HL-60 Differentiation Research Articles

Elastin-derived peptides (EDPs) as a potential pro-malignancy factor in human leukemia cell lines.

The extracellular matrix (ECM) is currently considered to be an important factor influencing the migration and progression of cancer cells. Therefore, the aim of our study was to investigate the mechanism of action of elastin-derived peptides in cancerous cells derived from the immunological system, i.e., HL-60, K562, and MEG-A2 cell lines. Moreover, an attempt to clarify the involvement of c-SRC kinase in EDP mechanism of action was also undertaken. Our data show that the VGVAPG and VVGPGA peptides are not toxic in the studied cell lines. Moreover, due to the involvement of KI67 and PCNA proteins in the cell cycle and proliferation, we can assume that neither peptide stimulates cell proliferation. Our data suggest that both peptides could initiate the differentiation process in all the studied cell lines. However, due to the different origins (HL-60 and K562-leukemic cell line vs. MEG-A2-megakaryoblastic origin) of the cell lines, the mechanism may differ. The increase in the ELANE mRNA expression noted in our experiments may also suggest enhancement of the migration of the tested cells. However, more research is needed to fully explain the mechanism of action of the VGVAPG and VVGPGA peptides in the HL-60, K562, and MEG-A2 cell lines. HIGHLIGHTS: • VGVAPG and VVGPGA peptides do not affect the metabolic activity of HL-60, K562, and MEG-A2 cells. • mTOR and PPARγ proteins are involved in the mechanism of action of VGVAPG and VVGPGA peptides. • Both peptides may initiate differentiation in HL-60, K562, and MEG-A2 cell lines.

Single-cell detection of DMSO promoted HL-60 differentiation toward granulocyte based on DC-iDEP for medicine screening.

The most common form of leukemia in adults is acute leukemia. Drug differentiation control is an extremely critical treatment for acute leukemia. Unfortunately, current techniques detecting differentiation control experience long time and complex steps of verification hindering the pace of medicine discovery: flow cytometry and RT-PCR are highly accurate and efficient at a cost of inconvenient fluorescent labeling or a high risk of contamination; conventional staining leads to cell death unavailable for further pharmacological tests. There is a great interest in developing simple, fast, and non-invasive techniques to screen medicine. DC-iDEP is an emerging label-free identification technique taking advantage of the whole cell native biophysical property for sorting cell populations. Here, HL-60 cell line has been used as a model to study the differentiation process toward granulocytes and medicine efficacy. The results showed that DEP succeeded in detecting the DMSO promoted HL-60 differentiation degree by the weighted average characterization factor. This factor is related to the single cell biophysical property, which accumulates to generate differences in each population with distinct constitutions. Furthermore, cichoric acid was investigated to be capable of promoting DMSO-induced differentiation efficiently. Using the change induced by cichoric acid, the HL-60 medicine screening application has been first attempted based on DEP. A rapid, label-free medicine screening method has been established to monitor HL-60 differentiation toward granulocyte and has great potential for medicine screening.

Dengue virus infection in mice induces bone marrow myeloid cell differentiation and generates Ly6Glow immature neutrophils with modulated functions.

While neutrophil activation during dengue virus infection is known, the effect of dengue virus infection on neutrophil biogenesis has not been studied. We demonstrate that dengue virus serotype 2 induces the differentiation of mice progenitor cells ex vivo toward the CD11b+Ly6C+Ly6G+ granulocyte population. We further observed an expansion of CD11b+Ly6CintLy6Glow myeloid cells in the bone marrow of dengue virus serotype 2-infected AG129 mice with low CXCR2 expression, implying an immature population. Additionally, dengue virus serotype 2 alone could induce the differentiation of promyelocyte cell line HL-60 into neutrophil-like cells, as evidenced by increased expression of CD10, CD66b, CD16, CD11b, and CD62L, corroborating the preferential shift toward neutrophil differentiation by dengue virus serotype 2 in the mouse model of dengue infection. The functional analysis showed that dengue virus serotype 2-induced neutrophil-like cells exhibited reduced phagocytic activity and enhanced NETosis, as evidenced by the increased production of myeloperoxidase, citrullinated histones, extracellular DNA, and superoxide. These neutrophil-like cells lose their ability to proliferate irreversibly and undergo arrest in the G0 to G1 phase of the cell cycle. Further studies show that myeloperoxidase-mediated signaling operating through the reactive oxygen species axis may be involved in dengue virus serotype 2-induced proliferation and differentiation of bone marrow cells as ABAH, a myeloperoxidase inhibitor, limits cell proliferation in vitro and ex vivo, affects the cell cycle, and reduces reactive oxygen species production. Additionally, myeloperoxidase inhibitor reduced NETosis and vascular leakage in dengue virus serotype 2-infected AG129 mice. Our study thus provides evidence that dengue virus serotype 2 can accelerate the differentiation of bone marrow progenitor cells into neutrophils through myeloperoxidase and modulate their functions.

