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Cytometry. Part B, Clinical Cytometry Sep 2021Flow cytometry immunophenotyping (FCIP) can improve diagnosis of myelodysplastic syndromes (MDS) and myelodysplastic/myeloproliferative neoplasms (MDS/MPN), although its...
Exploring blast composition in myelodysplastic syndromes and myelodysplastic/myeloproliferative neoplasms: CD45RA and CD371 improve diagnostic value of flow cytometry through assessment of myeloblast heterogeneity and stem cell aberrancy.
BACKGROUND
Flow cytometry immunophenotyping (FCIP) can improve diagnosis of myelodysplastic syndromes (MDS) and myelodysplastic/myeloproliferative neoplasms (MDS/MPN), although its application is challenging due to difficulties in standardization, complexity of antibody panels and subjective interpretation of data. Since blasts are invariably affected in these disorders, we developed a FCIP approach for detailed and objective analysis of the blast population.
METHODS
FCIP using a one-tube 10-color (13-marker) antibody panel was performed on bone marrow samples from 23 MDS and 8 MDS/MPN patients, 21 cytopenic patients non-diagnostic for MDS (Non-MDS), and 16 Control samples.
RESULTS
MDS and MDS/MPN cases demonstrated one to several immunophenotypic abnormalities including: increased myeloblasts, decreased stage-1 hematogones, aberrant stem cells, abnormal myeloblast heterogeneity/divergence from normal, increased or decreased CD45 intensity, increased CD117 or CD123 intensity, decreased CD38 intensity, and aberrant expression of lineage markers (CD5, CD19, CD56). A Blast score was developed that showed sensitivity of 80.6% and specificity of 90.5% for immunophenotypic diagnosis of MDS and MDS/MPN. Expression levels of CD45RA and CD371 were used to evaluate abnormal myeloblast heterogeneity and stem cell aberrancy. Both these features were, for the first time, incorporated into a scoring system and resulted in 19% increase in the sensitivity of the assay for lower-risk MDS.
CONCLUSION
Deep immunophenotypic analysis of the blast population is valuable for diagnosis of MDS and MDS/MPN and can potentially provide sensitivity and specificity figures comparable to those previously described using more comprehensive panels that assess maturing myelomonocytic and erythroid elements in addition to progenitor cells.
Topics: Aged; Aged, 80 and over; Female; Flow Cytometry; Granulocyte Precursor Cells; Humans; Lectins, C-Type; Leukocyte Common Antigens; Male; Middle Aged; Myelodysplastic Syndromes; Myelodysplastic-Myeloproliferative Diseases; Receptors, Mitogen; Stem Cells
PubMed: 33369070
DOI: 10.1002/cyto.b.21983 -
The Journal of Investigative Dermatology Jul 2024The pathological hallmark of psoriasis is the infiltration of neutrophils into the skin. Some neutrophil-derived microRNAs (miRNAs) serve as biomarkers for various...
The pathological hallmark of psoriasis is the infiltration of neutrophils into the skin. Some neutrophil-derived microRNAs (miRNAs) serve as biomarkers for various diseases, but none have been reported for psoriasis. In this study, we investigated the involvement of miRNAs released from neutrophils in psoriasis pathogenesis. We compared the expression of miRNAs in the sera of patients with psoriasis with that in healthy individuals and found that the expression of 2 miRNAs-miR-223 and miR-1290-was significantly upregulated in the sera of patients with psoriasis. The serum levels of these miRNAs positively correlated with the PASI and CRP levels. We used all-trans retinoic acid to induce the differentiation of human promyelocytic leukemia HL-60 cells into neutrophil-like cells and found that the release of both miRNAs increased during differentiation. Furthermore, the release of miR-1290 was increased by TNF-α in neutrophil-like cells and human neutrophils. Treatment with the miR-1290 precursor promoted the proliferation of human keratinocytes, increased the proportion of S-phase cells, and upregulated the phosphorylation of extracellular signal-regulated kinase 1/2. These results suggest that miR-1290 plays a vital role in regulating neutrophil differentiation and keratinocyte proliferation and could be a serum marker of psoriasis severity.
