Differentiation Of HL-60 Cells Research Articles

FITC characterization of a cathepsin B-responsive nanoprobe for report of differentiation of HL60 cells into macrophages.

A cathepsin B (Cat B)-responsive optical nanoprobe is designed and prepared for report of HL60 differentiation into macrophage. A peptide sequence FRFK is linked to fluorescein (FITC) via the distant amino group of its lysine and N-terminated with acrylic acid (AA) to yield a molecular fluorescent probe AA-FRFK (FITC). The molecular probe is further embedded in poly(lactic-co-glycolic acid) (PLGA) to form a fluorescent nanoprobe AA-FRFK (FITC)@PLGA. The resultant optical nanoprobe is degradable by lysosomal Cat B, which is expressed in macrophages with a level of 5-10 times of that in HL60 cells. As a result, a significant decrease in fluorescence intensity is associated with the differentiation process of HL60 to macrophage and can be used as an indication of the differentiation process. The findings may pave a way toward the development of a universal in vitro labeling strategy of exogenous stem cells for report of in vivo cell differentiation by a dual-mode imaging modality involving optical imaging and magnetic resonance imaging.

A splicing factor phosphorylated by protein kinase A is increased in HL60 cells treated with retinoic acid

Retinoic acid (RA) induces the differentiation of human promyelocytic leukemia HL60 cells into granulocytic cells and inhibits proliferation. Certain of actions of RA are mediated by RA nuclear receptors that regulate gene expression. However, it is also known that direct protein modification by RA (retinoylation) can occur. One such retinoylated protein in HL60 cells is a regulatory subunit of protein kinase A (PKA), which is increased in the nucleus following RA treatment and which then increases phosphorylation of other nuclear proteins. However, a complete understanding of which nuclear proteins are phosphorylated is lacking. In the current study, we employed mass spectrometry to identify one of the PKA-phosphorylated proteins as a serine/arginine-rich splicing factor 1 (SF2, SRSF1). We found that RA treatment increased the level of PKA-phosphorylated SF2 but decreased the level of SF2. While SF2 regulates myelogenous cell leukemia-1 (Mcl-1, anti-apoptotic factor), RA treatment reduced the level of Mcl-1L (full-length Mcl-1 long) and increased the level of Mcl-1S (Mcl-1 short; a short splicing variant of the Mcl-1). Furthermore, treatment with a PKA inhibitor reversed these effects on Mcl-1 and inhibited RA-induced cell differentiation. In contrast, treatment with a Mcl-1L inhibitor enhanced RA-induced cell differentiation. These results indicate that RA activates PKA in the nucleus, increases phosphorylation of SF2, raises levels of Mcl-1S and lowers levels of Mcl-1L, resulting in the induction of differentiation. RA-modified PKA may play an important role in inducing cell differentiation and suppressing cell proliferation.

Mitofusin-2 regulates leukocyte adhesion and β2 integrin activation.

Neutrophils are critical for inflammation and innate immunity, and their adhesion to vascular endothelium is a crucial step in neutrophil recruitment. Mitofusin-2 (MFN2) is required for neutrophil adhesion, but molecular details are unclear. Here, we demonstrated that β2 -integrin-mediated slow-rolling and arrest, but not PSGL-1-mediated cell rolling, are defective in MFN2-deficient neutrophil-like HL60 cells. This adhesion defect is associated with reduced expression of fMLP (N-formylmethionyl-leucyl-phenylalanine) receptor FPR1 as well as the inhibited β2 integrin activation, as assessed by conformation-specific monoclonal antibodies. MFN2 deficiency also leads to decreased actin polymerization, which is important for β2 integrin activation. Mn2+ -induced cell spreading is also inhibited after MFN2 knockdown. MFN2 deficiency limited the maturation of β2 integrin activation during the neutrophil-directed differentiation of HL60 cells, which is indicated by CD35 and CD87 markers. MFN2 knockdown in β2-integrin activation-matured cells (CD87high population) also inhibits integrin activation, indicating that MFN2 directly affects β2 integrin activation. Our study illustrates the function of MFN2 in leukocyte adhesion and may provide new insights into the development and treatment of MFN2 deficiency-related diseases.

