Acute Monocytic Leukemia Research Articles

LPS induces inflammatory chemokines via TLR-4 signalling and enhances the Warburg Effect in THP-1 cells.

The Warburg Effect has emerged as a potential drug target because, in some cancer cell lines, it is sufficient to subvert it in order to kill cancer cells. It has also been shown that the Warburg Effect occurs in innate immune cells upon infection. Innate immune cells play critical roles in the tumour microenvironment but the Warburg Effect is not fully understood in monocytes. Furthermore, it is important to understand the impact of infections on key players in the tumour microenvironment because inflammatory conditions often precede carcinogenesis and mutated oncogenes induce inflammation. We investigated the metabolic programme in the acute monocytic leukaemia cell line, THP-1 in the presence and absence of lipopolysaccharide, mimicking bacterial infections. We found that stimulation of THP-1 cells by LPS induces a subset of pro-inflammatory chemokines and enhances the Warburg Effect. Surprisingly, perturbation of the Warburg Effect in these cells does not lead to cell death in contrast to what was observed in non-myeloid cancer cell lines in a previous study. These findings indicate that the Warburg Effect and inflammation are activated by bacterial lipopolysaccharide and may have a profound influence on the microenvironment.

A case of acute monocytic leukemia complicated with multiple myeloma

A case of acute monocytic leukemia complicated with multiple myeloma

MicroRNA‑20a negatively regulates the growth and osteoclastogenesis of THP‑1 cells by downregulating PPARγ.

The present study aimed to explore the mechanisms through which microRNA (miR)‑20a may be involved in the differentiation of THP‑1 human acute monocytic leukemia cells into osteoclasts. THP‑1 cells were differentiated into macrophages (osteoclast precursors) and subsequently into osteoclast cells. The expression levels of miR‑20a in THP‑1 cells were significantly reduced in a time‑dependent manner during phorbol‑12‑myristate‑13‑acetate (PMA), macrophage colony‑stimulating factor (M‑CSF) and receptor activator of nuclear factor‑κB ligand RANKL‑induced osteoclastogenesis. Following transfection with a miR‑20a mimics, the levels of miR‑20a in PMA‑treated THP‑1 cells increased more than 40‑fold as compared with expression in the control cells. In addition, the overexpression of miR‑20a inhibited proliferation, initiated S phase cell cycle arrest and induced apoptosis of PMA‑treated THP‑1 cells. Additionally, miR‑20a mimics treatment notably decreased the levels of tartrate‑resistant acid phosphatase, nuclear factor of activated T‑cells, cytoplasmic 1 and peroxisome proliferator‑activated receptor γ (PPARγ) during THP‑1 cell further differentiation progress. In summary, miR‑20a may negatively regulate the proliferation and osteoclastogenesis of THP‑1 cells during its osteoclast differentiation progress by downregulating PPARγ.

The value of detecting MLL gene rearrangement in children with acute monocytic leukemia

To assess the value of detecting the rearrangement of mixed lineage leukemia (MLL) gene in children with acute mononuclear leukemia (AML). Dual-color fluorescence in situ hybridization (FISH) probe was used to detect MLL gene rearrangement in 68 children with AML by interphase FISH. The results were compared with that of conventional G banding chromosomal analysis. Among the 68 children, 28 were detected by FISH with positive hybridization signals, with a detection rate for MLL gene rearrangement being 41.2%. Twelve (17.6%) reciprocal translocations and interruption of 11q23 were detected by G banding analysis. The difference in the detection rates between the two methods was statistically significant (P< 0.05). The sensitivity of FISH assay for MLL gene rearrangement was significantly higher than that of G banding chromosomal karyotyping. Combined use of both methods for children with AML can improve the detection rate of MLL gene rearrangements and provide crucial clues for clinical diagnosis, treatment and prognosis.

MicroRNA‑217 is involved in the progression of atherosclerosis through regulating inflammatory responses by targeting sirtuin 1.

