p < 0.001) (S cells: 42.3% vs 2.3%, n = 6, p < 0.001). Furthermore, both M and S cells were able to engraft in immunodeficient mice after they were cocultured on AHESS-5.
Results suggest that angiopoietin-1 supports SRC activities in human CD34(-) BM cells, as murine studies demonstrated. Furthermore, identification of previously undetected subpopulations of BM CD34(-) HSCs unveils heterogenous components in the stem cell pool.
[Show abstract][Hide abstract] ABSTRACT: To characterize human hematopoietic stem cells (HSCs), xenotransplantation techniques such as the severe combined immunodeficiency (SCID) mouse repopulating cell (SRC) assay have proven the most reliable methods thus far. While SRC quantification by limiting dilution analysis (LDA) is the gold standard for measuring in vitro expansion of human HSCs, LDA is a statistical method and does not directly establish that a single HSC has self-renewed in vitro. This would require a direct clonal method and has not been done. By using lentiviral gene marking and direct intra-bone marrow injection of cultured CD34+ CB cells, we demonstrate here the first direct evidence for self-renewal of individual SRC clones in vitro. Of 74 clones analyzed, 20 clones (27%) divided and repopulated in more than 2 mice after serum-free and stroma-dependent culture. Some of the clones were secondary transplantable. This indicates symmetric self-renewal divisions in vitro. On the other hand, 54 clones (73%) present in only 1 mouse may result from asymmetric divisions in vitro. Our data demonstrate that current ex vivo expansion conditions result in reliable stem cell expansion and the clonal tracking we have employed is the only reliable method that can be used in the development of clinically appropriate expansion methods.
[Show abstract][Hide abstract] ABSTRACT: Recent studies have shown that bone marrow (BM) contains cells capable of differentiating into myocytes in vivo. However, addition of demethylation drugs has been necessary to induce myocyte differentiation from BM cells in vitro, and precise mechanisms of BM cells' conversion to myocytes and the origin of those cells have not been established. We investigated the expression of myogenic markers during differentiation and maturation of myocytes from BM-derived multipotent adult progenitor cells (MAPC) under physiological culture condition.
Frozen BM samples from 21 healthy donors were used as a source of MAPC. To induce myocyte differentiation MAPC was cultured in the presence of 5% FCS, VEGF, bFGF, and IGF-1, and the expressions of myocyte markers were examined at various time points. We also investigated engraftment and differentiation of MAPC-derived myocytes in vivo.
Frozen BM-derived MAPC, cultured under the physiological myogenic condition, demonstrated spatial expression patterns of several myocyte markers similar to that of authentic myocyte differentiation. When injected into murine muscles, MAPC treated with the myogenic condition engrafted and differentiated into myocyte marker-positive cells and myotubes in vivo.
For the first time, we were able to induce myocyte formation from BM cells under the physiological condition in vitro and demonstrated that treating cells with this condition prior to intramuscular injection increased efficiency of engraftment and differentiation in vivo.
[Show abstract][Hide abstract] ABSTRACT: Putative myogenic and endothelial (myo-endothelial) cell progenitors were identified in the interstitial spaces of murine skeletal muscle by immunohistochemistry and immunoelectron microscopy using CD34 antigen. Enzymatically isolated cells were characterized by fluorescence-activated cell sorting on the basis of cell surface antigen expression, and were sorted as a CD34+ and CD45- fraction. Cells in this fraction were approximately 94% positive for Sca-1, and mostly negative (<3% positive) for CD14, 31, 49, 144, c-kit, and FLK-1. The CD34+/45- cells formed colonies in clonal cell cultures and colony-forming units displayed the potential to differentiate into adipocytes, endothelial, and myogenic cells. The CD34+/45- cells fully differentiated into vascular endothelial cells and skeletal muscle fibers in vivo after transplantation. Immediately after sorting, CD34+/45- cells expressed only c-met mRNA, and did not express any other myogenic cell-related markers such as MyoD, myf-5, myf-6, myogenin, M-cadherin, Pax-3, and Pax-7. However, after 3 d of culture, these cells expressed mRNA for all myogenic markers. CD34+/45- cells were distinct from satellite cells, as they expressed Bcrp1/ABCG2 gene mRNA (Zhou et al., 2001). These findings suggest that myo-endothelial progenitors reside in the interstitial spaces of mammalian skeletal muscles, and that they can potentially contribute to postnatal skeletal muscle growth.
The Journal of Cell Biology 05/2002; 157(4):571-7. · 10.82 Impact Factor