Role of S100A8/A9 for Cytokine Secretion, Revealed in Neutrophils Derived from ER-Hoxb8 Progenitors

S100A9, a Ca2+-binding protein, is tightly associated to neutrophil pro-inflammatory functions when forming a heterodimer with its S100A8 partner. Upon secretion into the extracellular environment, these proteins behave like damage-associated molecular pattern molecules, which actively participate in the amplification of the inflammation process by recruitment and activation of pro-inflammatory cells. Intracellular functions have also been attributed to the S100A8/A9 complex, notably its ability to regulate nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activation. However, the complete functional spectrum of S100A8/A9 at the intracellular level is far from being understood. In this context, we here investigated the possibility that the absence of intracellular S100A8/A9 is involved in cytokine secretion. To overcome the difficulty of genetically modifying neutrophils, we used murine neutrophils derived from wild-type and S100A9−/− Hoxb8 immortalized myeloid progenitors. After confirming that differentiated Hoxb8 neutrophil-like cells are a suitable model to study neutrophil functions, our data show that absence of S100A8/A9 led to a dysregulation of cytokine secretion after lipopolysaccharide (LPS) stimulation. Furthermore, we demonstrate that S100A8/A9-induced cytokine secretion was regulated by the nuclear factor kappa B (NF-κB) pathway. These results were confirmed in human differentiated HL-60 cells, in which S100A9 was inhibited by shRNAs. Finally, our results indicate that the degranulation process could be involved in the regulation of cytokine secretion by S100A8/A9.

Transcriptome and Literature Mining Highlight the Differential Expression of ERLIN1 in Immune Cells during Sepsis.

Simple SummarySepsis is a disease affecting 19 million people worldwide, and accounts for 5 million deaths annually. Efforts in finding predictive markers of sepsis development have been difficult due to the complex clinical features of the disease. Public data repositories are valuable resources for mining gene expression changes across different studies. Using such resources, we observed a consistent increase in ERLIN1—a gene coding for an ER membrane prohibitin and regulator of cholesterol—in whole blood, and across a variety of immune cells, during sepsis or sepsis-like conditions. We verified this finding by exposing the whole blood of healthy volunteers to a combination of lipopolysaccharide and peptidoglycan in order to simulate sepsis. We observed an increase in ERLIN1 in whole-blood neutrophils and HL60 cell lines during sepsis; however, the protein was expressed differently in other immune blood cells. The current available studies on ERLIN1 and sepsis indicate a knowledge gap between the functions of ERLIN1, calcium balance, and cholesterol and fatty acid synthesis, and sepsis. Together with experimental data, we think that ERLIN1 is modulated differently in immune cells in response to infection, and has important implications for ER functions and/or ER membrane protein components during sepsis.Sepsis results from the dysregulation of the host immune system. This highly variable disease affects 19 million people globally, and accounts for 5 million deaths annually. In transcriptomic datasets curated from public repositories, we observed a consistent upregulation (3.26–5.29 fold) of ERLIN1—a gene coding for an ER membrane prohibitin and a regulator of inositol 1, 4, 5-trisphosphate receptors and sterol regulatory element-binding proteins—under septic conditions in healthy neutrophils, monocytes, and whole blood. In vitro expression of the ERLIN1 gene and proteins was measured by stimulating the whole blood of healthy volunteers to a combination of lipopolysaccharide and peptidoglycan. Septic stimulation induced a significant increase in ERLIN1 expression; however, ERLIN1 was differentially expressed among the immune blood cell subsets. ERLIN1 was uniquely increased in whole blood neutrophils, and confirmed in the differentiated HL60 cell line. The scarcity of ERLIN1 in sepsis literature indicates a knowledge gap between the functions of ERLIN1, calcium homeostasis, and cholesterol and fatty acid biosynthesis, and sepsis. In combination with experimental data, we bring forth the hypothesis that ERLIN1 is variably modulated among immune cells in response to cellular perturbations, and has implications for ER functions and/or ER membrane protein components during sepsis.

