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MAbs 2020Neutrophils can release DNA and granular cytoplasmic proteins that form smooth filaments of stacked nucleosomes (NS). These structures, called neutrophil extracellular...
Neutrophils can release DNA and granular cytoplasmic proteins that form smooth filaments of stacked nucleosomes (NS). These structures, called neutrophil extracellular traps (NETs), are involved in multiple pathological processes, and NET formation and removal are clinically significant. The monoclonal antibody 2C5 has strong specificity toward intact NS but not to individual NS components, indicating that 2C5 could potentially target NS in NETs. In this study, NETs were generated using neutrophils and HL-60 cells differentiated into granulocyte-like cells. The specificity of 2C5 toward NETs was evaluated by ELISA, which showed that it binds to NETs with the specificity similar to that for purified nucleohistone substrate. Immunofluorescence showed that 2C5 stains NETs in both static and perfused microfluidic cell cultures, even after NET compaction. Modification of liposomes with 2C5 dramatically enhanced liposome association with NETs. Our results suggest that 2C5 could be used to identify and visualize NETs and serve as a ligand for NET-targeted diagnostics and therapies.
Topics: Animals; Antibodies, Monoclonal, Murine-Derived; Antibody Specificity; Extracellular Traps; HL-60 Cells; Humans; Mice; Mice, Inbred BALB C
PubMed: 33323006
DOI: 10.1080/19420862.2020.1850394 -
European Journal of Cell Biology Jun 2024We analyzed actin cytoskeleton alterations during NET extrusion by neutrophil-like dHL-60 cells and human neutrophils in the absence of DNase1 containing serum to avoid...
We analyzed actin cytoskeleton alterations during NET extrusion by neutrophil-like dHL-60 cells and human neutrophils in the absence of DNase1 containing serum to avoid chromatin degradation and microfilament disassembly. NET-formation by dHL-60 cells and neutrophils was induced by Ionomycin or phorbol-12-myristat-13-acetate (PMA). Subsequent staining with anti-actin and TRITC-phalloidin showed depolymerization of the cortical F-actin at spatially confined areas, the NET extrusion sites, effected by transient activation of the monooxygenase MICAL-1 supported by the G-actin binding proteins cofilin, profilin, thymosin ß4 and probably the F-actin fragmenting activity of gelsolin and/or its fragments, which also decorated the formed NETs. MICAL-1 itself appeared to be proteolyzed by neutrophil elastase possibly to confine its activity to the NET-extrusion area. The F-actin oxidization activity of MICAL-1 is inhibited by Levosimendan leading to reduced NET-formation. Anti-gasdermin-D immunohistochemistry showed a cytoplasmic distribution in non-stimulated cells. After stimulation the NET-extrusion pore displayed reduced anti-gasdermin-D staining but accumulated underneath the plasma membrane of the remaining cell body. A similar distribution was observed for myosin that concentrated together with cortical F-actin along the periphery of the remaining cell body suggesting force production by acto-myosin interactions supporting NET expulsion as indicated by the inhibitory action of the myosin ATPase inhibitor blebbistatin. Isolated human neutrophils displayed differences in their content of certain cytoskeletal proteins. After stimulation neutrophils with high gelsolin content preferentially formed "cloud"-like NETs, whereas those with low or no gelsolin formed long "filamentous" NETs.
Topics: Humans; Extracellular Traps; Neutrophils; Actin Cytoskeleton; HL-60 Cells; Actins; Gelsolin
PubMed: 38555846
DOI: 10.1016/j.ejcb.2024.151407 -
BMJ Case Reports Jan 2024Myeloid sarcoma is a very rare extramedullary malignant tumour, most often associated with acute myeloid leukaemia. We report the case of a man in his early 20s who...
