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Oncotarget Sep 2015Recent discoveries have led to the testing of novel targeted therapies for the treatment of acute myeloid leukemia (AML). To better inform the results of clinical...
Recent discoveries have led to the testing of novel targeted therapies for the treatment of acute myeloid leukemia (AML). To better inform the results of clinical trials, there is a need to identify and systematically assess biomarkers of response and pharmacodynamic markers of successful target engagement. Spleen tyrosine kinase (SYK) is a candidate therapeutic target in AML. Small-molecule inhibitors of SYK induce AML differentiation and impair leukemia progression in preclinical studies. However, tools to predict response to SYK inhibition and to routinely evaluate SYK activation in primary patient samples have been lacking. In this study we quantified phosphorylated SYK (P-SYK) in AML cell lines and establish that increasing levels of baseline P-SYK are correlated with an increasing sensitivity to small-molecule inhibitors targeting SYK. In addition, we found that pharmacological inhibition of SYK activity extinguishes P-SYK expression as detected by an immunohistochemical (IHC) test. Quantitative analysis of P-SYK expression by the IHC test in a series of 70 primary bone marrow biopsy specimens revealed a spectrum of P-SYK expression across AML cases and that high P-SYK expression is associated with unfavourable outcome independent of age, cytogenetics, and white blood cell count. This study thus establishes P-SYK as a critical biomarker in AML that identifies tumors sensitive to SYK inhibition, identifies an at-risk patient population, and allows for the monitoring of target inhibition during treatment.
Topics: Antineoplastic Agents; Biomarkers, Tumor; Bone Marrow; Bone Marrow Examination; Dose-Response Relationship, Drug; Enzyme Activation; HL-60 Cells; Humans; Immunohistochemistry; Intracellular Signaling Peptides and Proteins; Kaplan-Meier Estimate; Leukemia, Myeloid, Acute; Molecular Targeted Therapy; Phosphorylation; Predictive Value of Tests; Prognosis; Protein Kinase Inhibitors; Protein-Tyrosine Kinases; Risk Factors; Signal Transduction; Syk Kinase; U937 Cells; Up-Regulation
PubMed: 26315286
DOI: 10.18632/oncotarget.4669 -
Cytometry. Part a : the Journal of the... Jul 2012Multiple wavelength operation in a flow cytometer is an exciting way for cell analysis based on both fluorescence and optical scattering processing. For example, this...
Multiple wavelength operation in a flow cytometer is an exciting way for cell analysis based on both fluorescence and optical scattering processing. For example, this multiparametric technique is currently used to differentiate blood cells subpopulations. The choice of excitation wavelengths matching fluorochrome spectra (it is currently the opposite) and the use of a broader range of fluorochromes can be made by taking advantage of a filtered supercontinuum white light source. In this study, we first wished to validate the use of a specific triggered supercontinuum laser in a flow cytometer based on white light scattering and electric sizing on human blood cells. Subsequently, to show the various advantages of this attractive system, using scattering effect, electrical detections, and fluorescence analysis, we realized cells sorting based on DNA/RNA stained by thiazole orange. Discrimination of white blood cells is efficiently demonstrated by using a triggered supercontinuum-based flow cytometer operating in a "one cell-one shot" configuration. The discriminated leukocyte populations are monocytes, lymphocytes, granulocytes, immature granulocytes, and cells having a high RNA content (monoblasts, lymphoblasts, and plasma cells). To the best of our knowledge, these results constitute the first practical demonstration of flow cytometry based on triggered supercontinuum illumination. This study is the starting point of a series of new experiments fully exploiting the spectral features of such a laser source. For example, the large flexibility in the choice of the excitation wavelength allows to use a larger number of fluorochromes and to excite them more efficiently. Moreover, this work opens up new research directions in the biophotonics field, such as the combination of coherent Raman spectroscopy and flow cytometry techniques.
Topics: Benzothiazoles; DNA; Flow Cytometry; Fluorescent Dyes; Humans; Lasers; Leukocyte Count; Leukocytes, Mononuclear; Light; Nucleic Acids; Quinolines; RNA; Scattering, Radiation
PubMed: 22573492
DOI: 10.1002/cyto.a.22065 -
Scientific Reports Jul 2020Loss of estrogens at menopause is a major cause of osteoporosis and increased fracture risk. Estrogens protect against bone loss by decreasing osteoclast number through...
Loss of estrogens at menopause is a major cause of osteoporosis and increased fracture risk. Estrogens protect against bone loss by decreasing osteoclast number through direct actions on cells of the myeloid lineage. Here, we investigated the molecular mechanism of this effect. We report that 17β-estradiol (E) decreased osteoclast number by promoting the apoptosis of early osteoclast progenitors, but not mature osteoclasts. This effect was abrogated in cells lacking Bak/Bax-two pro-apoptotic members of the Bcl-2 family of proteins required for mitochondrial apoptotic death. FasL has been previously implicated in the pro-apoptotic actions of E. However, we show herein that FasL-deficient mice lose bone mass following ovariectomy indistinguishably from FasL-intact controls, indicating that FasL is not a major contributor to the anti-osteoclastogenic actions of estrogens. Instead, using microarray analysis we have elucidated that ERα-mediated estrogen signaling in osteoclast progenitors decreases "oxidative phosphorylation" and the expression of mitochondria complex I genes. Additionally, E decreased the activity of complex I and oxygen consumption rate. Similar to E, the complex I inhibitor Rotenone decreased osteoclastogenesis by promoting osteoclast progenitor apoptosis via Bak/Bax. These findings demonstrate that estrogens decrease osteoclast number by attenuating respiration, and thereby, promoting mitochondrial apoptotic death of early osteoclast progenitors.
