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Journal of Leukocyte Biology Sep 2023Advantages of cloned Hoxb8 neutrophil-like cells are discussed and contrasted with weaknesses of human HL-60 and PLB-985 neutrophil-like cell lines, and shared and...
Advantages of cloned Hoxb8 neutrophil-like cells are discussed and contrasted with weaknesses of human HL-60 and PLB-985 neutrophil-like cell lines, and shared and distinct features of primary murine and human neutrophils are summarized.
Topics: Animals; Mice; Humans; Neutrophils; HL-60 Cells; NADPH Oxidases
PubMed: 37403206
DOI: 10.1093/jleuko/qiad078 -
Leukemia Sep 2023The transcription factor CCAAT-enhancer binding factor alpha (C/ebpα) is a master controller of myeloid differentiation that is expressed as long (p42) and short (p30)...
The transcription factor CCAAT-enhancer binding factor alpha (C/ebpα) is a master controller of myeloid differentiation that is expressed as long (p42) and short (p30) isoform. Mutations within the CEBPA gene selectively deleting p42 are frequent in human acute myeloid leukemia. Here we investigated the individual genomics and transcriptomics of p42 and p30. Both proteins bound to identical sites across the genome. For most targets, they induced a highly similar transcriptional response with the exception of a few isoform specific genes. Amongst those we identified early growth response 1 (Egr1) and tribbles1 (Trib1) as key targets selectively induced by p42 that are also underrepresented in CEBPA-mutated AML. Egr1 executed a program of myeloid differentiation and growth arrest. Oppositely, Trib1 established a negative feedback loop through activation of Erk1/2 kinase thus placing differentiation under control of signaling. Unexpectedly, differentiation elicited either by removal of an oncogenic input or by G-CSF did not peruse C/ebpα as mediator but rather directly affected the cell cycle core by upregulation of p21/p27 inhibitors. This points to functions downstream of C/ebpα as intersection point where transforming and differentiation stimuli converge and this finding offers a new perspective for therapeutic intervention.
Topics: Humans; Granulocyte Precursor Cells; Leukemia, Myeloid, Acute; Cell Differentiation; Protein Isoforms; Mutation; CCAAT-Enhancer-Binding Protein-alpha
PubMed: 37532789
DOI: 10.1038/s41375-023-01989-8 -
Nature Communications Sep 2021Acute myeloid leukemia (AML) remains incurable, largely due to its resistance to conventional treatments. Here, we find that increased abundance of the ubiquitin ligase...
Acute myeloid leukemia (AML) remains incurable, largely due to its resistance to conventional treatments. Here, we find that increased abundance of the ubiquitin ligase RNF5 contributes to AML development and survival. High RNF5 expression in AML patient specimens correlates with poor prognosis. RNF5 inhibition decreases AML cell growth in culture, in patient-derived xenograft (PDX) samples and in vivo, and delays development of MLL-AF9-driven leukemogenesis in mice, prolonging their survival. RNF5 inhibition causes transcriptional changes that overlap with those seen upon histone deacetylase (HDAC)1 inhibition. RNF5 induces the formation of K29 ubiquitin chains on the histone-binding protein RBBP4, promoting its recruitment to and subsequent epigenetic regulation of genes involved in AML maintenance. Correspondingly, RNF5 or RBBP4 knockdown enhances AML cell sensitivity to HDAC inhibitors. Notably, low expression of both RNF5 and HDAC coincides with a favorable prognosis. Our studies identify an ERAD-independent role for RNF5, demonstrating that its control of RBBP4 constitutes an epigenetic pathway that drives AML, and highlight RNF5/RBBP4 as markers useful to stratify patients for treatment with HDAC inhibitors.
Topics: Acute Disease; Animals; Cell Line, Tumor; DNA-Binding Proteins; Female; Genetic Predisposition to Disease; HEK293 Cells; HL-60 Cells; Histone Deacetylase Inhibitors; Humans; K562 Cells; Leukemia, Myeloid; Membrane Proteins; Mice, Inbred C57BL; Mice, Inbred NOD; Mice, Knockout; Mice, SCID; U937 Cells; Ubiquitin-Protein Ligases; Xenograft Model Antitumor Assays; Mice
PubMed: 34518534
DOI: 10.1038/s41467-021-25664-7 -
Frontiers in Immunology 2023T-cell immunoglobulin and mucin domain-3 (TIM-3) is a transmembrane molecule first identified as an immunoregulator. This molecule is also expressed on leukemic cells in...