Myeloid sarcoma is a very rare extramedullary malignant tumour, most often associated with acute myeloid leukaemia. We report the case of a man in his early 20s who presented with chronic headache, raised intracranial pressure and progressive vision loss of 2 years duration with no systemic manifestations. He had a history of myeloid sarcoma of the left thigh 15 years ago, treated with external beam radiotherapy and in complete remission for more than 13 years. However, the progressive blindness remained unexplained for 2 years, and he was eventually diagnosed with isolated meningeal relapse without marrow or systemic involvement. Imaging revealed subarachnoid haemorrhage, diffuse leptomeningeal enhancement and involvement of lower dorsal cord and conus, and cerebrospinal fluid cytology showed myeloid blasts. He was managed with intrathecal chemotherapy and craniospinal irradiation, after which he had mild improvement in vision.
Topics: Male; Humans; Sarcoma, Myeloid; Neoplasm Recurrence, Local; Meningeal Neoplasms; Blindness; Granulocyte Precursor Cells
PubMed: 38191225
DOI: 10.1136/bcr-2023-256821 -
Optics Express Jan 2020Optofluidic time-stretch quantitative phase imaging (OTS-QPI) is a powerful tool as it enables high-throughput (>10,000 cell/s) QPI of single live cells. OTS-QPI is...
Optofluidic time-stretch quantitative phase imaging (OTS-QPI) is a powerful tool as it enables high-throughput (>10,000 cell/s) QPI of single live cells. OTS-QPI is based on decoding temporally stretched spectral interferograms that carry the spatial profiles of cells flowing on a microfluidic chip. However, the utility of OTS-QPI is troubled by difficulties in phase retrieval from the high-frequency region of the temporal interferograms, such as phase-unwrapping errors, high instrumentation cost, and large data volume. To overcome these difficulties, we propose and experimentally demonstrate frequency-shifted OTS-QPI by bringing the phase information to the baseband region. Furthermore, to show its boosted utility, we use it to demonstrate image-based classification of leukemia cells with high accuracy over 96% and evaluation of drug-treated leukemia cells via deep learning.
Topics: Antineoplastic Agents; HL-60 Cells; Humans; Imaging, Three-Dimensional; K562 Cells; Leukemia; Microfluidics; Optics and Photonics; Time Factors
PubMed: 32118978
DOI: 10.1364/OE.380679 -
Anticancer Research Apr 2022As part of our continuing investigation in coumarin derivatives as potential anticancer substances, a series of alkylpsoralens were synthesized, and their...
BACKGROUND/AIM
As part of our continuing investigation in coumarin derivatives as potential anticancer substances, a series of alkylpsoralens were synthesized, and their antiproliferative activity was evaluated in leukemic HL60 cells.
MATERIALS AND METHODS
Alkylpsoralens were systematically synthesized from the combination of several chloroketones and 7-hydroxycoumarin derivatives.
RESULTS
Among the compounds synthesized, 4,4',8-trimethylpsoralen demonstrated the most potent activity (IC=6.6 μM).
CONCLUSION
The correlation between the alkylation pattern and antiproliferative activity showed the importance of the C4-methyl and C8-methyl moieties in the psoralen nucleus as well as the importance of lipophilicity for their antiproliferative activity.
Topics: Antineoplastic Agents; Coumarins; HL-60 Cells; Humans
PubMed: 35346996
DOI: 10.21873/anticanres.15654 -
Zhongguo Shi Yan Xue Ye Xue Za Zhi Feb 2024To investigate the effect of expression on the proliferation and the accumulation of intracellular drug of HL60/A and HL60 cells and its possible molecular mechanism.
OBJECTIVE
To investigate the effect of expression on the proliferation and the accumulation of intracellular drug of HL60/A and HL60 cells and its possible molecular mechanism.
METHODS
Lentiviral transfection technology was used to construct HL60/A and HL60 cells with knocked down or overexpressed and their control cells. The efficiency of knockdown and overexpression was evaluated by Western blot. The cell proliferation was detected by CCK-8 assay. The intracellular drug accumulation was detected by laser confocal detection and flow cytometry. The expression levels of MRP1, P-gP and p-AKT were evaluated by flow cytometry and Western blot.
RESULTS
After was knocked down,the proliferation ability of HL60/A cells was significantly reduced, the accumulation of intracellular drug was significantly increased and the expression of MRP1 and P-gP protein were decreased. After was overexpressed, the proliferation ability of HL60 was significantly increased, the accumulation of intracellular drug was significantly decreased and the expression of MRP1 and P-gP protein were increased. Intervention of LY294002 significantly antagonized the promotion on cell proliferation, the inhibition on intracellular drug accumulation and the expression of MRP1 and P-gP mediated by overexpressing in HL60 cells.