Atherosclerosis is a chronic inflammatory disease, and it is a global clinical problem. The development of new and effective therapeutic targets for atherosclerosis is necessary. A number of microRNAs (miRNAs) have been demonstrated to serve a crucial role in atherosclerosis. However, the role of miRNA (miR)-217 in atherosclerosis remains unclear. Therefore, the aim of the present study was to investigate the role and mechanism of miR-217 in atherosclerosis. The level of miR-217 was detected in the blood of patients with atherosclerosis using reverse transcription-quantitative PCR. THP-1 acute monocytic leukemia cells were treated with oxidized low-density lipoprotein (ox-LDL) to develop an atherosclerotic cell model of macrophages. The relationship between miR-217 and sirtuin 1 (SIRT1) was determined by TargetScan and dual luciferase reporter assay. Cell apoptosis was measured by flow cytometry. Production of pro-inflammatory factors and triglyceride (TG) and total cholesterol (TC) levels were also determined. The results demonstrated that miR-217 was significantly upregulated in atherosclerosis. SIRT1 was demonstrated to be a direct target of miR-217 and was downregulated in atherosclerosis. Downregulation of miR-217 significantly inhibited ox-LDL-induced TG and TC level increase, cell apoptosis and the upregulation of the pro-inflammatory factors tumor necrosis factor α, interleukin (IL)-6 and IL-1β. Additionally, the SIRT1/AMP-activated protein kinase α/NF-κB pathway was at least partially involved in modulating the effects of miR-217 inhibition on THP-1 cells treated with ox-LDL. In addition, the effects of miR-217 downregulation on ox-LDL-treated THP-1 cells were eliminated by SIRT1 silencing. In conclusion, the results of the present study indicated that miR-217 downregulation may relieve atherosclerosis through the inhibition of macrophage apoptosis and inflammatory response by targeting SIRT1.

The regulation of inflammation-related genes after palmitic acid and DHA treatments is not mediated by DNA methylation.

Fatty acids (FAs) are known to participate in body inflammatory responses. In particular, saturated FAs such as palmitic acid (PA) induce inflammatory signals in macrophages, whereas polyunsaturated FAs, including docosahexaenoic acid (DHA), have been related to anti-inflammatory effects. Several studies have suggested a role of fatty acids on DNA methylation, epigenetically regulating gene expression in inflammation processes. Therefore, this study investigated the effect of PA and DHA on the inflammation-related genes on human macrophages. In addition, a second aim was to study the epigenetic mechanism underlying the effect of FAs on the inflammatory response. For these purposes, human acute monocytic leukaemia cells (THP-1) were differentiated into macrophages with 12-O-tetradecanoylphorbol-13-acetate (TPA), followed by an incubation with PA or DHA. At the end of the experiment, mRNA expression, protein secretion, and CpG methylation of the following inflammatory genes were analysed: interleukin 1 beta (IL1B), tumour necrosis factor (TNF), plasminogen activator inhibitor-1 (SERPINE1) and interleukin 18 (IL18). The results showed that the treatment with PA increased IL-18 and TNF-α production. Contrariwise, the supplementation with DHA reduced IL-18, TNF-α and PAI-1 secretion by macrophages. However, the incubation with these fatty acids did not apparently modify the DNA methylation status of the investigated genes in the screened CpG sites. This research reveals that PA induces important pro-inflammatory markers in human macrophages, whereas DHA decreases the inflammatory response. Apparently, DNA methylation is not directly involved in the fatty acid-mediated regulation of the expression of these inflammation-related genes.

Characteristic genes in THP‑1 derived macrophages infected with Mycobacterium tuberculosis H37Rv strain identified by integrating bioinformatics methods.