Topics: Humans; Psoriasis; MicroRNAs; Keratinocytes; Cell Proliferation; Neutrophils; Male; Female; Cell Differentiation; HL-60 Cells; Middle Aged; Adult; Biomarkers; Up-Regulation; Case-Control Studies; Tumor Necrosis Factor-alpha
PubMed: 38157932
DOI: 10.1016/j.jid.2023.10.042 -
Environmental Science and Pollution... Jul 2023Phytol (Pyt), a diterpenoid, possesses many important bioactivities. This study evaluates the anticancer effects of Pyt on sarcoma 180 (S-180) and human leukemia (HL-60)...
Phytol (Pyt), a diterpenoid, possesses many important bioactivities. This study evaluates the anticancer effects of Pyt on sarcoma 180 (S-180) and human leukemia (HL-60) cell lines. For this purpose, cells were treated with Pyt (4.72, 7.08, or 14.16 μM) and a cell viability assay was performed. Additionally, the alkaline comet assay and micronucleus test with cytokinesis were also performed using doxorubicin (6 μM) and hydrogen peroxide (10 mM) as positive controls and stressors, respectively. Results revealed that Pyt significantly reduced the viability and rate of division in S-180 and HL-60 cells with IC values of 18.98 ± 3.79 and 1.17 ± 0.34 μM, respectively. Pyt at 14.16 μM exerted aneugenic and/or clastogenic effects in S-180 and HL-60 cells, where the number of micronuclei and other nuclear abnormalities (e.g., nucleoplasmic bridges and nuclear buds) were frequently observed. Moreover, Pyt at all concentrations induced apoptosis and showed necrosis at 14.16 μM, suggesting its anticancer effects on the tested cancer cell lines. Taken together, Pyt showed promising anticancer effects, possibly through inducing apoptosis and necrosis mechanisms, and it exerted aneugenic and/or clastogenic effects on the S-180 and HL-60 cell lines.
Topics: Animals; Humans; HL-60 Cells; Sarcoma 180; Phytol; Apoptosis; Necrosis; Micronucleus Tests; Sarcoma
PubMed: 37308630
DOI: 10.1007/s11356-023-28036-4 -
Cell Communication and Signaling : CCS Oct 2023Neutrophils depend heavily on glycolysis for energy production under normal conditions. In contrast, neutrophils require energy supplied by mitochondrial oxidative...
LRRK2 is involved in the chemotaxis of neutrophils and differentiated HL-60 cells, and the inhibition of LRRK2 kinase activity increases fMLP-induced chemotactic activity.
BACKGROUND
Neutrophils depend heavily on glycolysis for energy production under normal conditions. In contrast, neutrophils require energy supplied by mitochondrial oxidative phosphorylation (OXPHOS) during chemotaxis. However, the mechanism by which the energy supply changes from glycolysis to OXPHOS remains unknown. Leucine-rich repeat kinase 2 (LRRK2) is partially present in the outer mitochondrial membrane fraction. Lrrk2-deficient cells show mitochondrial fragmentation and reduced OXPHOS activity. We have previously reported that mitofusin (MFN) 2 is involved in chemotaxis and OXPHOS activation upon chemoattractant N-formyl-Met-Leu-Phe (fMLP) stimulation in differentiated HL-60 (dHL-60) cells. It has been previously reported that LRRK2 binds to MFN2 and partially colocalizes with MFN2 at the mitochondrial membranes. This study investigated the involvement of LRRK2 in chemotaxis and MFN2 activation in neutrophils and dHL-60 cells.
METHODS
Lrrk2 knockout neutrophils and Lrrk2 knockdown dHL-60 cells were used to examine the possible involvement of LRRK2 in chemotaxis. Lrrk2 knockdown dHL-60 cells were used a tetracycline-inducible small hairpin RNA (shRNA) system to minimize the effects of LRRK2 knockdown during cell culture. The relationship between LRRK2 and MFN2 was investigated by measuring the GTP-binding activity of MFN2 in Lrrk2 knockdown dHL-60 cells. The effects of LRRK2 kinase activity on chemotaxis were examined using the LRRK2 kinase inhibitor MLi-2.