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

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.

Transcription Factor Activation Profiles (TFAP) identify compounds promoting differentiation of Acute Myeloid Leukemia cell lines

Repurposing of drugs for new therapeutic use has received considerable attention for its potential to limit time and cost of drug development. Here we present a new strategy to identify chemicals that are likely to promote a desired phenotype. We used data from the Connectivity Map (CMap) to produce a ranked list of drugs according to their potential to activate transcription factors that mediate myeloid differentiation of leukemic progenitor cells. To validate our strategy, we tested the in vitro differentiation potential of candidate compounds using the HL-60 human cell line as a myeloid differentiation model. Ten out of 22 compounds, which were ranked high in the inferred list, were confirmed to promote significant differentiation of HL-60. These compounds may be considered candidate for differentiation therapy. The method that we have developed is versatile and it can be adapted to different drug repurposing projects.

Tyrosol, at the Concentration Found in Maltese Extra Virgin Olive Oil, Induces HL-60 Differentiation towards the Monocyte lineage

Tyrosol is a phenolic found in extra virgin olive oil (EVOO). In a Maltese monocultivar EVOO, it was present at a concentration of 9.23 ppm. The HL-60 acute myeloid leukaemia cell line, which can be differentiated to both monocytes and neutrophils, was exposed to tyrosol at this concentration and analysed for evidence of differentiation and effects of cytotoxicity. The polyphenol induced a 1.93-fold increase in cellular oxidative activity (p-value 0.044) and enhanced surface expression of CD11b and CD14. This indicates that tyrosol induces monocytic-like differentiation. An RNA-seq analysis confirmed the upregulation of monocyte genes and the loss of neutrophil genes concomitant with the bi-potential promyelocyte precursor moving down the monocytic pathway. A cell cycle analysis showed an accumulation of cells in the Sub G0/G1 phase following tyrosol exposure for 5 days, which coincided with an increase in apoptotic and necrotic markers. This indicates differentiation followed by cell death, unlike the positive monocyte differentiation control PMA. This selective cytotoxic effect following differentiation indicates therapeutic potential against leukaemia.

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.

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.

Cell Adhesion Expelling Analysis for Discriminating the State of Differentiation in HL60 Using Dielectrophoretic Force

