Differentiation Of HL-60 Cells Research Articles

O-GlcNAc-Mediated Regulation of Galectin Expression and Secretion in Human Promyelocytic HL-60 Cells Undergoing Neutrophilic Differentiation.

In this study, we have tested the hypothesis that the expression and secretion of galectins are driven through mechanisms globally impacted by homeostatic regulation involving the post-translational modification of intracellular proteins with O-linked N-acetylglucosamine (O-GlcNAc). We showed that neutrophilic differentiation of HL-60 cells induced by all-trans retinoic acid (ATRA) and 6-diazo-5-oxo-L-norleucine (DON) was associated with a significant drop of cellular O-GlcNAc levels in serum-contained and serum-free cell culture media. Galectin gene and protein expression profiles in HL-60 cells were specifically modified by ATRA and by inhibitors of O-GlcNAc cycle enzymes, however overall trends for each drug were similar between cells growing in the presence or absence of serum except for LGALS9 and LGALS12. The secretion of four galectins (-1, -3, -9, and -10) by HL-60 cells in a serum-free medium was stimulated by O-GlcNAc-reducing ATRA and DON while O-GlcNAc-elevating thiamet G (O-GlcNAcase inhibitor) failed to change the basal levels of extracellular galectins. Taken together, these results demonstrate that O-GlcNAc homeostasis is essential not only for regulation of galectin expression in cells but also for the secretion of multiple members of this protein family, which can be an important novel aspect of unconventional secretion mechanisms.

CDDO Induces Differentiation through PTEN Expression in HL-60 Cells

Acute promyelocytic leukemia (APL) represents a group of haematological malignancies characterized by blocked differentiation. Cell differentiation has been as a strong target of treatment of APL. Peroxisome proliferator-activated receptors gamma (PPAR-γ) play an important role in the differentiation of APL cell lines. Previous study has shown that PPAR-γ ligands induce neutrophil/ monocytic differentiation in human acute promyelocytic leukemia HL-60 cells. It has recently been shown that the novel synthetic triterpenoid 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid (CDDO) induces differentiation in leukemia HL-60 cells. In this study we established a CDDO-induced differentiation model for the treatment of APL. We evaluated for the surface antigen expression associated with myeloid maturation, CD11b and CD14 by flow cytometric analysis and RT-PCR. We demonstrated that CD11b and CD14 increased as CDDO -induced differentiation. In addition, CDDO induced a HL-60 differentiation through the regulation of PTEN expression. PTEN also has various cellular function such as cell differentiation. PPAR-γ could synergistically up-regulate PTEN in human leukemia cells and consequently stimulate the differentiation of these cells We confirmed that suppression of PTEN with the inhibitor (SF1760) caused reduction of differentiated HL-60 cells. Our results suggest PPAR-γ an important role in controlling the differentiation of leukemia cells via the regulation of PTEN. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

The characteristics of neutrophil extracellular traps produced by all-trans retinoic acid-induced dHL-60 under PMA stimulation

Extracellular traps released by neutrophils (neutrophil extracellular traps, NETs) are a double-edged sword, and understanding the mechanism of NET formation is of great significance for disease treatment. However, the short lifespan, the large individual differences, and the inability to perform gene editing render it difficult to decipher NET formation using neutrophils. It is necessary to find a model cell to replace neutrophils to study the mechanism of NET formation. In this study, we used different concentrations (0, 0.1, 1, and 10 μmol/L) of all-trans retinoic acid (ATRA) to differentiate HL-60 cells for different days (1, 3, 5, and 7 days). By detecting the cell viability and nuclear morphology of cells, we confirmed that HL-60 cells were differentiated to neutrophil-like cells (dHL-60) after treated with ATRA for at least 5 days. Using immunofluorescence staining to detect the formation of NETs, we demonstrated that dHL-60 cells differentiated for 5 days with 1 μmol/L ATRA could generate NETs comparable to those produced by neutrophils upon phorbol 12-myristate 13-acetate (PMA) stimulation, without histone H3 citrullination. Furthermore, the formation of NETs by dHL-60 cells were NADPH-dependent and PAD4-independent, consistent with neutrophils. Taken together, these observations suggest that dHL-60 cells differentiated with 1 μmol/L ATRA for 5 days can be used as a model cell for neutrophils to study the mechanism of NET formation.