Mycobacterium tuberculosis (M. tb) is a highly successful pathogen that has co-existed with humans for 1,000's of years. As the cornerstone of the immune system, macrophages are a key part of innate immunity. They ingest and degrade foreign substances including aging cells and microorganisms, coordinate the inflammatory process, and are the first line of defense against M. tb infection. Recent advances in cellular mycobacteriology have indicated that M. tb uses an remarkably complex strategy to disrupt macrophage function, in order to counteract the antimicrobial mechanisms of the innate and adaptive immune responses, thereby achieving immune escape. With the popularity of microarray technology, a variety of public platforms have provided a variety of gene expression data associated with physiological and disease conditions. Meta-analysis can systematically and quantitatively analyze multiple independent data concerning the same disease, greatly improving the statistical significance and credibility of the gene expression data analysis performed. In the present study, 6 microarray expression datasets of human acute monocytic leukemia THP-1 cell line infected by M. tb H37Rv strain were collected from the GEO database. A total of 4 high-quality datasets were identified using meta-analysis methods in R language, and 306 differentially expressed genes with statistical significance were obtained. Then, a protein-protein interaction (PPI) network of these differentially expressed genes was constructed on the Search Tool for the Retrieval of Interacting Genes/Proteins Database online tool and visualized by Cytoscape v. 3.6.1 software. Using CentiScape and MCODE plugin in the Cytoscape software to mine the functional modules associated with M. tb infection process, 32 characteristic genes were identified. Gene ontology and Kyoto Encyclopedia of Genes and Genomes analysis was performed on the 32 characteristic genes, and it was demonstrated that these genes were primarily associated with the type I interferon (IFN) pathway. In the established model of THP-1-derived macrophages infected by M. tb, the actual differential expression levels of IFN-stimulated gene 15 (ISG15), 2′-5-oligoadenylate synthetase like (OASL), IFN regulatory factor 7 (IRF7) and DExD/H-box helicase 58 (DDX58), the first 4 genes of the 32 characteristic genes, were verified by reverse transcription quantitative polymerase chain reaction. The results were consistent with the results of microarray analysis. The association between ISG15, OASL and IRF7 and TB infection was also verified. Although a number of studies have identified that the type I IFN pathway may assist M. tb to achieve immune escape, the present study used a meta-analysis of microarray data and PPI network analysis to examine some of the novel genes identified in the IFN pathway. The results furthered the understanding of the molecular mechanisms of the TB immune response and provided a novel perspective for future therapeutic goals.

Synthetic 3-alkylpyridine alkaloid analogues as a new scaffold against leukemic cell lines: cytotoxic evaluation and mode of action

Leukemia is a malignant disease that originates in the bone marrow, local of blood cells production. Chemotherapy treatment is one of the alternatives to improve the survival of patients. In this context, the search for new compounds with potential cytotoxic action is justified. The present work evaluated the cytotoxic activity of 16 new synthetic analogs of 3-alkylpyridine alkaloids. The cytotoxic profiles were determined by MTT in vitro assay against THP-1 (acute monocytic leukemia), K562 (chronic myeloid leukemia) and PBMC (peripheral blood mononuclear) human cells. To investigate the mechanism of action, cell cycle analysis and alterations of TP53, p21, Bax, Bcl2, NOX-1, NOX-2, NOX-4 and p47-phox gene expressions were performed by qPCR, along with the measurement of reactive oxygen species (2’,7’-dichlorodihydrofluorescein diacetate and dihydroethidium). Compounds 4c, 5b, 5c and 6d were the most active and selective for THP-1 and compounds 7c and 11 were found to be more active for K562. All of this induced apoptosis in the tested strains. Concerning the investigation of the mechanism of action, it was observed that the pathway being activated is a p53-independent pathway. The data presented in this work indicate that 3-alkylpyridine alkaloid analogues are a potential class of compounds with cytotoxic action.

Molecular hydrogen suppresses free-radical-induced cell death by mitigating fatty acid peroxidation and mitochondrial dysfunction.

Molecular hydrogen (H2) was believed to be an inert and nonfunctional molecule in mammalian cells; however, we overturned the concept by reporting the therapeutic effects of H2 against oxidative stress. Subsequently, extensive studies revealed multiple functions of H2 by exhibiting the efficacies of H2 in various animal models and clinical studies. Here, we investigated the effect of H2 on free-radical-induced cytotoxicity using tert-butyl hydroperoxide in a human acute monocytic leukemia cell line, THP-1. Cell membrane permeability was determined using lactate dehydrogenase release assay and Hoechst 33342 and propidium iodide staining. Fatty acid peroxidation and mitochondrial viability were measured using 2 kinds of fluorescent dyes, Liperfluo and C11-BODIPY, and using the alamarBlue assay based on the reduction of resazurin to resorufin by mainly mitochondrial succinate dehydrogenase, respectively. Mitochondrial membrane potential was evaluated using tetramethylrhodamine methyl ester. As a result, H2 protected the cultured cells against the cytotoxic effects induced by tert-butyl hydroperoxide; H2 suppressed cellular fatty acid peroxidation and cell membrane permeability, mitigated the decline in mitochondrial oxidoreductase activity and mitochondrial membrane potential, and protected cells against cell death evaluated using propidium iodide staining. These results suggested that H2 suppresses free-radical-induced cell death through protection against fatty acid peroxidation and mitochondrial dysfunction.