We provide a novel method to discriminate the cells binding with molecules immobilized on a glass substrate by using the repulsive force of negative dielectrophoresis. The device consists of a lower substrate with interdigitated (IDA) patterns (12 μm in width and 50 μm in gap) of indium-tin oxide (ITO) and an upper ITO substrate disposed on the lower substrate. When an AC voltage is applied to a pair of microband electrodes comprising IDA to the upper ITO substrate connected to the ground, the cells are pressed onto gaps between the microband electrodes, and adhesion of the cells to the substrate is promoted (1st patterning in Fig. A). Subsequently, when an AC voltage is microband electrode “a” against the microband “b” and the upper ITO connected to the ground, the weakest region of the electric field is formed just above the microband “b”, and the cells move there by the repulsive force of negative dielectrophoresis (2nd patterning in Fig. B). At this time, if the cells adhered to the substrate via binding to molecules immobilized on the lower substrate, the cells remain on the gaps. Cells that do not interact with the molecules are expelled on the microband electrode. These procedures provide a rapid method for evaluating the expression of surface proteins that interact with molecules on the lower substrate in as little as 2 min; 60 sec in an adhesion and 60 sec in an expelling.The proposed method was applied to identify the differentiation status of HL60. Undifferentiated HL60 is known to express CD13 but not CD11b, while HL60 differentiated into neutrophils and monocytic lineages is known to up-regulate CD11b expression and down-regulate CD13 expression. In the device pretreated with anti-CD11b antibody, almost all HL60 formed well-defined linear patterns on the gaps between the microband electrodes at the 1st patterning by applying the ac voltage (100 kHz in frequency and 20 Vpp in voltage) for 60 seconds and aligned on the microband electrode “b” after 2nd patterning (day 0 in Fig. B). This result suggests that CD11b rarely expressed in HL60 at the state of undifferentiation. It was also confirmed that the expression levels of CD11b were extremely low by immunobiological fluorescent imaging. Conversely, HL60s differentiated with 1 μM all-trans retinoic acid (ATRA), which is well known as a inducer of HL60 to neutrophil, were randomly adhered on the gaps, and only a few of the adhered HL60s moved onto the microband electrodes after 2nd patterning (day 4 inf Fig. B). The increase of number of cells remaining on the gaps in differentiated HL60 represented the upregulation of CD11b expression. The expression of CD11b in differentiated cells cultured in ATRA-containing medium for 4 days was confirmed by immunostaining. There was a good correlation between the expression levels of CD11b obtained by the present method and those obtained by conventional fluorescent method. In addition, the expression of CD13 was investigated using the device pretreated with anti-CD13. In this case, the majority of undifferentiated cells adhered on the gap between the microband electrode and the differentiated cells were expelled from the gap onto the microband electrode “b”. Thus, using the repulsive force of n-DEP to expel cells unbound to the substrate, the differentiation status can be elucidated in as little as 120 seconds. Figure 1

Inhibition of Wnt/Beta-Catenin Pathway Impairs Natural Killer (NK) Cells Induced Apoptosis of Myeloid Leukemia Cells

Introduction:NK cells play an important role in the immune response to cancer due to their ability to induce T-cell independent apoptosis of neoplastic cells. NK cells recognize tumor cells through the interaction of several distinct cell surface receptors, with the net result of inducing NK cell activation and cytotoxic attack on tumor cells. It is hypothesized that a failure of NK surveillance is a contributing factor in the development of acute myeloid leukemia (AML). The discovery of pathways that regulate AML cell apoptosis in response to NK cells activation may represent a potential therapeutic target. It is known that the Wnt/beta-catenin pathway regulates cellular proliferation, differentiation and resistance to apoptosis in AML cells. However, its contribution to the resistance of AML cells in response to NK cell-induced apoptosis resistance is currently unknown. Objective:The objective of the current study is to determine if inhibition of the Wnt/beta catenin pathway impacts NK cell-induced apoptosis of AML cells. Methods:HL-60 leukemia cells were treated with different concentrations of the Wnt/beta-catenin pathway inhibitor XAV-939 (tankyrase inhibitor) to determine the lowest dose capable of inhibiting the Wnt/beta-catenin pathway without inducing cell apoptosis. Inhibition of the pathway was confirmed by evaluating beta-catenin protein by intracellular flow cytometry and real-time PCR for downstream factors MYC and TCF4. Next, differentiation of HL-60 cells into mature granulocytes was evaluated by analyzing expression of CD11b by flow cytometry after stimulation with dimethyl sulfoxide (DMSO), as another marker of Wnt/Beta-catenin pathway inhibition. After confirming pathway inhibition, HL-60 cells were incubated with healthy donor derived NK cells in a 2:1 ratio for 24 hours in the presence or absence of XAV-939. Apoptosis was evaluated by annexin staining. Expression of NK cell inhibitory proteins were determined by flow cytometry. A panel of 84 genes for hematopoietic transcription factors was evaluated by PCR array (RT2 Profiler PCR Array Gene Expression - Qiagen) in HL-60 cells after exposure to XAV-939 for 2 and 6 hours. Expression of apoptosis related genes in HL-60 cells after exposure to XAV-939 was determined by real-time PCR. Results:The dose of 1mM XAV-939 was selected to evaluate is effect in the HL-60 cell line. Suppression of pathway signaling was confirmed by a decrease in expression of Beta-catenin,MYC,TCF4and blockade of HL-60 differentiation into neutrophils. After coincubation with NK cells, there was a decrease in HL-60 cell death with concomitant exposure to XAV-939 (70% [untreated] vs. 30% [XAV-939]; p<0.05) (figure 1). There was no change in NK cell surface receptors or their putative ligands in HL-60 cells as evaluated by flow cytometry. Analysis of expression of hematopoietic transcription factor in HL-60 cells showed a downregulation of several transcription factors after exposure to XAV-939, includingJUNDandTCF7L2. Since there was a decrease in apoptotic cell death, we next evaluated expression of genes related to apoptotic pathways and could demonstrate a decrease in expression ofFASandTNFR1after exposure to XAV-939 (p<0.05) (figure 2A and 2B). Conclusions:These results suggest that inhibition of the Wnt/beta-catenin pathway decreases apoptosis of myeloid leukemia cells mediated by NK cells, and this effect may be secondary to a decreased expression ofFASandTNFR1in the leukemic cells. Figure Disclosures Santos: Novartis:Other: Speaker fee;Bristol-Myers Squibb:Other: Speaker fee.