INTRODUCTION
T-cell immunoglobulin and mucin domain-3 (TIM-3) is a transmembrane molecule first identified as an immunoregulator. This molecule is also expressed on leukemic cells in acute myeloid leukemia and master cell survival and proliferation. In this study, we aimed to explore the effect of TIM-3 interaction with its ligand galectin-9 (Gal-9) on glucose and lipid metabolism in AML cell lines.
METHODS
HL-60 and THP-1 cell lines, representing M3 and M5 AML subtypes, respectively, were cultured under appropriate conditions. The expression of TIM-3 on the cell surface was ascertained by flow cytometric assay. We used real-time PCR to examine the mRNA expression of GLUT-1, HK-2, PFKFB-3, G6PD, ACC-1, ATGL, and CPT-1A; colorimetric assays to measure the concentration of glucose, lactate, GSH, and the enzymatic activity of G6PD; MTT assay to determine cellular proliferation; and gas chromatography-mass spectrometry (GC-MS) to designate FFAs.
RESULTS
We observed the significant upregulated expression of , , , , , and and the enzymatic activity of G6PD in a time-dependent manner in the presence of Gal-9 compared to the PMA and control groups in both HL-60 and THP-1 cell lines ( > 0.05). Moreover, the elevation of extracellular free fatty acids, glucose consumption, lactate release, the concentration of cellular glutathione (GSH) and cell proliferation were significantly higher in the presence of Gal-9 compared to the PMA and control groups in both cell lines (p < 0.05).
CONCLUSION
TIM-3/Gal-9 ligation on AML cell lines results in aerobic glycolysis and altered lipid metabolism and also protects cells from oxidative stress, all in favor of leukemic cell survival and proliferation.
Topics: Humans; Galectins; Hepatitis A Virus Cellular Receptor 2; HL-60 Cells; Lactates; Leukemia, Myeloid, Acute; Lipid Metabolism
PubMed: 38022614
DOI: 10.3389/fimmu.2023.1267578 -
PloS One 2023Tumor-associated neutrophils are found in many types of cancer and are often reported to contribute to negative outcomes. The presence of transforming growth factor-beta...
Tumor-associated neutrophils are found in many types of cancer and are often reported to contribute to negative outcomes. The presence of transforming growth factor-beta (TGF-β) in the tumor microenvironment reportedly contributes to the skewing of neutrophils to a more pro-tumor phenotype. The effects of TGF-β on neutrophil signaling and migration are, however, unclear. We sought to characterize TGF-β signaling in both primary human neutrophils and the neutrophil-like cell line HL-60 and determine whether it directly induces neutrophil migration. We found that TGF-β1 does not induce neutrophil chemotaxis in transwell or underagarose migration assays. TGF-β1 does activate canonical signaling through SMAD3 and noncanonical signaling through ERK1/2 in neutrophils in a time- and dose-dependent manner. Additionally, TGF-β1 present in the tumor-conditioned media (TCM) of invasive breast cancer cells results in SMAD3 activation. We discovered that TCM induces neutrophils to secrete leukotriene B4 (LTB4), which is a lipid mediator important for amplifying the range of neutrophil recruitment. However, TGF-β1 alone does not induce secretion of LTB4. RNA-sequencing revealed that TGF-β1 and TCM alter gene expression in HL-60 cells, including the mRNA levels of the pro-tumor oncostatin M (OSM) and vascular endothelial growth factor A (VEGFA). These new insights into the role and impact of TGF-β1 on neutrophil signaling, migration, and gene expression have significant implications in the understanding of the changes in neutrophils that occur in the tumor microenvironment.
Topics: Humans; Transforming Growth Factor beta1; Neutrophils; Vascular Endothelial Growth Factor A; Leukotriene B4; Transforming Growth Factor beta; Culture Media, Conditioned; HL-60 Cells; Gene Expression
PubMed: 37682817
DOI: 10.1371/journal.pone.0290886 -
Signal Transduction and Targeted Therapy May 2021Tyrosine kinase inhibitors (TKIs) targeting FLT3 have shown activity but when used alone have achieved limited success in clinical trials, suggesting the need for...