The N-acetylgalactosamine-transferase11 (GALNT11) activation in leukemia and the role of cyclophosphamide and cytarabine in expression of this enzyme

Background: We investigated the activation of N-acetylgalactosamine-transferase11 (GALNT11) and the role of cyclophosphamide and cytarabine in expression of this enzyme in AML and CML patients. Methods: GALNT11 activity and expression levels were evaluated in human chronic myelogenous leukemia cells (K562) and human acute monocytic leukemia cells (THP1) after incubation with different concentrations of cyclophosphamide (Cyclo) and cytarabine (Cyta). Anti-proliferative effects of Cyclo and Cyta were examined by MTT assay. Real time-PCR and ELISA assays were applied to investigate the expression and activity of GALNT11, respectively. Results: The IC50 values for Cyclo were 34.73±0.12 μM for K562 and 36.16±0.23 µM for THP1 cells. These valuses for Cyta were also 0.38±0.15 μM for K562 and 0.52±0.18 µM for THP1 cells . Conclusion: The activation and expression of GALNT11 increased in acute and chronic leukemia as glycosylation disorders. Also cyclophosphamide and specially cytarabine cause significant increase in activation and expression of GALNT11 which inhibits glycosylation and tumorogenesis dependent signaling pathway.

MiRNA143 Downregulated the Expression of Cyclophilin A to Inhibit the Progression of Atherosclerosis

To investigate the influence of microRNA (miR)-143 on atherosclerosis and its working mechanism via cyclophilin A. Human acute monocytic leukemia cells (human acute monocytic leukemia cells, human THP-1 monocytes) were treated with ox-LDL 80 mg/L for 48 h to establish a foam cell model. The modeling was evaluated by oil red O staining. The expressions levels of miRNA-143 and CyPA were detected by q-PCR. RNA extracted was confirmed by SiO2 nano magnetic bead. Dual-luciferase reporter gene assay was used to verify the targeted regulatory relationship between miRNA-143 and CyPA. Next, the cells were transfected with miR-143 mimics and treated with a CyPA inhibitor (cyclophilin A). The expression levels of CyPA and downstream matrix metalloproteinases-9 (MMP-9) in the downstream were detected by q-PCR and western blot, respectively. No or few lipid droplets were observed in the normal THP-1 cells. After treatment with ox-LDL for 48 h, a large amount of red-stained lipid was observed in the cytoplasm, indicating that the THP-1 derived foam cell models were successfully constructed. q-PCR results indicated that miRNA-143 was significantly downregulated in the THP-1-derived foam cell model, whereas CyPA was upregulated (P &lt; 0.05); the dual luciferase reporter gene assay was performed for miRNA-143 and 3'-UTR of CyPA, and the fluorescence intensity of the 3'-UTR vector group was significantly lower than that of the empty vector group (P &lt; 0.05). Fluorescence intensity detection of mutations at the target site showed that it had no effect was produced on the fluorescence intensity. This implied a negative regulatory effect conferred of miRNA-143 on CyPA; As compared to the model group, transfection with miRNA-143 mimics led to a significant downregulation of CyPA mRNA (P &lt; 0.05), which was accompanied by significant downregulation of both MMP-9 mRNA and protein (P &lt; 0.05). MMP-9 was also significantly downregulated upon the addition of the CyPA inhibitor cyclosporine A (P &lt; 0.05).