Trametinib enhances ATRA-induced differentiation in AML cells

All-trans retinoic acid (ATRA) is only clinically useful in acute promyelocytic leukemia (APL), but not other subtypes of acute myeloid leukemia (AML). In the present study, a clinically achievable concentration of trametinib, a highly selective inhibitor of MEK, enhanced ATRA-induced differentiation in AML cell lines, HL-60 and U937 as well as AML primary cells. Moreover, trametinib–ATRA (tra–ATRA) co-treatment restored ATRA sensitivity in ATRA-resistant AML cell line, HL-60Res. The protein level of STAT3 and the phosphorylation of Akt or JNK were enhanced with tra–ATRA treatment in HL-60, U937, and HL-60Res cells, respectively. Furthermore, tra–ATRA-induced differentiation in HL-60, U937, and HL-60Res cells was inhibited by STAT3, PI3K, and JNK inhibitors, respectively. Therefore, STAT3, Akt, and JNK signaling pathways were involved in tra–ATRA-induced differentiation in HL-60, U937, and HL-60Res cells, respectively. Taken together, our findings may provide novel therapeutic strategies for AML patients.

Uremic Serum Damages Endothelium by Provoking Excessive Neutrophil Extracellular Trap Formation

Background: Cardiovascular disease (CVD) is the leading cause of death in patients with chronic kidney disease (CKD). Endothelial cell (EC) dysfunction is a key CKD-specific risk factor; however, the mechanisms by which uremia harms the endothelium are still unclear. We report a role for excessive neutrophil extracellular trap (NET) formation induced by uremic serum on EC injury. Methods: Level of plasma nucleosome and myeloperoxidase-DNA, established in vivo markers of NETs, as well as intracellular adhesion molecule (ICAM)-1 were measured in hemodialysis (HD) patients and healthy volunteers (HV) and their prognostic role evaluated. For in vitro studies, we differentiated HL-60 cells into neutrophil-like cells (dHL-60) by applying retinoic acid and determined the effect of uremic serum on these dHL-60 and human umbilical vein EC (HUVEC). Findings: The level of in vivo NETs was significantly higher in incident HD patients compared to HV, and these markers were strongly associated with ICAM-1. Specifically, nucleosome and ICAM-1 levels were independent predictors of a composite endpoint, all-cause mortality or vascular access failure. In vitro, HD-derived serum significantly increased NET formation by dHL-60, and these NETs decreased EC viability and induced their apoptosis. In addition, the ICAM-1 level in HUVEC supernatant was significantly increased by uremic serum-induced NETs compared to control serum-induced NETs. Interpretation: Dysregulated neutrophil activities in the uremic milieu may play a key role in vascular inflammatory responses. The high mortality and CVD rates in ESRD may be explained in part by excessive NET formation leading to EC damage and dysfunction. Funding: This research was supported by National Research Foundation of Korea (NRF) grant funded by the Korea government (2020R1A2C110138611). Declaration of Interests: No conflicts of interest in this study. Ethics Approval Statement: This study was approved by the Institutional Ethics Committee and informed consent was obtained from the HD patients and HVs.