An Evaluation of the Activity of Histidine-Rich Glycoprotein on Differentiated Neutrophil-Like Cells from Human Cell Lines.

Histidine-rich glycoprotein (HRG) treatment ameliorated the survival rate of septic mice by suppressing excess immunothrombus formation. Although such findings suggested that HRG may be one of the most useful drugs for sepsis, obtaining a stable experimental system to standardize the HRG drug product is difficult to achieve using neutrophils isolated from volunteers. This is due to the short survival time and individual differences of human neutrophils. In the present study, we determined whether the differentiated neutrophil-like cell lines exhibited similar responses to HRG compared with human purified neutrophils. All-trans retinoic acid (ATRA) was employed to induce the differentiation of the human myeloid leukemia cell lines HL-60 and NB-4. Thereafter, the cells were treated with Hank's balanced salt solution, human serum albumin, or HRG. The effects of HRG on these cells were evaluated according to cell shape, microcapillary passage, reactive oxygen species (ROS) production, neutrophil extracellular traps (NETs) formation, the expression of activated CD11b, and cell viability. HRG maintained the round shape of differentiated neutrophil-like cells, decreased the time required by cells to pass through the microcapillaries, and inhibited ROS production, NETs formation, and the expression of activated CD11b on the cell surface. Moreover, the cells could survive longer in the presence of HRG than the control. The ATRA-induced differentiated cell lines could be used as alternatives to neutrophils to investigate the effects of HRG on neutrophils. This method can thus be used as an essential standardization test in pharmaceutical development. SIGNIFICANCE STATEMENT: Human neutrophils exhibit varying responses to histidine-rich glycoprotein (HRG); however, all-trans retinoic acid-induced differentiated neutrophil-like cell lines can be used as reliably proxies to investigate the effects of HRG on neutrophils. Additionally, these cell lines can be employed in the development of therapies for the treatment of sepsis.

Role of Hexokinase 2 and 3 in Energy Metabolism, Myeloid Differentiation and Cancer Development