Abstract 4556: Targeting the TAM receptors on prostate cancer tumor-associated macrophages

Abstract Tumor growth and progression is influenced by the composition of the tumor microenvironment including host stromal cells and immune cells. In prostate cancer, a large proportion of the tumor volume is made up of macrophages, and high macrophage infiltrate correlates with poor prognosis. Macrophages can be polarized to various subtypes including M1 and M2 in response to stimuli in their environment. M1 macrophages have anti-tumor functions such as immune stimulation. M2 macrophages have pro-tumor functions such as promoting angiogenesis, extracellular matrix remodeling and immune suppression. The majority of prostate cancer tumor-associated macrophages exhibit M2-like characteristics. Thus, targeting M2 tumor-associated macrophages is a promising strategy for cancer therapy. M2 macrophages engage in efferocytosis, or phagocytosis of apoptotic cells. This process is an anti-inflammatory, immunosuppressive event that promotes tissue remodeling and tumor growth. The TAM receptors (Tyro3, Axl, MerTK) are a family of receptor tyrosine kinases whose role in efferocytosis has been well described in the literature. The TAM receptors bind phosphatidylserine on apoptotic cells using their ligands Gas6 and Protein S as bridging proteins. Following binding to phosphatidylserine, TAM receptor signaling induces a cytoskeleton rearrangement to engulf the apoptotic cell. Since efferocytosis is a tumor promoting process, we hypothesize targeting the TAM receptors on M2 macrophages may be a beneficial anti-cancer strategy. We are characterizing TAM receptor expression on M1 and M2 macrophages using two complementary in vitro methods: macrophage polarization of monocytes derived from healthy human donors and of the acute monocytic leukemia cell line THP-1. Cell surface markers of M1 macrophages (CD86) and M2 macrophages (CD163 and CD206) were measured to assess polarization by flow cytometry. We are currently further confirming the degree of polarization by measuring cytokine secretion and mRNA expression of M1- and M2-associated genes. We used flow cytometry analysis to assess TAM receptor expression on the in vitro polarized M1 and M2 macrophages. In the human monocyte derived model, we detected higher levels of expression of MerTK and Tyro3 on M2 macrophages in several biological replicates. We also found that M2 THP-1 macrophages express higher levels of MerTK and Tyro3 than M1 THP-1 macrophages, consistent with macrophages from the monocyte derived model. In conclusion, these data support the TAM receptors as a relevant therapeutic target for inhibiting M2 pro-tumor functions. Further work by qRT-PCR, western blot and immunofluorescence staining will be used to confirm higher levels of the MerTK and Tyro3 on M2 macrophages and further investigate expression of Axl on M1 and M2 macrophages. Citation Format: Kayla V. Myers, Kenneth J. Pienta. Targeting the TAM receptors on prostate cancer tumor-associated macrophages [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4556.

Pd(II) complexes with N-heteroaromatic hydrazone ligands: Anticancer activity, in silico and experimental target identification

Anticancer activity of Pd complexes 1–5 with bidentate N-heteroaromatic hydrazone ligands was investigated on human acute monocytic leukemia (THP-1; cells in a suspension) and human mammary adenocarcinoma (MCF-7; two-dimensional layer and three-dimensional spheroid tumor model) cell lines. For the Pd(II) complexes with condensation products of ethyl hydrazainoacetate and quinoline-8-carboxaldehyde (complex 1) and 2-formylpyridine (complex 3), for which apoptosis was determined as a mechanism of anticancer activity, further investigation revealed that they arrest the cell cycle in G0/G1 phase, induce generation of reactive oxygen species and inhibit Topoisomerase I in vitro. In silico studies corroborate experimental findings that these complexes show topoisomerase inhibition activity in the micromolar range and indicate binding to a DNA's minor groove as another potential target. Based on the results obtained by circular dichroism and fluorescence spectroscopy measurements, the most active complexes are suitable to be delivered to a blood stream via human serum albumin.

Curcumin can reverse the impairment of phagocytic function of macrophages induced by exosomes from patients with systemic lupus erythematosus