Tyrosine kinase inhibitors (TKIs) targeting FLT3 have shown activity but when used alone have achieved limited success in clinical trials, suggesting the need for combination with other drugs. We investigated the combination of FLT3 TKIs (Gilteritinib or Sorafenib), with Venetoclax, a BCL-2 selective inhibitor (BCL-2i), on FLT3/ITD leukemia cells. The combination of a FLT3 TKI and a BCL-2i synergistically reduced cell proliferation and enhanced apoptosis/cell death in FLT3/ITD cell lines and primary AML samples. Venetoclax also re-sensitized FLT3 TKI-resistant cells to Gilteritinib or Sorafenib treatment, mediated through MAPK pathway inhibition. Gilteritinib treatment alone dissociated BIM from MCL-1 but increased the binding of BIM to BCL-2. Venetoclax treatment enhanced the binding of BIM to MCL-1 but dissociated BIM from BCL-2. Treatment with the drugs together resulted in dissociation of BIM from both BCL-2 and MCL-1, with an increased binding of BIM to the cell death mediator BAX, leading to increased apoptosis. These findings suggest that Venetoclax mitigates the unintended pro-survival effects of FLT3 TKI mainly through the dissociation of BIM and BCL-2 and also decreased BIM expression. This study provides evidence that the addition of BCL-2i enhances the effect of FLT3 TKI therapy in FLT3/ITD AML treatment.
Topics: Bcl-2-Like Protein 11; Bridged Bicyclo Compounds, Heterocyclic; HL-60 Cells; Humans; Leukemia, Myeloid, Acute; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-bcl-2; Sorafenib; Sulfonamides; THP-1 Cells; fms-Like Tyrosine Kinase 3
PubMed: 34024909
DOI: 10.1038/s41392-021-00578-4 -
Journal of Hematology & Oncology Jul 2020MicroRNAs (miRNAs) play important roles in cell proliferation, differentiation, and survival and may be useful for acute myeloid leukemia (AML) diagnosis and prognosis.... (Review)
Review
MicroRNAs (miRNAs) play important roles in cell proliferation, differentiation, and survival and may be useful for acute myeloid leukemia (AML) diagnosis and prognosis. In this study, we defined a novel miRNA, hsa-miR-12462, through small RNA sequencing of the bone marrow (BM) cells from 128 AML patients. Overexpression of hsa-miR-12462 in AML cells (U937 and HL-60) significantly decreased their growth rate when compared with those of the wild-type and MOCK controls. In a xenograft mouse model, tumor weight and size in the mice bearing the U937 cells with hsa-miR-12462 overexpression were significantly reduced when compared with those bearing the mock cells. The AML cells overexpressing hsa-miR-12462 had increased sensitivity to cytarabine chemotherapy. Combining the data from the MiRDB, an online microRNA database ( http://mirdb.org ), with the RNA-sequencing results, SLC9A1 was predicted to be one of the targets of hsa-miR-12462. hsa-miR-12462 was further confirmed to bind exclusively to the 3'UTR of SLC9A1 in U937 cells, leading to downregulation of SLC9A1. In summary, a higher level of hsa-miR-12462 in AML cells is associated with increased sensitivity to cytarabine chemotherapy via downregulation of SLC9A1.
Topics: 3' Untranslated Regions; Animals; Cytarabine; Drug Resistance, Neoplasm; Gene Expression Regulation; HL-60 Cells; Humans; Leukemia, Myeloid, Acute; Mice; Neoplasm Proteins; RNA, Neoplasm; Sodium-Hydrogen Exchanger 1; Tumor Burden; Tumor Cells, Cultured; U937 Cells; Xenograft Model Antitumor Assays
PubMed: 32703317
DOI: 10.1186/s13045-020-00935-w -
International Journal of Molecular... Oct 2022The quantity of aquaporin 5 protein in neutrophil granulocytes is associated with human sepsis-survival. The C-allele of the aquaporin ()-1364A/C polymorphism was shown...