RESULTS
fMLP-induced chemotactic activity was reduced in Lrrk2 knockout neutrophils in vitro and in vivo. Lrrk2 knockdown in dHL-60 cells expressing Lrrk2 shRNA also reduced fMLP-induced chemotactic activity. Lrrk2 knockdown dHL-60 cells showed reduced OXPHOS activity and suppressed mitochondrial morphological change, similar to Mfn2 knockdown dHL-60 cells. The amount of LRRK2 in the mitochondrial fraction and the GTP-binding activity of MFN2 increased upon fMLP stimulation, and the MFN2 GTP-binding activity was suppressed in Lrrk2 knockdown dHL-60 cells. Furthermore, the kinase activity of LRRK2 and Ser935 phosphorylation of LRRK2 were reduced upon fMLP stimulation, and LRRK2 kinase inhibition by MLi-2 increased the migration to fMLP.
CONCLUSIONS
LRRK2 is involved in neutrophil chemotaxis and the GTP-binding activity of MFN2 upon fMLP stimulation. On the other hand, the kinase activity of LRRK2 shows a negative regulatory effect on fMLP-induced chemotactic activity in dHL-60 cells. Video Abstract.
Topics: Humans; Chemotaxis; Neutrophils; HL-60 Cells; Oxidative Phosphorylation; RNA, Small Interfering; Guanosine Triphosphate; Leucine-Rich Repeat Serine-Threonine Protein Kinase-2
PubMed: 37904222
DOI: 10.1186/s12964-023-01305-y -
Biochemistry and Cell Biology =... Aug 2022The 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...
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.
Topics: Cell Cycle Proteins; Cell Differentiation; Cell Proliferation; HL-60 Cells; Humans; Intracellular Signaling Peptides and Proteins; LIM Domain Proteins; Leukemia, Myeloid, Acute; Muscle Proteins
PubMed: 35916339
DOI: 10.1139/bcb-2021-0507 -
Mitochondrion Nov 2021The weightlessness or microgravity, a physical factor in space, may adversely affect the health of the space travellers or astronauts. The knowledge about the effect of...
The weightlessness or microgravity, a physical factor in space, may adversely affect the health of the space travellers or astronauts. The knowledge about the effect of microgravity on human cancer cells is very limited and poorly understood. Here, we employed rotary cell culture system (RCCS) to induce simulated microgravity (SMG) and examined its effects on human promyelocytic leukemic HL-60 cells. These cells were grown in normal gravity condition (1g) for control purpose. The 72 h exposure of cells to SMG decreased cell proliferation and viability which were accompanied by the reduced expression of PCNA and phosphorylated ERK1/2 and AKT proteins. SMG increased the DNA damage as well as the expression of DNA damage sensing proteins including ATM, ATR, Chk1, Chk2 and γH2A.X. The expression of AP1, XRCC1 and APEX1 regulating BER, XPC regulating NER and MLH1 and PMS2 regulating MMR were downregulated. However, SMG increased the expression of Ku70/80, DNA-PK and Rad51, regulating NHEJ and HR. SMG induced apoptosis and increased the levels of cleaved-poly-(ADP-ribose) polymerase and cleaved-caspase-3. An increase in Bax/Bcl-2 ratio and dissipation of mitochondrial membrane potential were also observed. SMG enhanced reactive oxygen species (ROS) formation which led to the enhanced DNA damage and apoptotic cell death. Overall, SMG induced ROS, DNA damage and differential expression of DNA repair genes, and altered the overall DNA repair capacity which may activate ATM/ATR-Chk1/2 and Ku70/80 and DNA-PK-mediated apoptotic cell death.