Spliceosomal Dysfunction Is a Critical Mediator of IDH2 Mutant Leukemogenesis

Mutations affecting RNA splicing factors are commonly found in leukemias and mutations in SF3B1, SRSF2, and U2AF1 impart specific changes to global splicing. We analyzed RNA-seq data from acute myeloid leukemia (AML) patients from The Cancer Genome Atlas (TCGA) to evaluate for spliceosomal alterations characteristic of mutant SRSF2, SF3B1, and U2AF1 . Although only 1 patient with an SRSF2 mutation was reported in the TCGA AML publication, we identified 19 additional patients to have the characteristic splicing changes of mutant SRSF2 . Mutational analysis of the RNA-seq data identified the SRSF2 proline 95 hotspot mutation in each of these 19 patients (19/178, 11%), making SRSF2 amongst the most commonly mutated genes in the AML TCGA dataset. Interestingly, 47% of SRSF2 mutant patients also had a co-existing IDH2 mutation (and conversely, 50% of IDH2 mutant patients had a co-existing SRSF2 mutation). These data are similar to recent observation that SRSF2 mutations were the most common accompanying mutation in IDH2 mutant myeloid leukemia patients treated on the phase I clinical trial of AG-221.We next sought to understand the functional contribution of SRSF2 mutations to co-existing IDH2 mutations. Overexpression of IDH2 R140Q and IDH2 R172K in Vav -cre Srsf2 P95H or Vav- cre Srsf2 wildtype(WT) backgrounds revealed a clear collaborative effect of mutant IDH2 and mutant Srsf2 (Figure A-B). As early as 4 weeks post-transplant, the peripheral blood of Srsf2 mutant mice transplanted with IDH2 R140Q or IDH2 R172K forms had a substantially greater percentage of GFP+ cells than seen in an Srsf2 WT background. Moreover, these mice exhibited significant myeloid skewing, macrocytic anemia, and thrombocytopenia of greater magnitude than seen with mutant IDH2 expressed in the SRSF2 WT background. IDH2 / Srsf2 double-mutant mice developed a lethal, mixed myelodysplastic/myeloproliferative neoplasm that was serially transplantable (Figure B). Effects from overexpression of mutant IDH2 were recapitulated in mice expressing Idh2 R140Q and Srsf2 P95H from endogenous loci(Mx1 -cre Idh2R140Q/+Srsf2P95H/+ double knock-in mice) (Figure C), providing the first model where spliceosomal gene mutations promote leukemogenesis in vivo .Given prior data identifying inhibition of TET2 as a mediator of IDH2 -mutant leukemogenesis, we evaluated if loss of TET2 might promote transformation of SRSF2 mutant cells. However, deletion of TET2 in an SRSF2 mutant background (Mx1 -cre Tet2fl/flSrsf2P95H/+) was insufficient to promote transformation (Figure D). We next analyzed RNA-seq data from the TCGA, Leucegene, and our own unpublished AML cohort using a novel analysis method to detect unannotated as well as complex splicing events that might explain the basis for the collaboration of mutant IDH2 and SRSF2 . This revealed that IDH2/SRSF2 double-mutant AML had increased aberrant splicing events compared to SRSF2 single-mutant AML and also that IDH2 -mutations are associated with a small but reproducible change in RNA splicing (Figure E). The vast majority of splicing changes affected by SRSF2 and/or IDH2 mutations were a change in cassette exon splicing with SRSF2 mutations promoting inclusion of C-rich containing mRNA sequences, regardless of the presence of concomitant IDH2 mutations (Figure F). One of the most robust mis-splicing events in IDH2/SRSF2 mutant AML was an intron retention event in INTS3 resulting in its reduced mRNA and protein expression (Figure G-H). INTS3 encodes a component of the integrator complex that participates in small nuclear RNA (snRNA) processing. Consistent with this, IDH2 / SRSF2 double-mutant cells exhibited alterations in processing of U2 and U4 snRNAs similar to that seen with direct INTS3 downregulation. Biologically, attenuation of INTS3 expression caused a blockade of myeloid differentiation in HL-60 cells upon ATRA treatment, an effect which was further enhanced in an IDH2 mutant background.These data uncover an important role for spliceosomal gene mutations in the pathogenesis of IDH2 mutant leukemogenesis and identify perturbations in the integrator complex as a novel mediator driving the transformation of IDH2 and SRSF2 mutant cells. The frequent coexistence of IDH2 and SRSF2 mutations underscores the enormous therapeutic potential for modulation of splicing in the ~50% of IDH2 -mutant leukemia patients who also harbor a spliceosomal gene mutation. [Display omitted] DisclosuresDe Botton:Servier: Honoraria; Agios: Honoraria, Research Funding; Celgene: Honoraria; Novartis: Honoraria; Pfizer: Honoraria. Stein:Pfizer: Consultancy, Other: Travel expenses; Novartis: Consultancy, Research Funding; Agios Pharmaceuticals, Inc.: Consultancy, Research Funding; Celgene Corporation: Consultancy, Other: Travel expenses, Research Funding; Seattle Genetics: Research Funding; GSK: Other: Advisory Board, Research Funding; Constellation Pharma: Research Funding. Peng:H3 Biomedicine Inc.: Employment. Buonamici:H3 Biomedicine Inc.: Employment. Palacino:H3 Biomedicine Inc.: Employment. Smith:H3 Biomedicine Inc.: Employment. Thompson:Merck: Membership on an entity's Board of Directors or advisory committees; Charles River Laboratories: Membership on an entity's Board of Directors or advisory committees; Agios Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Other: Founder. Levine:Roche: Research Funding; Roche: Research Funding; Qiagen: Equity Ownership; Qiagen: Equity Ownership; Celgene: Research Funding; Celgene: Research Funding.