EGCG Prevents the Transcriptional Reprogramming of an Inflammatory and Immune-Suppressive Molecular Signature in Macrophage-like Differentiated Human HL60 Promyelocytic Leukemia Cells.

Simple SummaryScientists are increasingly harnessing the power of the immune system to prevent cancer. While macrophages are a major component of the tumor microenvironment where they orchestrate various aspects of immunity, dysregulated immune and inflammatory responses will play a key role in cancer initiation and progression. Depending on their activation status, macrophages can have a dual impact on tumorigenesis by either antagonising cytotoxic immune cells or enhancing antitumor responses. Increased infiltration of tumor-associated macrophages has long been associated with poor patient prognosis in most solid cancers, highlighting their value as potential diagnostic and prognostic biomarkers in cancer. A variety of macrophage-centered approaches to cancer therapy have been investigated, including strategies to prevent tumor-promoting activities. In this study, we highlight the chemopreventive properties of EGCG derived from green tea that underpin its ability to re-program macrophage molecular signature and function.Background: The promyelocytic leukemia cell differentiation process enables recapitulation of the polarized M1 or M2 macrophage-like phenotype with inflammatory and immune-suppressive properties. While evidence supports the anti-inflammatory effect of dietary-derived epigallocatechin-3-gallate (EGCG), its impact on the onset of immune phenotype molecular signature remains unclear. Methods: Human HL60 promyelocytic cells grown in suspension were differentiated into CD11bHigh/CD14Low adherent macrophages with phorbol 12-myristate 13-acetate (PMA). Gelatin zymography was used to assess the levels of matrix metalloproteinase (MMP)-9, and total RNA was isolated for RNAseq and RT-qPCR assessment of differentially expressed gene levels involved in inflammation and immunity. Protein lysates were used to assess the phosphorylation status of signaling intermediates involved in macrophage-like cell differentiation. Results: Cell adhesion and induction of MMP-9 were indicative of HL60 cell differentiation into a macrophage-like phenotype. The extracellular signal-regulated kinase (ERK), glycogen synthase kinase (GSK)-3, p90 ribosomal S6 kinases (RSK), and cAMP-response-element-binding protein (CREB) were all phosphorylated, and EGCG reduced such phosphorylation status. Increases in inflammation and immunity genes included, among others, CCL22, CSF1, CSF2, IL1B, and TNF, which inductions were prevented by EGCG. This was corroborated by unbiased transcriptomic analysis which further highlighted the capacity of EGCG to downregulate the hematopoietic stem cell regulator CBFA2T3. Conclusion: EGCG inhibits inflammatory signaling crosstalk and prevents the onset of an immune phenotype in macrophage-like differentiated cells.

Anti-leukemic principle(s) from Momordica charantia seeds induce differentiation of HL-60 cells through ERK/MAPK signalling pathway.

The online version contains supplementary material available at 10.1007/s10616-022-00547-x.

A Reactive Metabolite of Clozapine Induces Hematopoietic Toxicity in HL-60 Cells Undergoing Granulocytic Differentiation through Its Effect on Glutathione Metabolism.