Objective To evaluate the effect of curcumin on phagocytic function of macrophages after the stimulation with exosomes from patients with systemic lupus erythematosus (SLE) . Methods From February 2016 to November 2017, 18 patients with active SLE and 18 healthy controls were enrolled from Guangzhou Institute of Dermatology and Sun Yat-sen Memorial Hospital, Sun Yat-sen University, and serum exosomes were extracted from these subjects. The exosomes from the healthy controls (CON-exo) and SLE patients (SLE-exo) as well as different concentrations (1, 5, 20 μmol/L) of curcumin were used alone or in combination to stimulate the acute monocytic leukemia cell line THP-1-derived macrophages. After coincubation of the stimulated macrophages with pHrodo™ Red indicator, flow cytometry was performed to determine the average fluorescence intensity, immunofluorescence staining to calculate the proportion of pHrodo™ Red-positive macrophages, and then the phagocytic activity of macrophages was assessed. Western blot analysis was conducted to determine the protein expression of CD14 in the stimulated macrophages. Statistical analysis was carried out by Student-t test for comparison between two groups, one-way analysis of variance for comparison among several groups, and least significant difference (LSD) -t test for multiple comparisons. Results Flow cytometry showed that there were significant differences in the relative fluorescence intensity of macrophages among the CON-exo group, CON-exo + 20 μmol/L curcumin group, SLE-exo group and SLE-exo + 20 μmol/L curcumin group (101.3% ± 14.05%, 94.27% ± 14.13%, 41.02% ± 9.54% and 87.33% ± 15.01%, respectively; F = 10.81, P < 0.01) , and immunofluorescence staining revealed that the proportion of pHrodo Red-positive macrophages significantly differed among the above 4 groups (82.16% ± 5.20%, 81.33% ± 4.51%, 54.20% ± 9.31% and 71.23% ± 5.43% respectively; F = 12.42, P < 0.01) . The fluorescence intensity of macrophages and proportion of pHrodo-positive macrophages were both significantly lower in the SLE-exo group than in the CON-exo group (t = 5.26, 5.35 respectively, both P < 0.01) and SLE-exo + 20 μmol/L curcumin group (t = 3.97, 3.26 respectively, both P < 0.05) . Western blot analysis showed that there were significant differences in the protein expression of CD14 among the CON-exo group, SLE-exo group, SLE-exo + 1 μmol/L curcumin group, SLE-exo + 5 μmol/L curcumin group and SLE-exo + 20 μmol/L curcumin group (96.33% ± 13.65%, 30.67% ± 5.86%, 45.24% ± 8.89%, 72.81% ± 6.62% and 90.67% ± 12.66% respectively; F = 24.57, P < 0.01) . The protein expression of CD14 was significantly lower in the SLE-exo group than in the CON-exo group (t = 8.06, P < 0.001) , SLE-exo + 5 μmol/L curcumin group and SLE-exo + 20 μmol/L curcumin group (t = 5.08, 7.38, both P < 0.001) , and the CD14 level showed an increasing trend along with the increase in the concentration of curcumin. Conclusion Exosomes from SLE patients markedly inhibit the phagocytosis of macrophages, while curcumin can reverse this inhibitory effect. Key words: Lupus erythematosus, systemic; Exosomes; Curcumin; Macrophages; Cytophagocytosis

Red blood cells participate in reverse cholesterol transport by mediating cholesterol efflux of high-density lipoprotein and apolipoprotein A-I from THP-1 macrophages

High-density lipoprotein (HDL) plays a main role in reverse cholesterol transport (RCT), one of the most important functions for preventing atherosclerosis. Recent reports have shown that red blood cells (RBCs) can be associated with RCT, an interaction facilitated by albumin. However, the RCT function of RBCs has not been thoroughly elucidated. In this study, the RCT function of RBCs was assessed using cholesterol efflux capacity (CEC) assays, in which [3H]-labeled cholesterol-loaded human acute monocytic leukemia (THP-1) macrophages were incubated with RBCs as a cholesterol acceptor in the presence or absence of HDL or its main component protein apolipoprotein A-I (apoA-I). The CEC of RBCs was found to be dose dependent, enabling uptake of cholesterol from THP-1 macrophages through apoA-I and HDL, and directly from apoA-I and HDL in medium without the presence THP-1 macrophages. Moreover, RBCs could exchange cholesterol with HDL in a bidirectional manner but could only exchange cholesterol with apoA-I in a single direction. Although albumin promoted the movement of cholesterol, synergistic effects were not observed for both apoA-I and HDL, in contrast to previous findings. These results strongly suggested that RBCs may play important roles in RCT by mediating cholesterol efflux as temporary cholesterol storage.