IntroductionTight control of metabolic processes is crucial for normal hematopoietic cell development, proliferation and cancer cell expansion. These processes all require variable bioenergetic demands, which are in part met by an upregulation of glycolytic activity. Hexokinases (HK1, 2, 3 and 4) catalyze the first and irreversible step of glycolysis. Recent evidence implicates a tumor promoting role for HK2 in KRas‐driven lung and ErbB2‐driven breast cancer. In contrast to the ubiquitously expressed HK1 and 2 in mammalian cells, HK3 is predominantly expressed in hematopoietic cells. We have identified HK3 as a transcriptional target of the hematopoietic‐specific transcription factor, PU.1 (SPI‐1). Moreover, HK3 is downregulated in acute myeloid leukemia (AML) patient samples and upregulated during terminal differentiation of selected acute myeloid leukemia cell lines concomitant with PU.1 upregulation.Methods and ResultsIn primary human CD34+ hematopoietic stem and early progenitor cells (HSPCs), we found HK1‐3 to be present at similar levels. While HK1 and HK2 mRNA levels remained unchanged during HSPC myeloid differentiation, HK3 mRNA levels significantly increased. Given the oncogenic role of HK2 and our findings of HK3 in AML cell lines, we next evaluated whether unique roles exist for HK2 and 3 in myeloid cells. Using shRNA or CRISPR/CAS9 technology, we generated HK2 and HK3 knockdowns (KD) and knockouts (KO) in HL60 and NB4 AML cell lines. Assessment of glycolytic and oxidative metabolism in HK altered cells revealed that loss of HK3 had no effect on steady state glycolytic activity, while loss of HK2 drastically reduced steady state glycolysis. In contrast, loss of HK3 expression resulted in increased cell death during all‐trans retinoic acid (ATRA) induced differentiation of HL60 (86±2% vs 36±3% viable cells at day 4, p<0.0001). Furthermore, investigation of HK3 subcellular localization point towards both cytosolic and nuclear localization for ectopically expressed HK3 in HL60 cells by western blot analysis. Mass spectrometric (MS) analysis of HL60 HK2 KO lines identified upregulation of proteins involved in key signaling pathways (Wnt, EGF and PDGF), as well as macromolecular metabolic processes, Fig. 1A. In contrast, HK3 KO HL60 cells showed upregulation of proteins involved in cellular homeostatic processes (ubiquitin proteasome pathway) and cancer‐related signaling pathways (Wnt, Ras) (p‐value<0.05), Fig. 1B.DiscussionTogether our findings implicate HK2 as the major glycolytically active isoform of hexokinases in myeloid cell lines, whereas HK3 exerts non‐canonical regulatory functions impacting normal myeloid differentiation and cancer regulation. Investigations are underway characterizing the metabolic phenotype of HK KO AML cells as well as defining the precise roles of HK2 and HK3 in normal HSPC differentiation, proliferation and metabolic activity.Support or Funding InformationSwiss Cancer ResearchPanther Pathway analyses of significantly upregulated proteins identified by MS analyses upon loss of either HK2 (A) or HK3 (B) in HL60 cell lines evaluated at steady state.Figure 1

Triterpenes from Poria cocos are revealed as potential retinoid X receptor selective agonists based on cell and in silico evidence.

Poria cocos is an edible and medicinal fungus that is widely used in Traditional Chinese Medicines as well as in modern applications. Retinoid X receptor (RXR) occupies a central place in nuclear receptor signaling, and a pharmacological RXR-dependent pathway is involved in myeloid cell function. Here, structural information for 82 triterpenes from P.cocos and 17 known RXR agonists was collected in a compound library and retrieved for a molecular docking study. Three triterpenes, 16α-hydroxytrametenolic acid (HTA), pachymic acid (PA), and polyporenic acid C (PPAC), were identified as novel RXR-specific agonists based on luciferase reporter assays and in silico evidence. Treatment with HTA, PA, and PPAC significantly induced differentiation of the human promyelocytic leukemia cell line HL-60 with EC50 values of 21.0±0.52, 6.7±0.37, and 9.4±0.65μM, respectively. These effects were partly blocked by the RXR antagonist UVI3003, suggesting that an RXR-dependent pathway may play an important role in their anti-acute promyelocytic leukemia (APL) effects. Taken together, triterpenes from P.cocos are revealed as naturally occurring RXR selective agonists with the potential for anti-cancer activity. These results suggest a novel approach to the treatment or prevention of APL.