Clozapine is an atypical antipsychotic with several advantages over conventional antipsychotics, in addition to its well-known efficacy in treatment-resistant schizophrenia. However, the high risk of agranulocytosis associated with clozapine therapy limits its clinical application. Clozapine bioactivation to an unstable protein-reactive metabolite, identified as a nitrenium intermediate, has been implicated in cytotoxicity toward neutrophils. Clozapine affects myeloid precursor cells rather than neutrophils; however, the impact of its reactive metabolite on myeloid precursor cells undergoing granulocytic differentiation remains unclear. Herein, we used hydrogen peroxide (H2O2) to generate the reactive metabolite and compared reactive metabolite-induced cytotoxicity between HL-60 cells undergoing granulocytic differentiation and differentiated HL-60 cells. In addition, we examined the role of oxidative stress in this type of cytotoxicity. The reactive metabolite of clozapine induced rapid cytotoxicity in HL-60 cells undergoing granulocytic differentiation, but not in differentiated HL-60 cells, with the metabolite exhibiting more potent cytotoxicity than clozapine. No cytotoxicity was observed following incubation with olanzapine, a structural analog of clozapine, even after exposure of the drug to H2O2. The reactive metabolite of clozapine decreased the levels of reduced glutathione, while addition of reduced glutathione attenuated the reactive metabolite-induced cytotoxicity. These findings indicate that glutathione metabolism plays a role in the hematopoietic toxicity induced by the reactive metabolite of clozapine. Oxidative stress may potentially increase susceptibility to the hematopoietic toxicity induced by the reactive metabolite of clozapine.

ELK1-Induced upregulation of long non-coding TNK2-AS1 promotes the progression of acute myeloid leukemia by EZH2-mediated epigenetic silencing of CELF2

ABSTRACT Acute myeloid leukemia (AML) is the second most common hematological malignancy after lymphoma in the world. Long non-coding RNAs (LncRNAs) have been suggested as key regulators of cancer development and progression in AML. As a member of lncRNA family, the biological role and mechanisms of tyrosine kinase non receptor 2 antisense RNA 1 (TNK2-AS1) in AML is still unclear. The expression of TNK2-AS1 was measured with RT-qPCR in AML cell lines. The changes of the proliferation, apoptosis, and differentiation in TNK2-AS1 shRNA-transfected HL-60 and THP-1 cells were detected with CCK-8, EdU, flow cytometry, Western blot, and NBT assays. Molecular control of TNK2-AS1 on CUGBP Elav-like family member 2 (CELF2) and ETS domain-containing protein-1 (ELK1) on TNK2-AS1 was assessed by chromatin immunoprecipitation (ChIP), RT-qPCR, Western blot, and RNA immunoprecipitation (RIP) assays. TNK2-AS1 expression was upregulated in AML cell lines and negatively correlated with survival patients. Knockdown of TNK2-AS1 markedly reduced AML cell proliferation and promoted apoptosis and differentiation. Likewise, TNK2-AS1 knockdown significantly suppressed tumor growth in vivo. Mechanistically, the upregulation of TNK2-AS1 was activated by transcription factor ELK1. We also uncovered that TNK2-AS1 exerted tumor-promoting effect through silencing CELF2 via binding with EZH2, thus activating PI3K/Akt pathway in AML cells. Elevated expression of TNK2-AS1 was induced by ELK1 and facilitated AML progression by suppressing CELF2 expression via EZH2-mediated epigenetic silencing, suggesting TNK2-AS1 may be a promising therapeutic target and prognostic marker for AML patients.

Targeting FHL1 impairs cell proliferation and differentiation of acute myeloid leukemia cells.

The four and a half LIM domains 1 (FHL1) is considered to play important roles in tumors. This study aims to investigate the role and precise mechanisms of FHL1 in acute myeloid leukemia (AML). Here, we found that FHL1 was highly expressed in AML. CCK8, flow cytometry, and Western blot analysis of cell cycle-related proteins showed that overexpression of FHL1 promoted proliferation and accelerated cell cycle progression in HL-60 cells. Conversely, knockdown of FHL1 inhibited the proliferation and induced cell cycle arrest in KG-1 cells. Furthermore, knockdown of FHL1 promoted cell differentiation, while overexpression of FHL1 restrained all-trans retinoic acid induced cell differentiation in HL-60 cells, revealed by Wright-Giemsa staining and cell surface antigen analysis. Moreover, in vivo experiments revealed that depletion of FHL1 inhibited tumor growth and led to increased levels of CD11b and CD14. Here, we first identify an unexpected and important role of FHL1 that contributes to the AML progression, indicating that FHL1 may be a potential therapeutic target for AML.