CONCLUSION
can promote cell proliferation, improve the expression of MRP1 and P-gP by activating PI3K/AKT signal, and reduce intracellular drug accumulation.
Topics: Humans; HL-60 Cells; Proto-Oncogene Proteins c-akt; Drug Resistance, Neoplasm; Phosphatidylinositol 3-Kinases; Cell Proliferation; Chaperonin Containing TCP-1
PubMed: 38387902
DOI: 10.19746/j.cnki.issn.1009-2137.2024.01.012 -
Nature Communications May 2020Microfluidics by soft lithography has proven to be of key importance for biophysics and life science research. While being based on replicating structures of a master...
Microfluidics by soft lithography has proven to be of key importance for biophysics and life science research. While being based on replicating structures of a master mold using benchtop devices, design modifications are time consuming and require sophisticated cleanroom equipment. Here, we introduce virtual fluidic channels as a flexible and robust alternative to microfluidic devices made by soft lithography. Virtual channels are liquid-bound fluidic systems that can be created in glass cuvettes and tailored in three dimensions within seconds for rheological studies on a wide size range of biological samples. We demonstrate that the liquid-liquid interface imposes a hydrodynamic stress on confined samples, and the resulting strain can be used to calculate rheological parameters from simple linear models. In proof-of-principle experiments, we perform high-throughput rheology inside a flow cytometer cuvette and show the Young's modulus of isolated cells exceeds the one of the corresponding tissue by one order of magnitude.
Topics: Algorithms; Dimethylpolysiloxanes; Elastic Modulus; Equipment Design; Flow Cytometry; HEK293 Cells; HL-60 Cells; Humans; Hydrodynamics; Microfluidic Analytical Techniques; Microfluidics; Models, Theoretical; Polyethylene Glycols; Rheology; Spheroids, Cellular
PubMed: 32366850
DOI: 10.1038/s41467-020-15813-9 -
Journal of Natural Products Feb 2022Harringtonine (HT), produced from species, is known to exhibit potent antiproliferative activity against myeloid leukemia cells by inhibiting protein synthesis. A...
Harringtonine (HT), produced from species, is known to exhibit potent antiproliferative activity against myeloid leukemia cells by inhibiting protein synthesis. A previous study using acute promyelocytic leukemia (HL-60) cells raised the possibility that the C-5' methyl group of HT plays an important role in regulating leukemia cell line antiproliferative activity. In order to investigate the effect of hydrocarbon chains at C-5' on the resultant activity, the C-5' methyl group was replaced with various straight- and branched-chain hydrocarbons using the corresponding alcohols, and their antiproliferative activity against HL-60 and HeLa cells was investigated. As a result, 4'--heptyl-4'-demethylharringtonine (, -heptyl derivative) showed the most potent cytotoxicity among the HT ester derivatives produced, with IC values of 9.4 nM and 0.4 μM for HL-60 and HeLa cells, respectively. Interestingly, the cytotoxicity of derivative against HL-60 and HeLa cells respectively was ∼5 (IC = 50.5 nM) and ∼10 times (IC = 4.0 μM) those of HT and ∼2 (IC = 21.8 nM) and ∼4 times (IC = 1.7 μM) more than homoharringtonine (HHT). These results demonstrate the potential of the derivative as a lead compound against leukemia.
Topics: Esters; HL-60 Cells; Harringtonines; HeLa Cells; Humans; Leukemia, Promyelocytic, Acute
PubMed: 35148094
DOI: 10.1021/acs.jnatprod.1c00888 -
IUBMB Life Jan 2021A little number of current autophagy inhibitors may have beneficial effects on the acute myeloid leukemia (AML) patients. However, there is a strong need to figure out...