Nicotinamide phosphoribosyltransferase inhibitor is a novel therapeutic candidate in LPS-induced neutrophil extracellular traps.

Neutrophil extracellular traps (NETs) are beneficial antibacterial defense structures. However, excessive NETs have also been linked to tissue damage and organ dysfunction. LPS and Gram-negative bacteria induce the formation of reactive oxygen species (ROS)-dependent NETs via the JNK pathway. It was found previously that knockdown of nicotinamide phosphoribosyltransferase (NAMPT) upregulates surfactant protein B (SFTPB or SP-B) and attenuates LPS-induced acute lung injury (ALI) via inhibiting JNK activation. This study investigated the effect of FK866, an intracellular NAMPT inhibitor, on the formation of LPS-induced NETs in mouse bronchoalveolar lavage (BAL) neutrophils and in differentiated HL-60 cells. The results show that inhibition of NAMPT by FK866 suppresses NETs formation in BAL neutrophils from the mice exposed to LPS. FK866 also suppresses NETs formation in the differentiated HL-60 cells stimulated with LPS. Additional data indicate that these effects are mediated by suppressing ROS production at least partly via inhibiting JNK activation and depleting NAD(P)H. The utility of inhibition of intracellular NAMPT may be a potential therapy for LPS-induced NETs-related diseases.

Inhibition of miR-139-5p by topical JTXK gel promotes healing of Staphylococcus aureus-infected skin wounds.

The inflammatory skin wound response is regulated by argonaute 2-bound microRNAs (Ago2-miRNAs) such as miR-139-5p, which inhibit transcription of their target mRNAs. Jiang Tang Xiao Ke (JTXK) is a traditional Chinese medicine that reduces miR-139-5p expression, suggesting that topical application of JTXK may have effects on wound healing. miR-139-/- mice and wild-type (WT) mice were employed to characterize the in vivo effects of miR-139-5p on sterile wound healing. Neutrophil migration and activation into the wound site were examined by live imaging analysis in lys-EGFP mice and myeloperoxidase/aminophenyl fluorescein assays, respectively. In silico and in vitro studies in differentiated HL60 cells were performed to identify miR-139-5p's downstream mediator(s). miR-139-/- neutrophil transplantation (with or without Eif4g2-knockdown rescue) or a topical JTXK gel preparation (with or without miR-139-5p mimic rescue) were employed to characterize the in vivo effects of miR-139-5p and JTXK, respectively, on Staphylococcus aureus (S. aureus)-infected wound healing. miR-139-/- mice display impaired sterile wound healing but improved S. aureus-infected wound healing. Eif4g2, a protein that supports neutrophil proliferation and differentiation, was identified as a key downstream mediator of miR-139-5p. miR-139-/- mice show elevated neutrophilic activation and Eif4g2 upregulation. miR-139-/- neutrophils enhanced S. aureus-infected wound healing in an Eif4g2-dependent manner. Moreover, topical JTXK gel therapy also enhanced S. aureus-infected wound healing in a miR-139-5p-dependent manner. miR-139-5p negatively regulates the neutrophilic response during S. aureus-infected wound healing, suggesting that JTXK or other miR-139-5p suppressants may be effective for treating infected skin wounds.

Securinine Induces Differentiation of Human Promyelocytic Leukemic HL-60 Cells through JNK-Mediated Signaling Pathway

Acute myeloid leukemia is characterized by abnormal differentiation of hematopoietic stem cells, leading to the accumulation of immature myeloid cells. Differentiation therapy has been a successful treatment option for acute promyelocytic leukemia but suffers from adverse effects. Therefore, search for novel differentiation-inducing agents with minimal side effects is desirable. Securinine, a naturally-occurring alkaloid, induces differentiation in various leukemic cells and apoptosis in other types of cancers. However, the underlying molecular mechanism(s) remain elusive. Our study aimed to elucidate the possible molecular mechanism(s) and signaling events involved in securinine-induced differentiation of HL-60 cells. Securinine inhibited proliferation in a time- and dose-dependent manner and triggered differentiation. A higher CD14+ population indicated maturation toward monocytic lineage. Securinine caused cell cycle arrest at the G0/G1 phase and enhanced ROS generation. Quantitative gene expression analysis showed significant down-regulation of C/EBP-α, C/EBP-ε, GAΤΑ, and c-myc and up-regulation of the PU.1 gene. The expression of distinct protein kinases Lyn, Chk-2, Yes, FAK, c-Jun, and JNK were enhanced. Use of specific inhibitors of crucial intracellular signaling proteins indicated that JNK and ERK blockade resulted in a significant decline in differentiation. These data thus confirm that securinine induces differentiation through the activation of the JNK-ERK signaling pathway in HL-60 cells.