The Effect of Honey Bee Venom on CD14 Protein Expression as Monocyte Marker in D-Alpha-Tocopheryl Succinate (Vitamin E)-Treated HL-60 Cells

Background: The membrane form of a Cluster of Differentiation 14 (CD14) is anchored to the phospholipid bilayer via glycosylphosphatidylinositol anchor. This molecule is expressed on the intrinsic surface of monocytes and neutrophils. This protein is not expressed on the HL60 cell surface. It is believed that the differentiation of HL-60 cells stops in the promyelocytic stage. The differentiation of HL-60 cells with compounds such as vitamin A, D, E results in membrane CD14 expression.
 Objectives: The objective of this study was an evaluation of CD14 expression by Honey Bee Venom (HBV) in HL60 cells treated by D-alpha-Tocopheryl Succinate (D-α-TS). Methods: HL-60 cells were cultured in RPMI Media 1640 medium and treated with different concentrations of D-α-TS and HBV. Cellular differentiation was tested by Nitro Blue Tetrazolium (NBT) staining, immunocytochemistry, and flow cytometry. The studied data were analyzed using one-way analysis of variance and InStat 3 software.
 Methods: HL-60 cells were cultured in RPMI medium and treated with different concentrations of D-α-TS and HBV. Cellular differentiation was tested by NBT staining, immunocytochemistry, and flow cytometry. The studied data were analyzed using one-way analysis of variance and InStat 3 software.
 Results: MTT assay demonstrated that HBV and D-alpha-tocopheryl induce death in HL-60 cell lines in a time and dose-dependent manner. Also, Wright-Giemsa, NBT staining showed morphologically differentiation. Immunocytochemistry and flow cytometry analysis shows that cells treated with 6 µg/mL D-α-TS and 2.5 µg/mL HBV for 5 days significantly increase the expression of CD14 in HL-60 compared to cells treated with D-α-TS.
 Conclusion: HBV can induce cell death and inhibit cell proliferation. Also, increase differentiation potency of D-α-TS via HBV can increase the differentiation by inhibiting NFκB and COX-2 and increasing the expression of P21 that plays an essential role in increasing CD14 protein expression and subsequently induce differentiation in HL-60 cells to monocytes.

Temperature-sensitive migration dynamics in neutrophil-differentiated HL-60 cells

Cell migration plays an essential role in wound healing and inflammatory processes inside the human body. Peripheral blood neutrophils, a type of polymorphonuclear leukocyte (PMN), are the first cells to be activated during inflammation and subsequently migrate toward an injured tissue or infection site. This response is dependent on both biochemical signaling and the extracellular environment, one aspect of which includes increased temperature in the tissues surrounding the inflammation site. In our study, we analyzed temperature-dependent neutrophil migration using differentiated HL-60 cells. The migration speed of differentiated HL-60 cells was found to correlate positively with temperature from 30 to 42 °C, with higher temperatures inducing a concomitant increase in cell detachment. The migration persistence time of differentiated HL-60 cells was higher at lower temperatures (30–33 °C), while the migration persistence length stayed constant throughout the temperature range. Coupled with the increased speed observed at high temperatures, this suggests that neutrophils are primed to migrate more effectively at the elevated temperatures characteristic of inflammation. Temperature gradients exist on both cell and tissue scales. Taking this into consideration, we also investigated the ability of differentiated HL-60 cells to sense and react to the presence of temperature gradients, a process known as thermotaxis. Using a two-dimensional temperature gradient chamber with a range of 27–43 °C, we observed a migration bias parallel to the gradient, resulting in both positive and negative thermotaxis. To better mimic the extracellular matrix (ECM) environment in vivo, a three-dimensional collagen temperature gradient chamber was constructed, allowing observation of biased neutrophil-like differentiated HL-60 migration toward the heat source.