A little number of current autophagy inhibitors may have beneficial effects on the acute myeloid leukemia (AML) patients. However, there is a strong need to figure out which settings should be activated or inhibited in autophagy pathway to prevail drug resistance and also to improve current treatment options in leukemia. Therefore, this study aimed to compare the effects of well-known inhibitors of autophagy (as 3-MA, BafA1, and HCQ) in leukemia KG-1 and HL-60 cells exposed to arsenic trioxide (ATO) and/or all-trans retinoic acid (ATRA). Cell proliferation and cytotoxicity of cells were examined by MTT assay. Autophagy was studied by evaluating the development of acidic vesicular organelles, and the autophagosomes formation was investigated by acridine orange staining and transmission electron microscopy. Moreover, the gene and protein expressions levels of autophagy markers (ATGs, p62/SQSTM1, and LC-3B) were also performed by qPCR and western blotting, respectively. The rate of apoptosis and cell cycle were evaluated using flow cytometry. We compared the cytotoxic and apoptotic effects of ATO and/or ATRA in both cell lines and demonstrated that some autophagy markers upregulated in this context. Also, it was shown that autophagy blockers HCQ and/or BafA1 could potentiate the cytotoxic effects of ATO/ATRA, which were more pronounced in KG-1 cells compared to HL-60 cell line. This study showed the involvement of autophagy during the treatment of KG-1 and HL-60 cells by ATO/ATRA. This study proposed that therapy of ATO/ATRA in combination with HCQ can be considered as a more effective strategy for targeting leukemic KG-1 cells.
Topics: Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Arsenic Trioxide; Autophagy; Cell Proliferation; HL-60 Cells; Humans; Leukemia, Myeloid, Acute; Leukemia, Promyelocytic, Acute; Tretinoin; Tumor Cells, Cultured
PubMed: 33205598
DOI: 10.1002/iub.2411 -
Blood Mar 2021Heterozygous de novo missense variants of SRP54 were recently identified in patients with congenital neutropenia (CN) who display symptoms that overlap with...
Heterozygous de novo missense variants of SRP54 were recently identified in patients with congenital neutropenia (CN) who display symptoms that overlap with Shwachman-Diamond syndrome (SDS). Here, we investigate srp54 knockout zebrafish as the first in vivo model of SRP54 deficiency. srp54-/- zebrafish experience embryonic lethality and display multisystemic developmental defects along with severe neutropenia. In contrast, srp54+/- zebrafish are viable, fertile, and show only mild neutropenia. Interestingly, injection of human SRP54 messenger RNAs (mRNAs) that carry mutations observed in patients (T115A, T117Δ, and G226E) aggravated neutropenia and induced pancreatic defects in srp54+/- fish, mimicking the corresponding human clinical phenotypes. These data suggest that the various phenotypes observed in patients may be a result of mutation-specific dominant-negative effects on the functionality of the residual wild-type SRP54 protein. Overexpression of mutated SRP54 also consistently induced neutropenia in wild-type fish and impaired the granulocytic maturation of human promyelocytic HL-60 cells and healthy cord blood-derived CD34+ hematopoietic stem and progenitor cells. Mechanistically, srp54-mutant fish and human cells show impaired unconventional splicing of the transcription factor X-box binding protein 1 (Xbp1). Moreover, xbp1 morphants recapitulate phenotypes observed in srp54 deficiency and, importantly, injection of spliced, but not unspliced, xbp1 mRNA rescues neutropenia in srp54+/- zebrafish. Together, these data indicate that SRP54 is critical for the development of various tissues, with neutrophils reacting most sensitively to the loss of SRP54. The heterogenic phenotypes observed in patients that range from mild CN to SDS-like disease may be the result of different dominant-negative effects of mutated SRP54 proteins on downstream XBP1 splicing, which represents a potential therapeutic target.
Topics: Animals; Congenital Bone Marrow Failure Syndromes; Disease Models, Animal; Gene Deletion; Gene Expression Regulation, Developmental; Gene Knockout Techniques; HL-60 Cells; Humans; Models, Molecular; Mutation; Neutropenia; RNA Splicing; RNA, Messenger; Signal Recognition Particle; X-Box Binding Protein 1; Zebrafish; Zebrafish Proteins
PubMed: 33227812
DOI: 10.1182/blood.2020008115