Topics: Apoptosis; Ataxia Telangiectasia Mutated Proteins; Cell Proliferation; Cell Survival; Checkpoint Kinase 1; Checkpoint Kinase 2; DNA Damage; HL-60 Cells; Humans; Mitochondria; Reactive Oxygen Species; Weightlessness
PubMed: 34571251
DOI: 10.1016/j.mito.2021.09.006 -
Scientific Reports Jul 2020In this study, we compared the effect of tricarbonyldichlororuthenium (II) dimer (CORM-2) and its CO-depleted molecule (iCORM-2) on human peripheral blood mononuclear...
In this study, we compared the effect of tricarbonyldichlororuthenium (II) dimer (CORM-2) and its CO-depleted molecule (iCORM-2) on human peripheral blood mononuclear cells (PBMCs) and human promyelocytic leukemia HL-60 cells. We determined cell viability, DNA damage and DNA repair kinetics. We also studied the effect of both compounds on DNA oxidative damage, free radical level and HO-1 gene expression. We showed that at low concentrations both CORM-2 and iCORM-2 stimulate PBMCs viability. After 24-h incubation, CORM-2 and iCORM-2, at the concentration of 100 µM, reduce the viability of both PBMCs and HL-60 cells. We also demonstrated that CORM-2 and iCORM-2, in the 0.01-100 µM concentration range, cause DNA damage such as strand breaks and alkaline labile sites. DNA damage was repaired efficiently only in HL-60 cells. CORM-2 significantly reduces oxidative stress induced by 1 mM HO in normal and cancer cells. On the contrary, iCORM-2 in HL-60 cells increases the level of free radicals in the presence of 1 and 5 mM HO. We also revealed that both CORM-2 and iCORM-2 induce HO-1 gene expression. However, CORM-2 induces this gene to a greater extent than iCORM-2, especially in HL-60 cells at 100 µM. Finally, we showed that CORM-2 and iCORM-2 reduce HO-induced DNA oxidative damage. Furthermore, CORM-2 proved to be a compound with stronger antioxidant properties than iCORM-2. Our results suggest that both active CORM-2 and inactive iCORM-2 exert biological effects such as cyto- and genotoxicity, antioxidant properties and the ability to induce the HO-1 gene. The released CO as well as iCORM-2 can be responsible for these effects.
Topics: Antioxidants; Carbon Monoxide; Cell Survival; DNA Damage; DNA Repair; Free Radicals; HL-60 Cells; Heme Oxygenase-1; Humans; Hydrogen Peroxide; Leukocytes, Mononuclear; Organometallic Compounds; Oxidative Stress; Up-Regulation
PubMed: 32699258
DOI: 10.1038/s41598-020-68948-6 -
Journal of Asian Natural Products... May 2021Two new pyrrolizidine alkaloids, sclerwalins A and B ( and ), and one known 9-O-E-hydroxysenecioylretronecine () were first isolated from the seeds of . Their chemical...
Two new pyrrolizidine alkaloids, sclerwalins A and B ( and ), and one known 9-O-E-hydroxysenecioylretronecine () were first isolated from the seeds of . Their chemical structures were elucidated by extensive 1 D NMR and 2 D NMR (HSQC, HMBC, COSY, and ROESY), MS and IR spectra. Cytotoxicities of all isolates were evaluated against five human tumor cell lines (HL-60, A-549, SMMC-7721, MCF-7 and SW480).[Formula: see text].
Topics: Cell Line, Tumor; HL-60 Cells; Humans; Molecular Structure; Pyrrolizidine Alkaloids; Seeds
PubMed: 32228193
DOI: 10.1080/10286020.2020.1740919 -
BMC Cancer May 2023Leukemic cell metabolism plays significant roles in their proliferation and survival. These metabolic adaptations are under regulation by different factors. Programmed...
BACKGROUND
Leukemic cell metabolism plays significant roles in their proliferation and survival. These metabolic adaptations are under regulation by different factors. Programmed Death Ligand -1 (CD-274) is one of the immune checkpoint ligands that do not only cause the immune escape of cancer cells, but also have some intracellular effects in these cells. PD-L1 is overexpressed on leukemic stem cells and relates with poor prognosis of AML. In this study, we investigated effects of PD-L1 stimulation on critical metabolic pathways of glucose and fatty acid metabolisms that have important roles in proliferation and survival of leukemic cells.