MAP kinase-dependent autophagy controls phorbol myristate acetate-induced macrophage differentiation of HL-60 leukemia cells

We investigated the mechanisms and the role of autophagy in the differentiation of HL-60 human acute myeloid leukemia cells induced by protein kinase C (PKC) activator phorbol myristate acetate (PMA). PMA-triggered differentiation of HL-60 cells into macrophage-like cells was confirmed by cell-cycle arrest accompanied by elevated expression of macrophage markers CD11b, CD13, CD14, CD45, EGR1, CSF1R, and IL-8. The induction of autophagy was demonstrated by the increase in intracellular acidification, accumulation/punctuation of autophagosome marker LC3-II, and the increase in autophagic flux. PMA also increased nuclear translocation of autophagy transcription factors TFEB, FOXO1, and FOXO3, as well as the expression of several autophagy-related (ATG) genes in HL-60 cells. PMA failed to activate autophagy inducer AMP-activated protein kinase (AMPK) and inhibit autophagy suppressor mechanistic target of rapamycin complex 1 (mTORC1). On the other hand, it readily stimulated the phosphorylation of mitogen-activated protein (MAP) kinases extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) via a protein kinase C-dependent mechanism. Pharmacological or genetic inhibition of ERK or JNK suppressed PMA-triggered nuclear translocation of TFEB and FOXO1/3, ATG expression, dissociation of pro-autophagic beclin-1 from its inhibitor BCL2, autophagy induction, and differentiation of HL-60 cells into macrophage-like cells. Pharmacological or genetic inhibition of autophagy also blocked PMA-induced macrophage differentiation of HL-60 cells. Therefore, MAP kinases ERK and JNK control PMA-induced macrophage differentiation of HL-60 leukemia cells through AMPK/mTORC1-independent, TFEB/FOXO-mediated transcriptional and beclin-1-dependent post-translational activation of autophagy.

Moraxella catarrhalis evades neutrophil oxidative stress responses providing a safer niche for nontypeable Haemophilus influenzae

SummaryMoraxella catarrhalis and nontypeable Haemophilus influenzae (NTHi) are pathogenic bacteria frequently associated with exacerbation of chronic obstructive pulmonary disease (COPD), whose hallmark is inflammatory oxidative stress. Neutrophils produce reactive oxygen species (ROS) which can boost antimicrobial response by promoting neutrophil extracellular traps (NET) and autophagy. Here, we showed that M. catarrhalis induces less ROS and NET production in differentiated HL-60 cells compared to NTHi. It is also able to actively interfere with these responses in chemically activated cells in a phagocytosis and opsonin-independent and contact-dependent manner, possibly by engaging host immunosuppressive receptors. M. catarrhalis subverts the autophagic pathway of the phagocytic cells and survives intracellularly. It also promotes the survival of NTHi which is otherwise susceptible to the host antimicrobial arsenal. In-depth understanding of the immune evasion strategies exploited by these two human pathogens could suggest medical interventions to tackle COPD and potentially other diseases in which they co-exist.

Decoding the signaling profile of hematopoietic progenitor kinase 1 (HPK1) in innate immunity: A proteomic approach.

Signaling via β2 integrins (CD11/CD18) as well as TCRs and BCRs involves similar pathways. However, the activation of the same signaling molecule can result in opposing effects. One such example is the hematopoietic progenitor kinase 1 (HPK1), which negatively regulates T and B cell activation but enforces neutrophil adhesion via β2 integrins. This difference may be defined by specific HPK1 interacting networks in different leukocyte subsets which have already been described in the adaptive immune system. Here, we set out to identify interacting proteins of HPK1 in neutrophil-like differentiated HL-60 cells exposed to immobilized fibrinogen and left nonactivated or Mn2+ -activated to allow β2 integrin-dependent adhesion. Co-IP experiments followed by mass spectrometry led to the identification of 115 HPK1-interacting proteins. A total of 58 proteins were found only in nonactivated cells and 39 proteins only in Mn2+ -activated adherent cells. From these results, we decoded a pre-existing signaling cluster of HPK1 in nonactivated cells encompassing proteins essential for β2 integrin-mediated signaling during neutrophil trafficking, namely DNAX-activation protein 12 (DAP12), spleen tyrosine kinase (Syk), and Rac1. Thus, our study provides novel insights into the complex architecture of the signaling processes during neutrophil activation and the complex signaling profile of HPK1 in leukocytes.