Mitofusin-2 regulates leukocyte adhesion through the maturation of beta2 integrin activation in differentiation

Abstract Neutrophils are critical in inflammation and innate immunity. Neutrophil adhesion to vascular endothelium is a crucial step in neutrophil recruitment. Mitofusin-2 is required for neutrophil adhesion, but molecular details are unclear. Here, we demonstrated that β2-integrin-mediated slow-rolling and arrest, but not PSGL-1-mediated cell rolling, are defective in mitofusin-2-deficient neutrophil-like HL60 cells. This adhesion defect is associated with reduced expression of fMLP receptors and β2 integrins as well as the inhibited β2 integrin activation, as assessed by conformational-specific antibodies. Mitofusin-2 deficiency limited the maturation of β2 integrin activation during the neutrophil-directed differentiation of HL60 cells, which is indicated by identified markers CD35 and CD87. Reversed to results in overall HL60 cells, mitofusin-2 knockdown in β2-integrin-activation-matured cells (CD87high population) enhanced integrin activation. Our study illustrates the function of mitofusin-2 in leukocyte adhesion and may provide new insights into the development and treatment of mitofusin-2-deficiency-related disease.

Mitofusin‐2 regulates leukocyte adhesion through the maturation of β2 integrin activation in differentiation

Neutrophils are critical in inflammation and innate immunity. Neutrophil adhesion to vascular endothelium is a crucial step in neutrophil recruitment. Mitofusin-2 is required for neutrophil adhesion, but molecular details are unclear. Here, we demonstrated that β2-integrin-mediated slow-rolling and arrest, but not PSGL-1-mediated cell rolling, are defective in mitofusin-2-deficient neutrophil-like HL60 cells. This adhesion defect is associated with reduced expression of fMLP receptors and β2 integrins as well as the inhibited β2 integrin activation, as assessed by conformational-specific antibodies. Mitofusin-2 deficiency limited the maturation of β2 integrin activation during the neutrophil-directed differentiation of HL60 cells, which is indicated by identified markers CD35 and CD87. Reversed to results in overall HL60 cells, mitofusin-2 knockdown in β2-integrin-activation-matured cells (CD87high population) enhanced integrin activation. Our study illustrates the function of mitofusin-2 in leukocyte adhesion and may provide new insights into the development and treatment of mitofusin-2-deficiency-related disease.

Mitocryptide-2: Identification of Its Minimum Structure for Specific Activation of FPR2-Possible Receptor Switching from FPR2 to FPR1 by Its Physiological C-terminal Cleavages.

Mitocryptides are a novel family of endogenous neutrophil-activating peptides originating from various mitochondrial proteins. Mitocryptide-2 (MCT-2) is one of such neutrophil-activating peptides, and is produced as an N-formylated pentadecapeptide from mitochondrial cytochrome b. Although MCT-2 is a specific endogenous ligand for formyl peptide receptor 2 (FPR2), the chemical structure within MCT-2 that is responsible for FPR2 activation is still obscure. Here, we demonstrate that the N-terminal heptapeptide structure of MCT-2 with an N-formyl group is the minimum structure that specifically activates FPR2. Moreover, the receptor molecule for MCT-2 is suggested to be shifted from FPR2 to its homolog formyl peptide receptor 1 (FPR1) by the physiological cleavages of its C-terminus. Indeed, N-terminal derivatives of MCT-2 with seven amino acid residues or longer caused an increase of intracellular free Ca2+ concentration in HEK-293 cells expressing FPR2, but not in those expressing FPR1. Those MCT-2 derivatives also induced β-hexosaminidase secretion in neutrophilic/granulocytic differentiated HL-60 cells via FPR2 activation. In contrast, MCT-2(1–4), an N-terminal tetrapeptide of MCT-2, specifically activated FPR1 to promote those functions. Moreover, MCT-2 was degraded in serum to produce MCT-2(1–4) over time. These findings suggest that MCT-2 is a novel critical factor that not only initiates innate immunity via the specific activation of FPR2, but also promotes delayed responses by the activation of FPR1, which may include resolution and tissue regeneration. The present results also strongly support the necessity of considering the exact chemical structures of activating factors for the investigation of innate immune responses.