Characterizing the molecular cytogenetics in acute monocytic leukemia

To characterize the molecular genetics of 81 patients with acute monocytic leukemia (AML). Fluorescence in situ hybridization (FISH) was employed to detect MLL gene rearrangements. Combined mutations of 17 genes were detected by DNA-based PCR and Sanger sequencing. Sixty seven patients were found to harbor at least one mutation. The most commonly mutated gene was NPM1 (n=18), which was followed by FLT3-ITD (n=16), NRAS (n=16), DNMT3A (n=15), TET2 (n=12), RUNX1 (n=11) and KRAS (n=9). Based on the functions of mutated genes, the most frequently involved genes were those involved in DNA methylation (38.27%), tyrosine kinase receptor signaling (32.1%), transcription regulation (28.4%), and RAS pathway (24.7%). Single gene mutation predominated in patient with cytogenetic abnormalities, while coexistence of 2 mutations have predominated in patient with normal cytogenetic findings. Stratified by cytogenetic findings, patients with single gene mutations (intermediate-risk group) had significantly higher complete remission (CR) rates than those with ≥2 gene mutations (unfavorable-risk group) (91.7% vs. 57.6% , 87.5% vs. 25.0%, P =0.0319, 0.0117, respectively). Over 80% of AML patients were found to harbor at least one mutation. Their clinical phenotype and prognosis may be impacted by the synergy of MLL gene rearrangement and multiple mutations. For patients under the same risk stratification, the number of mutations is reversely correlated with the CR rate.

Cloning of New Antigen Gene MLAA-34 Promoter and Identification of Core Region in Acute Monocytic Leukemia

To clone the promoter sequence of acute monocytic leukemia new antigen gene.MLAA-34 and identify its promoter core region. The full-length fragment of MLAA-34 gene promoter region was amplified by PCR, then was ligated into pGL3-Basic vector, and the recombinant plasmid was cloned. Constructed a series of MLAA-34 gene promoter 5' flanking region truncated plasmid. These recombinant plasmids were transfected into U937 and HEK293 cells, and the dual luciferase reporter gene was used to detect the promoter activity of each fragment to determine the minimum active region. Transcription factor binding sites were analyzed by bioinformatics methods. The recombinant plasmid containing MLAA-34 promoter sequence and its truncated plasmid were successfully constructed, and the promoter activity was significantly increased as compared with the empty vector (P＜0.001). The minimal active region of MLAA-34 located between 402 bp and 200 bp. It contained multiple transcription factor binding sites such as E2F1, MZF-1, SP1, USF2 and STAT3. The promoter of luciferase reporter gene has been successfully constructed with different deletion fragments of MLAA-34, and its core promoter region may contain multiple transcription factor sequence.

Effects of enhancer of Zeste homolog 2 inhibitor GSK126 on the cell proliferation and apoptosis of acute leukemia and lymphoma cells in vitro

Objective To investigate the effects of a novel enhancer of Zeste homolog 2 (EZH2) inhibitor GSK126 in vitro on the cell proliferation and apoptosis in acute leukemia and lymphoma cells. Methods CCK-8 assay was used to measure the effects of GSK126 with different concentrations and time on the cell proliferation in human T-cell acute lymphoblastic leukemia (T-ALL) CEM cell line, acute monocytic leukemia U937 cell line and Burkitt lymphoma Raji cell line. Annexin V/PI double staining method was used to determine the effects of GSK126 on the cell apoptosis. The effect of GSK126 on the mRNA levels of EZH2, bcl-2 and bcl-xL were measured by using quantitative polymerase chain reaction (qPCR). Results After the function for 24 h, 48 h and 72 h, compared to the negative control group (0 μmol/L), 5, 10, 15, 20, 25 μmol/L GSK126 treatment showed a significant dose-and time-dependent cell proliferation suppression in CEM cells; 5, 10, 15, 20 μmol/L GSK126 treatment showed a significant dose-and time-dependent cell proliferation suppression in U937 cells; 5, 10, 15, 20, 25, 30 μmol/L GSK126 treatment showed a significant time-dependent suppression in Raji cells (all P < 0.05). The 48 h 50% inhibitory concentration (IC50) of GSK126 on CEM, U937, Raji cells was (13.46±0.83), (11.65±1.02), (15.00±0.19) μmol/L, respectively. GSK126 treatment with the doses of 8, 12 and 16 μmol/L promoted the apoptosis of CEM and U937 cells compared with the negative control group (0 μmol/L), and there were statistical differences in the apoptosis rate (F = 167.995, P < 0.01; F = 158.400, P < 0.01). In Raji cells, only 16 μmol/L GSK126 treatment had a higher cell apoptosis rate compared with the control group (t = 47.998, P < 0.05). Furthermore, 8, 12 and 16 μmol/L GSK126 treatment suppressed the expressions of EZH2 mRNA in CEM, U937 and Raji cells compared with the control group (F = 82.035, P < 0.01; F = 252.712, P < 0.01; F = 690.536, P < 0.01), and the expressions of bcl-2 and bcl-xL mRNA (bcl-2: F = 1 900.525, P < 0.01; F = 431.324, P < 0.01; F = 216.184, P < 0.01; bcl-xL: F = 256.751, P < 0.01; F = 147.019, P < 0.01; F = 209.325, P < 0.01). Conclusion EZH2 plays an important role in the occurrence and development of acute leukemia and lymphoma. GSK126 as a high selective inhibitor of EZH2 might be a potential new drug in treatment of hematological malignancies. Key words: Hematologic neoplasms; Cell proliferation; Apoptosis; Enhancer of Zeste homolog 2; GSK126