Ursolic Acid Suppresses Oncostatin M Expression through Blockade of PI3K/Akt/NF-κB Signaling Processes in Neutrophil-like Differentiated HL-60 Cells

Cytokine oncostatin M (OSM) plays an important role in a variety of inflammatory reactions and is mainly produced in neutrophils in inflammatory diseases. While natural pentacyclic triterpenoid ursolic acid (UA) possesses a wide range of beneficial effects, such as anti-oxidant, anti-tumor, and anti-inflammatory, the regulatory processes of OSM suppression by UA in neutrophils are still poorly understood. This study was aimed at examining how UA regulates OSM expression in neutrophil-like differentiated (d)HL-60 cells. Enzyme-linked immunosorbent assay, quantitative polymerase chain reaction, and immunoblotting were employed to analyze the effects of UA. Whereas stimulation with granulocyte-macrophage colony-stimulating factor (GM-CSF) led to elevations of OSM production and mRNA expression, these elevations were lowered by treatment with UA in neutrophil-like dHL-60 cells. When the cells were exposed to GM-CSF, phosphorylated levels of phosphatidylinositol 3-kinase, Akt, and nuclear factor-kB were upregulated. However, the upregulations were diminished by treatment with UA in neutrophil-like dHL-60 cells. The results of this study proposed that UA might relieve inflammatory diseases via inhibition of OSM.

Resveratrol Downregulates Granulocyte-Macrophage Colony-Stimulating Factor-Induced Oncostatin M Production through Blocking of PI3K/Akt/NF-κB Signal Cascade in Neutrophil-like Differentiated HL-60 Cells.

Oncostatin M (OSM) is essential in a wide range of inflammatory responses, and most OSM is produced by neutrophils in respiratory diseases. While resveratrol (RES) is regarded as an anti-inflammatory agent in a variety of conditions, the mechanism of OSM inhibition by RES in neutrophils remains to be elucidated. In this study, we investigated whether RES could inhibit OSM production in neutrophil-like differentiated (d)HL-60 cells. The effects of RES were measured by means of an enzyme-linked immunosorbent assay, real-time polymerase chain reaction, and Western blotting. Increases in production and mRNA expression of OSM resulted from the addition of granulocyte-macrophage colony-stimulating factor (GM-CSF) in neutrophil-like dHL-60 cells; however, these increases were downregulated by RES treatment. Exposure to GM-CSF led to elevations of phosphorylation of phosphatidylinositol 3-kinase (PI3K), Akt, and nuclear factor (NF)-kB. Treatment with RES induced downregulation of the phosphorylated levels of PI3K, Akt, and NF-κB in neutrophil-like dHL-60 cells. These results suggest that RES could be applicable to prevent and/or treat inflammatory disorders through blockade of OSM.

Generation of Membrane-Derived Nanovesicles by Nitrogen Cavitation for Drug Targeting Delivery and Immunization.

Extracellular vesicles (EVs) derived from cell membranes act as therapeutics and targeted drug carriers. However, the production scalability and reproducibility of EVs limit their biomedical applications. In the past few years, our lab has developed a nitrogen cavitation approach to efficiently produce EVs from any types of eukaryotic cells and bacteria. We have demonstrated that EVs derived from differentiated HL-60 cells can improve the treatment of inflammatory diseases. In addition, we have showed an increased survival of animals from bacterial infections by Pseudomonas aeruginosa after the mice were immunized with the nanovesicles derived from Pseudomonas aeruginosa membrane.

