Effective phagocytosis is crucial for host defense against pathogens. Macrophages entrap pathogens into a phagosome and subsequently acidic lysosomes fuse to the phagosome. Previous studies showed ...the pivotal role of actin-remodeling mediated by phosphoinositide-related signaling in phagosome formation, but the mechanisms of phagosome-lysosome fusion remain unexplored. Here we show that in complement-mediated phagocytosis, phagosome-lysosome fusion requires the disappearance of F-actin structure surrounding the phagosome and a tyrosine kinase Syk plays a key role in this process. Using macrophage-like differentiated HL60 and Syk-knockout (Syk-KO) HL60 cells, we found that Syk-KO cells showed insufficient phagosome acidification caused by impaired fusion with lysosomes and permitted the survival of Candida albicans in complement-mediated phagocytosis. Phagosome tracking analysis showed that during phagosome internalization process, F-actin surrounding phagosomes disappeared in both parental and Syk-KO cells but this structure was reconstructed immediately only in Syk-KO cells. In addition, F-actin-stabilizing agent induced a similar impairment of phagosome-lysosome fusion. Collectively, Syk-derived signaling facilitates phagosome-lysosome fusion by regulating actin-remodeling.
Treatment outcomes for acute myeloid leukemia and myelodysplastic syndromes (MDS) remain unsatisfactory despite progress in various types of chemotherapy and hematopoietic stem cell transplantation. ...Therefore, there is a need for the development of new treatment options. We investigated the growth‐suppressive effects of withaferin A (WA), a natural plant steroidal lactone, on myelodysplasia and leukemia cell lines. WA exhibited growth‐suppressive effects on the cell lines, MDS‐L, HL‐60, THP‐1, Jurkat and Ramos, and induction of cell cycle arrest at G2/M phase at relatively low doses. Evaluation by annexin V/PI also confirmed the induction of partial apoptosis. Gene expression profiling and subsequent gene set enrichment analysis revealed increased expression of heme oxygenase‐1 (HMOX1). HMOX1 is known to induce autophagy during anticancer chemotherapy and is considered to be involved in the treatment resistance. Our study indicated increased HMOX1 protein levels and simultaneous increases in the autophagy‐related protein LC3A/B in MDS‐L cells treated with WA, suggesting increased autophagy. Combined use of WA with chloroquine, an autophagy inhibitor, enhanced early apoptosis and growth suppression. Together with the knowledge that WA had no apparent suppressive effect on the growth of human normal bone marrow CD34‐positive cells in the short‐term culture, this drug may have a potential for a novel therapeutic approach to the treatment of leukemia or MDS.
Withaferin A exhibited growth‐suppressive effects on MDS/leukemia cell lines and induction of cell cycle arrest at G2/M phase. Gene expression profiling and subsequent gene set enrichment analysis (GSEA) revealed increased expression of heme oxygenase‐1. As withaferin A had no apparent suppressive effect on the growth of human normal bone marrow CD34‐positive cells, this drug may have a potential for a novel therapeutic approach to the treatment of leukemia or MDS.
The prognosis of the patients with inoperable or advanced granulosa cell tumors (GCTs) is still poor, and therefore it is important to establish a novel treatment strategy. Here we investigated the ...in vitro effects of a histone deacetylase inhibitor, panobinostat (PS) on two GCT cell lines (KGN and COV434). GCT cell lines were found to be susceptible to PS treatment and it inhibited cell growth mainly by apoptosis. In cell cycle analysis, PS reduced only the ratio of S phase in GCT cell lines. Combined treatment of PS with a deubiquitinase inhibitor, VLX1570 enhanced the expression of p21, cleaved PARP, cleaved caspase-9, heme oxygenase-1, and the acetylation of histone H4 and alpha-tubulin, leading to an additive anti-proliferative effect on KGN and COV434. The gene set enrichment analysis revealed that PS treatment suppressed DNA replication- or cell cycle-related gene expression which led to chemotherapeutic cell death and in addition, this treatment induced activation of the gene set of adherens junction towards a normalized direction as well as activation of neuron-related gene sets that might imply unexpected differentiation potential due to epigenetic modification by a HDAC inhibitor in KGN cells. Exposure of KGN and COV434 cells to PS increased the expression of E-cadherin, one of the principal regulators associated with adherens junction in quantitative RT-PCR and immunoblotting analysis. In the present study, we indicate a basis of a novel therapeutic availability of a HDAC inhibitor for the treatment of GCTs and further investigations will be warranted.