Characterization of NADPH Oxidase Expression and Activity in Acute Myeloid Leukemia Cell Lines: A Correlation with the Differentiation Status.

In acute myeloid leukemia (AML), a low level of reactive oxygen species (ROS) is associated with leukemic stem cell (LSC) quiescence, whereas a high level promotes blast proliferation. ROS homeostasis relies on a tightly-regulated balance between the antioxidant and oxidant systems. Among the oxidants, NADPH oxidases (NOX) generate ROS as a physiological function. Although it has been reported in AML initiation and development, the contribution of NOX to the ROS production in AML remains to be clarified. The aim of this study was to investigate the NOX expression and function in AML, and to examine the role of NOX in blast proliferation and differentiation. First, we interrogated the NOX expression in primary cells from public datasets, and investigated their association with prognostic markers. Next, we explored the NOX expression and activity in AML cell lines, and studied the impact of NOX knockdown on cell proliferation and differentiation. We found that NOX2 is ubiquitously expressed in AML blasts, and particularly in cells from the myelomonocytic (M4) and monocytic (M5) stages; however, it is less expressed in LSCs and in relapsed AML. This is consistent with an increased expression throughout normal hematopoietic differentiation, and is reflected in AML cell lines. Nevertheless, no endogenous NOX activity could be detected in the absence of PMA stimulation. Furthermore, CYBB knockdown, although hampering induced NOX2 activity, did not affect the proliferation and differentiation of THP-1 and HL-60 cells. In summary, our data suggest that NOX2 is a marker of AML blast differentiation, while AML cell lines lack any NOX2 endogenous activity.

Isobavachalcone inhibits acute myeloid leukemia: Potential role for ROS-dependent mitochondrial apoptosis and differentiation.

Isobavachalcone (IBC) has been shown to induce apoptosis and differentiation of acute myeloid leukemia (AML) cells. However, the underlying molecular mechanisms are not fully understood. Herein, IBC exhibited significant inhibition on the cell viability, proliferation, and the colony formation ability of AML cells. Moreover, IBC induced mitochondrial apoptosis evidenced by reduced mitochondrial membrane potential, increased Bax level, decreased Bcl-2, Bcl-xL, and Mcl-1 levels, elevated cytochrome c level in the cytosol and increased cleavage of caspase-9, caspase-3, and PARP. Furthermore, IBC obviously promoted the differentiation of AML cells, accompanied by the increase of the phosphorylation of MEK and ERK and the C/EBPα expression as well as the C/EBPβ LAP/LIP isoform ratio, which was significantly reversed by U0126, a specific inhibitor of MEK. Notably, IBC enhanced the intracellular ROS level. More importantly, IBC-induced apoptosis and differentiation of HL-60 cells were significantly mitigated by NAC. In addition, IBC also exhibited an obvious anti-AML effect in NOD/SCID mice with the engraftment of HL-60 cells. Together, our study suggests that the ROS-medicated signaling pathway is highly involved in IBC-induced apoptosis and differentiation of AML cells.