Roles of MFN2 and MFN2 associated protein in chemotaxis of neutrophil-like differentiated HL-60 cells

Roles of MFN2 and MFN2 associated protein in chemotaxis of neutrophil-like differentiated HL-60 cells

Dexamethasone Attenuates Oncostatin M Production via Suppressing of PI3K/Akt/NF-κB Signaling in Neutrophil-like Differentiated HL-60 Cells.

Oncostatin M (OSM) plays a role in various inflammatory reactions, and neutrophils are the main source of OSM in pulmonary diseases. However, there is no evidence showing the mechanism of OSM production in neutrophils. While dexamethasone (Dex) has been known to exert anti-inflammatory activity in various fields, the precise mechanisms of OSM downregulation by Dex in neutrophils remain to be determined. Here, we examined how OSM is produced in neutrophil-like differentiated HL-60 cells. Enzyme-linked immunosorbent assay, real-time polymerase chain reaction, and Western blot analysis were utilized to assess the potential of Dex. Granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulation resulted in OSM elevation in neutrophil-like dHL-60 cells. OSM elevation induced by GM-CSF is regulated by phosphatidylinositol 3-kinase (PI3K)/Akt/nuclear factor (NF)-kB signal cascades. GM-CSF stimulation upregulated phosphorylated levels of PI3K or Akt or NF-κB in neutrophil-like dHL-60 cells. Treatment with Dex decreased OSM levels as well as the phosphorylated levels of PI3K or Akt or NF-κB in neutrophil-like dHL-60 cells. Our findings show the potential of Dex in the treatment of inflammatory diseases via blocking of OSM.

Evidence of off-target effects of bosutinib that promote retinoic acid-induced differentiation of non-APL AML cells

ABSTRACT In the present study, we determined the effects of the Src family kinase (SFK) inhibitor, Bosutinib, and the engineered loss of the Lyn SFK on all-trans retinoic acid-induced leukemic cell differentiation. Retinoic acid (RA) is an embryonic morphogen and dietary factor that demonstrates chemotherapeutic efficacy in inducing differentiation of a non-APL AML cell model, the HL-60 human myeloblastic (FAB-M2) leukemia cell line, via activation of a novel signalsome containing an ensemble of signaling molecules that drive differentiation. Bosutinib is an inhibitor of SFKs used to treat myeloid leukemias where prominent high expression of SFKs, in particular Lyn, has been observed. Using either Bosutinib or loss of Lyn expression due to shRNA promoted RA-induced phenotypic differentiation, G0 arrest, and respiratory burst (functional differentiation) of HL-60 cells. Signaling events putatively seminal to RA-induced differentiation, the expression of Fgr, Cbl, Slp-76 and Vav, and the phosphorylation of c-Raf (pS259), Vav (p-tyr), and Slp76 (p-tyr) were not inhibited by Bosutinib or loss of Lyn. Nor was RA-induced upregulation of p-tyr phosphorylation of p47phox, a member of the NADPH complex that produces ROS, a putative phosphorylation dependent signaling regulator. Surprisingly, Bosutinib still works in the absence of Lyn to enhance RA-induced differentiation and neither compromised RA-induced expression, nor phosphorylation of signaling molecules that drive differentiation. These findings suggested there is a novel, off-target, Lyn-independent effect of Bosutinib that is of therapeutic significance to differentiation therapy.

Uremic serum damages endothelium by provoking excessive neutrophil extracellular trap formation

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. 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, HV-derived neutrophils and differentiated HL-60 cells by retinoic acid were used to determine the effect of uremic serum-induced NETs on human umbilical vein EC (HUVEC). 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 uremic serum significantly increased NET formation both in dHL-60 and isolated neutrophils compared to control serum, and these NETs decreased EC viability and induced their apoptosis. In addition, the level of ICAM-1, E-selectin and von Willebrand factor in HUVEC supernatant was significantly increased by uremic serum-induced NETs compared to control serum-induced NETs. 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.