CdSe Quantum Dots Induce Cellular Differentiation in HL-60 Cells

Background: CdSe QDs, synthesized using a green micro-emulsion method, have been evaluated for their potential in inducing HL-60 differentiation by employing various biochemical assays and as cellular imaging agents. Methods: CdSe QDs have been found to effectively induce differentiation with lower rate of cell apoptosis as compared with the positive control (DMSO). Results: Our results exhibit effective induction of HL-60 differentiation by CdSe QDs implicating their therapeutic role in cancerous cells in addition to their usual application as cell-imaging agents. Conclusion: Our studies report that the chemo-preventive properties of CdSe exhibit great potential and further tailored-synthesis and cell-differen-tiations studies have a promising future in the preventive treatment of HL60 cancerous tissue.

Mechanism Inducing the HL-60 Cell Differentiation by Haishengsu Tegillarca Granosa Extract via Targeting ULK1 Protein

To investigate the proliferation, differentiation potential and autophagic mechanism HL-60 cells via ULK1 protein targeted by Haishengsu extracted from Tegillarca granosa. The HL-60 cells were divided into five groups: Haishengsu of 10,100,1000 mg/L-treated group, all-trans retinoic acid (ATRA) 5 μmol/L-treated group and control group. The cell proliferation rates were detected by MTT after culture 12,24,48 h and 72 h. The expression of differentiation and maturation markers CD11b and CD15 were detected by flow cytometry, and the expression of autophagy molecules ULK1 and LC-3 proteins were measured by Western blot. After cultured for 48 and 72 h the cell proliferation rates of HL-60 in Haishengsu 100 and 1000 mg/L groups were significantly lower than those of ATRA 5 μmol/L group and Haishengsu 10 mg/L group and control group (P<0.05). After 72 h, the levels of CD11b, CD15, ULK1 and LC-3 expression of HL-60 cell in Haishengsu 100 and 1000 mg/L groups were higher than those in ATRA 5 μmol/L group, Haishengsu 10 mg/L group and control group (P<0.05). While the HL-60 cells in Haishengsu 10, 100 and 1000 mg/L group were treated with ULK1 protein inhibitor and after cultured for 72 h, the proliferation rate of HL-60 cell increased, levels of CD11b and CD15 expression decreased at the same time, and the levels of ULK1 and LC-3 proteins in HL-60 cells also decreased (P<0.05). Haishengsu, which extracted from Tegillarca granosa, can induce HL-60 cell proliferation and differentiation by targeting ULK1 protein in a concentration and time dependent manner, its effective mochanism may be mediating autophagic activity, promoting cell differentiation and maturation.

Whole-genome fingerprint of the DNA methylome during chemically induced differentiation of the human AML cell line HL-60/S4.

ABSTRACTEpigenomic regulation plays a vital role in cell differentiation. The leukemic HL-60/S4 [human myeloid leukemic cell line HL-60/S4 (ATCC CRL-3306)] promyelocytic cell can be easily differentiated from its undifferentiated promyelocyte state into neutrophil- and macrophage-like cell states. In this study, we present the underlying genome and epigenome architecture of HL-60/S4 through its differentiation. We performed whole-genome bisulphite sequencing of HL-60/S4 cells and their differentiated counterparts. With the support of karyotyping, we show that HL-60/S4 maintains a stable genome throughout differentiation. Analysis of differential Cytosine-phosphate-Guanine dinucleotide methylation reveals that most methylation changes occur in the macrophage-like state. Differential methylation of promoters was associated with immune-related terms. Key immune genes, CEBPA, GFI1, MAFB and GATA1 showed differential expression and methylation. However, we observed the strongest enrichment of methylation changes in enhancers and CTCF binding sites, implying that methylation plays a major role in large-scale transcriptional reprogramming and chromatin reorganisation during differentiation. Correlation of differential expression and distal methylation with support from chromatin capture experiments allowed us to identify putative proximal and long-range enhancers for a number of immune cell differentiation genes, including CEBPA and CCNF. Integrating expression data, we present a model of HL-60/S4 differentiation in relation to the wider scope of myeloid differentiation.