Unprecedented progress continues to be made in the treatment strategies for the myelodysplastic syndromes (MDS). This review provides an introduction to DNA methyltransferase inhibitors. These agents ...include 5-azacytidine (azacitidine; AZA) and 5-aza-2'- deoxycytidine (decitabine; DAC). In particular, AZA was shown for the first time to significantly prolong the life expectancy of high-risk MDS patients in international multicenter clinical studies. The selection of responders and assessment of the long-term efficacy warrant further study.
A novel proteasome deubiquitinase inhibitor, VLX1570, has been highlighted as a promising therapeutic agent mainly for lymphoid neoplasms and solid tumors. We examined in vitro effects of VLX1570 on ...eight myeloid and three lymphoid leukemia cell lines. From cell culture studies, 10 out of 11 cell lines except K562 were found to be susceptible to VLX1570 treatment and it inhibited cell growth mainly by apoptosis. Next, to identify the signaling pathways associated with apoptosis, we performed gene expression profiling using HL‐60 with or without 50 nmol/L of VLX1570 for 3 hours and demonstrated that VLX1570 induced the genetic pathway involved in “heat shock transcription factor 1 (HSF1) activation”, “HSF1 dependent transactivation”, and “Regulation of HSF1 mediated heat shock response”. VLX1570 increased the amount of high molecular weight polyubiquitinated proteins and the expression of HSP70 as the result of the suppression of ubiquitin proteasome system, the expression of heme oxygenase‐1, and the amount of phosphorylation in JNK and p38 associated with the generation of reactive oxygen species (ROS) induced apoptosis and the amount of phosphorylation in eIF2α, inducing the expression of ATF4 and endoplasmic reticulum (ER) stress dependent apoptosis protein, CHOP, and the amount of phosphorylation slightly in IRE1α, leading to increased expression of XBP‐1s in leukemia cell lines. In the present study, we demonstrate that VLX1570 induces apoptosis and exerts a potential anti‐leukemic effect through the generation of ROS and induction of ER stress in leukemia cell lines.
VLX1570 induces apoptosis mainly through reactive oxygen (ROS) stress signaling and unfolded protein response pathway under endoplasmic reticulum (ER) stress. Regarding ER stress, VLX1570 activated the protein kinase RNA‐like endoplasmic reticulum kinase signaling pathway, leading to ER stress‐dependent apoptosis. As another pathway, excess accumulation of polyubiquitinated proteins on the mitochondria induces ROS generation, followed by phosphorylation of JNK and p38, leading to caspases‐dependent apoptosis.
Although it is known that red blood cell (RBC) parameters and platelet count depend on ethnicity and sex, their reference intervals in healthy Asian populations are limited. The aim of this study was ...to establish reference intervals for RBC parameters and platelet count for healthy adults in Japan. A total of 750 healthy adults (447 women and 303 men; median age 40 years (18–67 years) at seven Japanese centers who participated in regular medical checkups entered this study. Their RBC parameters and platelet count were measured using automated hematocytometers. The reference intervals of the RBC parameters and platelet count according to sex in healthy adults were determined. There was an age-specific decrease in RBC counts and an age-specific increase in mean corpuscular volume in men. This study emphasizes the need to consider sex and age in the clinical use of reference intervals of RBC parameters.
A microtubule-associated motor protein, kinesin-like family member 20A (KIF20A; also called MKlp2) is required for cytokinesis and contributes to intracellular vesicular trafficking. KIF20A plays a ...critical role in the development of several cancers, but its role in blood cells and hematological malignancies have not been studied. In the present study, we focused on the role of KIF20A in hematopoietic cells and possible involvement in myeloid neoplasms. We found that human leukemia cell lines and normal bone marrow CD34-positive cells stimulated by growth factors, but not mature peripheral blood cells, exhibit high KIF20A expression. We further found that HL60 cells, which originally express a large amount of KIF20A, showed decreased KIF20A expression in parallel with both neutrophil-like and macrophage-like differentiation-induction. KIF20A-knockdown using a lentivirus shRNA transfection system led to partial cell cycle arrest at the G2/M phase and frequent appearance of multinucleated cells. Treatment with a KIF20A-selective inhibitor, paprotrain enhanced the multinuclearity of KIF20A-knockdown cell clones and suppressed growth. The present study contributes to our understanding of the role of KIF20A in blood cells and leukemia cells in particular.