HDN-1 induces cell differentiation toward apoptosis in promyelocytic leukemia cells depending on its selective effect on client proteins of Hsp90

Heat Shock Protein 90 (Hsp90) is frequently upregulated in many cancers, and its inhibition simultaneously blocks multiple signaling pathways, resulting in cell differentiation or apoptosis. However, the complexity of Hsp90 in differentiation and its relation with apoptosis have remained unsettled. In this study, we demonstrated that HDN-1, a C-terminal inhibitor of Hsp90, induced the differentiation of HL-60 cells toward apoptosis. HDN-1 induced the differentiation of cells containing mutant AML1-ETO into mature granulocytes, which was related to its selective effect on client proteins of Hsp90. HDN-1 destabilized AML1-ETO and preserved C/EBPβ at the same time, thereby induced a total increase in C/EBPβ levels because of AML1-ETO negative regulation to C/EBPβ expression. Neither HDN-1 nor 17-AAG (an N-terminal inhibitor of Hsp90) led to the differentiation of NB4 cells because mutant PML-RARα was not affected as a client protein of Hsp90; thus, no additional expression of C/EBPβ was induced. 17-AAG did not affect the differentiation of HL-60 cells due to decreased AML1-ETO and C/EBPβ levels. These results indicate that HDN-1 drives cell differentiation toward apoptosis depending on its selective influence on client proteins of Hsp90, establishing the relationship between differentiation and apoptosis and uncovering the mechanism of HDN-1 in promyelocytic leukemia cell differentiation. Moreover, HDN-1 is very promising for the development of anticancer agents with the induction of differentiation.

Chemotaxis and swarming in differentiated HL-60 neutrophil-like cells

The human leukemia cell line (HL-60) is an alternative to primary neutrophils in research studies. However, because HL-60 cells proliferate in an incompletely differentiated state, they must undergo differentiation before they acquire the functional properties of neutrophils. Here we provide evidence of swarming and chemotaxis in differentiated HL-60 neutrophil-like cells (dHL-60) using precise microfluidic assays. We found that dimethyl sulfoxide differentiated HL-60 cells (DdHL-60) have a larger size, increased length, and lower ability to squeeze through narrow channels compared to primary neutrophils. They migrate through tapered microfluidic channels slower than primary neutrophils, but faster than HL-60s differentiated by other protocols, e.g., using all-trans retinoic acid. We found that dHL-60 can swarm toward zymosan particle clusters, though they display disorganized migratory patterns and produce swarms of smaller size compared to primary neutrophils.

IQF characterization of a cathepsin B-responsive nanoprobe for report of differentiation of HL60 cells into macrophages.

Tracking of in vivo fates of exogenous cell transplants in terms of viability, migration, directional differentiation and function delivery by a suitable method of medical imaging is of great significance in the development and application of various cell therapies. In this contribution directional differentiation of HL60 cells into macrophages and granulocytes, and a difference in the associated expression level of cathepsin B (Cat B) among the parent and daughter cells is used as a model to guide and evaluate the development of a Cat B-responsive Abz-FRFK-Dnp@PLGA nanoprobe for an optical report of the differentiation process. A well-documented internally quenched fluorescence (IQF) pair coupled with a peptide substrate FRFK of Cat B was synthesized and imbedded in PLGA to form the nanoprobe. The nanoprobe is resistant to leakage when dispersed in water for 10 days. Degradation of the nanoprobe is dominated by Cat B. HL60 cells were then labelled with the Abz-FRFK-Dnp@PLGA nanoprobe to track the differentiation process. Differentiation of labelled HL60 cells into macrophages exhibited a significantly higher fluorescence relative to the granulocytes or the labelled parent cells. The fluorescence difference allows the differentiation process to be followed. The established characterization and assessment procedure is to be used for the development and evaluation of nanoprobes for other imaging modalities.

Trdmt1 3'-untranslated region functions as a competing endogenous RNA in leukemia HL-60 cell differentiation.

Severe blockage in myeloid differentiation is the hallmark of acute myeloid leukemia (AML). Trdmt1 plays an important role in hematopoiesis. However, little is known about the function of Trdmt1 in AML cell differentiation. In the present study, Trdmt1 was up-regulated and miR-181a was down-regulated significantly during human leukemia HL-60 cell differentiation after TAT-CT3 fusion protein treatment. Accordingly, miR-181a overexpression in HL-60 cells inhibited granulocytic maturation. In addition, our “rescue” assay demonstrated that Trdmt1 3′-untranslated region promoted myeloid differentiation of HL-60 cells by sequestering miR-181a and up-regulating C/EBPα (a critical factor for normal myelopoiesis) via its competing endogenous RNA (ceRNA) activity on miR-181a. These findings revealed an unrecognized role of Trdmt1 as a potential ceRNA for therapeutic targets in AML.