The quantity of aquaporin 5 protein in neutrophil granulocytes is associated with human sepsis-survival. The C-allele of the aquaporin ()-1364A/C polymorphism was shown to be associated with decreased AQP5 expression, which was shown to be relevant in this context leading towards improved outcomes in sepsis. To date, the underlying mechanism of the C-allele-leading to lower AQP5 expression-has been unknown. Knowing the detailed mechanism depicts a crucial step with a target to further interventions. Genotype-dependent regulation of AQP5 expression might be mediated by the epigenetic mechanism of promoter methylation and treatment with epigenetic-drugs could maybe provide benefit. Hence, we tested the hypothesis that promoter methylation differs between genotypes in specific types of immune cells.: promoter methylation was quantified in cells of septic patients and controls by methylation-specific polymerase chain reaction and quantified by a standard curve. In cell-line models, AQP5 expression was analyzed after demethylation to determine the impact of promoter methylation on AQP5 expression. C-allele of -1364 A/C promoter polymorphism is associated with a five-fold increased promoter methylation in neutrophils ( = 0.0055) and a four-fold increase in monocytes ( = 0.0005) and lymphocytes ( = 0.0184) in septic patients and healthy controls as well. In addition, a decreased promoter methylation was accompanied by an increased AQP5 expression in HL-60 ( = 0.0102) and REH cells ( = 0.0102). The C-allele which is associated with lower gene expression in sepsis is accompanied by a higher methylation level of the promoter. Hence, promoter methylation could depict a key mechanism in genotype-dependent expression.
Topics: Aquaporin 5; DNA Methylation; HL-60 Cells; Humans; Promoter Regions, Genetic; Sepsis
PubMed: 36233114
DOI: 10.3390/ijms231911813 -
Cells Oct 2022Studies of induced granulocytic differentiation help to reveal molecular mechanisms of cell maturation. The nuclear proteome represents a rich source of regulatory...
Studies of induced granulocytic differentiation help to reveal molecular mechanisms of cell maturation. The nuclear proteome represents a rich source of regulatory molecules, including transcription factors (TFs). It is important to have an understanding of molecular perturbations at the early stages of the differentiation processes. By applying the proteomic quantitative profiling using isobaric labeling, we found that the contents of 214, 319, 376, 426, and 391 proteins were altered at 3, 6, 9, 12, and 72 h, respectively, compared to 0 h in the HL-60 cell nuclear fraction under all--retinoid acid (ATRA) treatment. From 1860 identified nuclear proteins, 231 proteins were annotated as proteins with transcription factor (TF) activity. Six TFs (RREB1, SRCAP, CCDC124, TRIM24, BRD7, and BUD31) were downregulated and three TFs EWSR1, ENO1, and FUS were upregulated at early time points (3-12 h) after ATRA treatment. Bioinformatic annotation indicates involvement of the HL-60 nuclear proteome in DNA damage recognition in the RUNX1-triggered pathway, and in the p53-regulation pathway. By applying scheduled multiple reaction monitoring using stable isotopically labeled peptide standards (MRM/SIS), we found a persistent increase in the content of the following proteins: PRAM1, CEPBP, RBPJ, and HIC1 in the HL-60 cell nuclear fraction during ATRA-induced granulocytic differentiation. In the case of STAT1, CASP3, PARP1, and PRKDC proteins, a transient increase in their content was observed at early time points (3-12 h) after the ATRA treatment. Obtained data on nuclear proteome composition and dynamics during granulocytic differentiation could be beneficial for the development of new treatment approaches for leukemias with the mutated gene.
Topics: Humans; Caspase 3; Cell Cycle Proteins; Cell Differentiation; Chromosomal Proteins, Non-Histone; Core Binding Factor Alpha 2 Subunit; Intracellular Signaling Peptides and Proteins; Leukemia, Promyelocytic, Acute; Nuclear Proteins; Proteome; Proteomics; Tretinoin; Tumor Suppressor Protein p53; HL-60 Cells; Granulocytes; Cell Nucleus
PubMed: 36291090
DOI: 10.3390/cells11203221 -
Scientific Reports Apr 2022Cell migration plays an essential role in wound healing and inflammatory processes inside the human body. Peripheral blood neutrophils, a type of polymorphonuclear...
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.
Topics: Cell Movement; HL-60 Cells; Humans; Inflammation; Neutrophils; Temperature
PubMed: 35488042
DOI: 10.1038/s41598-022-10858-w