Adenanthin Induces Differentiation of Acute Promyelocytic Leukemia Cells by Targeting Peroxiredoxin III

To investigate the differentiation of acute promyelocytic leukemia (APL) cells induced by adenosine targeting Prx III. HL-60 cells were divided into four groups: control group, all-trans retinoic acid (ATRA) group, adenanthin group and ATRA+adenanthin group. Cell morphologic changes were observed under optical microscope. The influence of adenanthin on the differentiation of HL-60 was observed by nitro blue tetrazolium chloride (NBT) test. Cell surface differentiation antigens CD11b expression was measured by flow cytometry. The protein expression of Prx III was detected by immunohistochemical assay. Adenanthin could induce the differentiation of HL-60 cells; the NBT reduction positive rate in ATRA+adenanthin group was significantly higher than that in ATRA group and adenanthin group (P＜0.05). The percentage of CD11b positive cells in ATRA+adenanthin group (43.62％±1.38％) was higher than that in adenanthin group (28.15％±1.78％), ATRA group (36.72％±1.33％) and control group (7.99％±1.78％) (P＜0. 05). The content of Prx Ⅲ protein in adenanthin group was significantly higher than that in control group and ATRA group (P＜0.05). Adenanthin and ATRA have a synergistic effect on the differentiation and maturation of HL-60 cells, and its mechanism may be related with regulation of Prx III expression.

Characterization of the human E2F4 promoter region and its response to 12-O-tetradecanoylphorbol-13-acetate.

The E2F transcription factors (TFs), which control the progression of the cell cycle in response to DNA-damage and various stresses, are known to interact with a tumor suppressor, Retinoblastoma 1 (RB1). We previously showed that the response of the human RB1 promoter to a 12-O-tetradecanoylphorbol-13-acetate (TPA) in HL-60 cells is mediated by a duplicated GGAA-motif, which is also present in the 5'-upstream of the E2F family genes. The motifs are especially rich in the 5'-upstream of the E2F4 gene. In the present study, we constructed Luciferase (Luc) expression vectors containing a 466-bp of the 5'-upstream of the human E2F4 gene. The transfection of this plasmid and deletion/mutation-introduced derivatives into HL-60 cells and a Luc reporter assay showed that duplicated and triplicated GGAA (TTCC) motifs in the E2F4 promoter respond to TPA. As expected, electrophoretic mobility shift assay (EMSA) indicated that SPI1 (PU.1) binds to the GGAA-motif containing element. A quantitative RT-PCR and Western blotting showed that the E2F4 transcripts and its encoding proteins accumulate during the differentiation of HL-60 into macrophage-like cells. In contrast, the expression of the E2F1 gene and the protein, which possibly acts as a cell cycle accelerator, was greatly diminished.

MiR-183-5p Inhibits Occurrence and Progression of Acute Myeloid Leukemia via Targeting Erbin.

Erbin has been shown to have significant effects on the development of solid tumors. However, little is known about its function and regulatory mechanism in hematological malignancies. The biological function of Erbin on cell proliferation was measured in vitro and in vivo. The predicted target of Erbin was validated by dual-luciferase reporter assay and rescue experiment. We found that overexpression of Erbin could inhibit the cell proliferation and promote the cell differentiation of acute myeloid leukemia (AML) cells, whereas depletion of Erbin could enhance the cell proliferation and block the cell differentiation in AML cells in vitro and in vivo. Besides, miR-183-5p was identified as the upstream regulator that negatively regulated the Erbin expression. The results were confirmed by dual-luciferase reporter and RNA pull-down assay. Furthermore, we found that miR-183-5p negatively regulated Erbin, resulting in enhanced cell proliferation of AML cells via activation of RAS/RAF/MEK/ERK and PI3K/AKT/FoxO3a pathways. The activation of RAS/RAF/MEK/ERK and PI3K/AKT/FoxO3a pathways was mediated by Erbin interacting with Grb2. These results were also validated by rescue experiments in vitro and in vivo. All above-mentioned findings indicated that the miR-183-5p/Erbin signaling pathway might represent a novel prognostic biomarker or therapeutic target for treatment of AML.