Priming with GM-CSF instead of G-CSF enhances CAG-induced apoptosis of acute monocytic leukemia cells in vitro.

High expression of granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor has been found in myelomonocytic or monocytic subtypes (M4/M5) of acute myeloid leukemia. Herein, we aimed to improve the effect of CAG [Ara-C, ACR, and G-CSF (granulocyte colony-stimulating factor)] regimen for acute monocytic leukemia by replacing G-CSF with GM-CSF. Results showed that the percentage of cells in S phase was higher with GM-CSF than with G-CSF treatment at 20ng/mL (P < 0.05). When THP-1 and SHI-1 cells were primed with 20ng/mL G-CSF or GM-CSF followed by Ara-C and ACR, cell proliferation rate in the CAGM (Ara-C, ACR, and GM-CSF) regimen was lower than in the CAG regimen (P < 0.05). Furthermore, CAGM regimen induced more obvious cell apoptosis than CAG regimen probably by reducing Bcl-2/Bax ratio (P < 0.05). Similar results were seen in primary cells from M5 patients. Collectively, our study suggests that priming with GM-CSF may be more effective than G-CSF in CAG regimen in acute monocytic leukemia.

Cytogenetics and associated mutation profile in patients with acute monocytic leukemia.

Cytogenetics and molecular testings for disease classifying and prognosis estimation are becoming routine in clinical practice. However, the molecular characteristics of acute monocytic leukemia (AML-M5) remain unclear. The aim of this study was to investigate the association between karyotypes and gene mutations, especially in AML-M5 patients with 11q23/KMT2A (MLL) rearrangement and normal karyotype. A total of 126 de novo AML-M5 patients were screened for mutations in the 51 genes known or suspected to have a role in myeloid malignancies or in monocytic differentiation using next-generation sequencing (NGS). Chromosome karyotype analysis was performed by R-banding method and further confirmed either by fluorescence in situ hybridization (FISH) and/or by multiple reverse transcription polymerase chain reaction (multiple RT-PCR). Of the 126 patients, one or more mutations were detected in 83.3% patients. FLT3-ITD and NRAS had the highest mutation frequency, followed by NPM1, DNMT3A, TET2, KRAS, and RUNX1. We also identified a significant difference in mutational spectrums between KMT2A-rearranged (KMT2Ar) patients and normal karyotype (NK) patients, as reflected in the average number of gene mutations per patient (1.66 vs 2.46), and in the frequencies of commonly mutated genes (FLT3-ITD: 6% vs 43.5%; NPM1: 0% vs 43.5%; RUNX1: 2.0% vs 15.2%; DNMT3A: 4% vs 26.1%; KRAS: 24.0% vs 4.35%). Patients harboring ≥3 mutations showed much lower complete remission rate than that with double mutations (P=0.043) in high-risk group. There was a significantly different mutation profile between KMT2Ar-patients and NK patients. Our research provided new insight into the molecular characteristics of AML-M5.