METHODS
After confirmation of PD-L1 expression by flow cytometry assay, we used recombinant protein PD-1 for stimulation of the PD-L1 on two AML cell lines, HL-60 and THP-1. Then we examined the effect of PD-L1 stimulation on glucose and fatty acid metabolism in cells at the genomic and metabolomic levels in a time dependent manner. We investigated expression changes of rate limiting enzymes of theses metabolic pathways (G6PD, HK-2, CPT1A, ATGL1 and ACC1) by qRT-PCR and also the relative abundance changes of free fatty acids of medium by GC.
RESULTS
We identified a correlation between PD-L1 stimulation and both fatty acid and glucose metabolism. The PD-L1 stimulated cells showed an influence in the pentose phosphate pathway and glycolysis by increasing expression of G6PD and HK-2 (P value = 0.0001). Furthermore, PD-L1 promoted fatty acid β-oxidation by increasing expression of CPT1A (P value = 0.0001), however, their fatty acid synthesis was decreased by reduction of ACC1 expression (P value = 0.0001).
CONCLUSION
We found that PD-L1 can promote proliferation and survival of AML stem cells probably through some metabolic changes in leukemic cells. Pentose phosphate pathway that has a critical role in cell proliferation and fatty acids β-oxidation that promote cell survival, both are increased by PD-L1 stimulation on AML cells.
Topics: Humans; B7-H1 Antigen; Glucose; Leukemia, Myeloid, Acute; HL-60 Cells; Cell Proliferation
PubMed: 37193972
DOI: 10.1186/s12885-023-10947-7 -
Molecules (Basel, Switzerland) Apr 2021Synthetic and natural ionophores have been developed to catalyze ion transport and have been shown to exhibit a variety of biological effects. We synthesized 24 aza- and...
Synthetic and natural ionophores have been developed to catalyze ion transport and have been shown to exhibit a variety of biological effects. We synthesized 24 aza- and diaza-crown ethers containing adamantyl, adamantylalkyl, aminomethylbenzoyl, and ε-aminocaproyl substituents and analyzed their biological effects in vitro. Ten of the compounds (, -, and ) increased intracellular calcium ([Ca]) in human neutrophils, with the most potent being compound (,'-[2-(1-adamantyl)acetyl]-4,10-diaza-15-crown-5), suggesting that these compounds could alter normal neutrophil [Ca] flux. Indeed, a number of these compounds (i.e., , -, and ) inhibited [Ca] flux in human neutrophils activated by -formyl peptide (MLF). Some of these compounds also inhibited chemotactic peptide-induced [Ca] flux in HL60 cells transfected with N-formyl peptide receptor 1 or 2 (FPR1 or FPR2). In addition, several of the active compounds inhibited neutrophil reactive oxygen species production induced by phorbol 12-myristate 13-acetate (PMA) and neutrophil chemotaxis toward MLF, as both of these processes are highly dependent on regulated [Ca] flux. Quantum chemical calculations were performed on five structure-related diaza-crown ethers and their complexes with Ca, Na, and K to obtain a set of molecular electronic properties and to correlate these properties with biological activity. According to density-functional theory (DFT) modeling, Ca ions were more effectively bound by these compounds versus Na and K. The DFT-optimized structures of the ligand-Ca complexes and quantitative structure-activity relationship (QSAR) analysis showed that the carbonyl oxygen atoms of the ,'-diacylated diaza-crown ethers participated in cation binding and could play an important role in Ca transfer. Thus, our modeling experiments provide a molecular basis to explain at least part of the ionophore mechanism of biological action of aza-crown ethers.
Topics: Aza Compounds; Calcium; Chemotaxis; Crown Ethers; Density Functional Theory; HL-60 Cells; Humans; Ligands; Models, Molecular; Neutrophils; Reactive Oxygen Species; Receptors, Formyl Peptide; Regression Analysis; Static Electricity; Thermodynamics
PubMed: 33921479
DOI: 10.3390/molecules26082225