Idiopathic pure red cell aplasia (PRCA) and secondary PRCA associated with thymoma and large granular lymphocyte leukemia are generally considered to be immune-mediated. The PRCA2004/2006 study ...showed that poor responses to immunosuppression and anemia relapse were associated with death. PRCA may represent the prodrome to MDS. Thus, clonal hematopoiesis may be responsible for treatment failure. We investigated gene mutations in myeloid neoplasm-associated genes in acquired PRCA. We identified 21 mutations affecting amino acid sequences in 11 of the 38 adult PRCA patients (28.9%) using stringent filtering of the error-prone sequences and SNPs. Four PRCA patients showed 7 driver mutations in TET2, DNMT3A and KDM6A, and 2 PRCA patients carried multiple mutations in TET2. Five PRCA patients had mutations with high VAFs exceeding 0.3. These results suggest that clonal hematopoiesis by stem/progenitor cells might be related to the pathophysiology of chronic PRCA in certain adult patients.
Myelodysplastic syndrome (MDS), a largely incurable hematological malignancy, is derived from aberrant clonal hematopoietic stem/progenitor cells (HSPCs) that persist after conventional therapies. ...Defining the mechanisms underlying MDS HSPC maintenance is critical for developing MDS therapy. The deacetylase SIRT1 regulates stem cell proliferation, survival, and self-renewal by deacetylating downstream proteins. Here we show that SIRT1 protein levels were downregulated in MDS HSPCs. Genetic or pharmacological activation of SIRT1 inhibited MDS HSPC functions, whereas SIRT1 deficiency enhanced MDS HSPC self-renewal. Mechanistically, the inhibitory effects of SIRT1 were dependent on TET2, a safeguard against HSPC transformation. SIRT1 deacetylated TET2 at conserved lysine residues in its catalytic domain, enhancing TET2 activity. Our genome-wide analysis identified cancer-related genes regulated by the SIRT1/TET2 axis. SIRT1 activation also inhibited functions of MDS HSPCs from patients with TET2 heterozygous mutations. Altogether, our results indicate that restoring TET2 function through SIRT1 activation represents a promising means to target MDS HSPCs.
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•SIRT1 depletion enhances MDS HSPC self-renewal and growth•SIRT1 insufficiency in MDS promotes TET2 hyperacetylation•SIRT1 agonist treatment blocks MDS HSPC maintenance•SIRT1 activation deacetylates TET2 and enhances its catalytic activity
An improved understanding of the mechanisms regulating myelodysplastic syndrome (MDS) hematopoietic stem/progenitor cell (HSPC) growth and self-renewal is critical for developing MDS therapy. Sun et al. report that SIRT1 deficiency-induced TET2 hyperacetylation promotes MDS HSPC function and provides an approach to target MDS HSPCs by activating SIRT1 deacetylase.
Vitamin K2 in the form of menatetrenone has clinical benefits for osteoporosis and cytopenia. Given the dominant role of mesenchymal-osteolineage cells in the regulation of hematopoiesis, we ...investigated whether menatetrenone alters the hematopoiesis-supportive capability of human bone marrow mesenchymal stromal/stem cells (BM-MSCs). Menatetrenone up-regulated fibronectin protein expression in BM-MSCs without affecting their proliferation and differentiation capabilities. In addition, menatetrenone treatment of BM-MSCs enhanced generation of the CD34
+
cell population in co-cultures through acceleration of the cell cycle. This effect was associated with cell–cell interactions mediated by VLA-4 and fibronectin. This proposal was supported by cytokine array and quantitative real-time PCR analyses, in which there were no significant differences between the expression levels of hematopoiesis-associated soluble factors in naïve and menatetrenone-treated BM-MSCs. Profiling of hematopoietic cells in co-cultures with menatetrenone-treated BM-MSCs demonstrated that they included significantly more CD34
+
CD38
+
hematopoietic progenitor cells and cells skewed toward myeloid and megakaryocytic lineages than those in co-cultures with untreated BM-MSCs. Notably, myelodysplastic syndrome-derived cells were induced to undergo apoptosis when co-cultured with BM-MSCs, and this effect was enhanced by menatetrenone. Overall, our findings indicate that pharmacological treatment with menatetrenone bestows a unique hematopoiesis-supportive capability on BM-MSCs, which may contribute to the clinical improvement of cytopenia.
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