Topics: Adenosine Triphosphate; Animals; Apoptosis; Biomarkers; Bone Density; Bone and Bones; Cell Count; Cell Differentiation; Cells, Cultured; Estrogens; Female; Gene Expression Regulation; Mice; Mice, Knockout; Mitochondria; Monocyte-Macrophage Precursor Cells; Osteoclasts; Osteogenesis; Oxidative Phosphorylation; Signal Transduction
PubMed: 32686739
DOI: 10.1038/s41598-020-68890-7 -
Journal of Experimental & Clinical... Nov 2012Epidemiological studies revealed significantly lower mortality rates in cancer patients receiving cardiac glycosides, which turned on interest in the anticancer...
BACKGROUND
Epidemiological studies revealed significantly lower mortality rates in cancer patients receiving cardiac glycosides, which turned on interest in the anticancer properties of these drugs. However, cardiac glycosides have also been shown to stimulate cell growth in several cell types. In the present investigation we analyzed the pro-death and pro-survival properties of ouabain in the human lymphoma derived cell line U937.
METHODS
ROS, intracellular Ca++, cell cycle were evaluated by loading the cells with fluorescent probes under cytofluorimetry. Cell counts and evaluation of trypan blue-excluding cells were performed under optic microscope. Protein detection was done by specific antibodies after protein separation from cellular lysates by SDS-PAGE and transfer blot.
RESULTS
High doses of ouabain cause ROS generation, elevation of [Ca++]i and death of lymphoma derived U937 cells. Lower doses of OUA activate a survival pathway in which plays a role the Na+/Ca++-exchanger (NCX), active in the Ca++ influx mode rather than in the Ca++ efflux mode. Also p38 MAPK plays a pro-survival role. However, the activation of this MAPK does not appear to depend on NCX.
CONCLUSION
This investigation shows that the cardiac glycoside OUA is cytotoxic also for the lymphoma derived cell line U937 and that can activate a survival pathway in which are involved NCX and p38 MAPK. These molecules can represent potential targets of combined therapy.
Topics: Calcium; Cell Death; Cell Survival; Flow Cytometry; Gene Expression Regulation, Neoplastic; Humans; Lymphoma; Ouabain; Reactive Oxygen Species; Sodium-Calcium Exchanger; U937 Cells; p38 Mitogen-Activated Protein Kinases
PubMed: 23153195
DOI: 10.1186/1756-9966-31-95 -
International Journal of Radiation... Aug 2015Radiation-induced heart disease (RIHD) is a delayed effect of radiotherapy for cancers of the chest, such as breast, esophageal, and lung. Kinins are small peptides with...
PURPOSE
Radiation-induced heart disease (RIHD) is a delayed effect of radiotherapy for cancers of the chest, such as breast, esophageal, and lung. Kinins are small peptides with cardioprotective properties. We previously used a rat model that lacks the precursor kininogen to demonstrate that kinins are involved in RIHD. Here, we examined the role of the kinin B2 receptor (B2R) in early radiation-induced signaling in the heart.
MATERIALS AND METHODS
Male Brown Norway rats received the B2R-selective antagonist HOE-140 (icatibant) via osmotic minipump from 5 days before until 4 weeks after 21 Gy local heart irradiation. At 4 weeks, signaling events were measured in left ventricular homogenates and nuclear extracts using western blotting and real-time polymerase chain reaction. Numbers of CD68-positive (monocytes/macrophages), CD2-positive (T-lymphocytes), and mast cells were measured using immunohistochemistry.
RESULTS
Radiation-induced c-Jun phosphorylation and nuclear translocation were enhanced by HOE-140. HOE-140 did not modify endothelial nitric oxide synthase (eNOS) phosphorylation or alter numbers of CD2-positive or mast cells, but enhanced CD68-positive cell counts in irradiated hearts.
CONCLUSIONS
B2R signaling may regulate monocyte/macrophage infiltration and c-Jun signals in the irradiated heart. Although eNOS is a main target for kinins, the B2R may not regulate eNOS phosphorylation in response to radiation.
Topics: Animals; Heart; Heart Diseases; Male; Myocardium; Radiation Dosage; Radiation Injuries; Radiotherapy; Rats; Receptor, Bradykinin B2
PubMed: 25955317
DOI: 10.3109/09553002.2015.1047041 -
Virology May 1999The ability of HIV to match levels of viral mRNA to the activation state of the host cell may play a role in its ability to persist as well as to replicate. This linkage...
The ability of HIV to match levels of viral mRNA to the activation state of the host cell may play a role in its ability to persist as well as to replicate. This linkage depends on the function of the viral transcriptional regulatory protein, Tat, which increases the efficiency of RNA elongation (transcriptional processivity) in response to cellular activation. To quantify levels of Tat function in vivo, a quantitative competitive RT-PCR assay was developed that reflects levels of TAR leader fragments (nonprocessive transcripts) and viral mRNA (processive transcripts), indicating low or high levels of Tat function, respectively. The abundance of these RNA species was measured in peripheral blood mononuclear cells (PBMC) of 22 HIV-1-positive individuals (CD4(+) T cell counts 63-934/mm3) and in established cell line models of HIV constitutive replication (H9IIIB) and reversible latency (U1 and ACH-2). In PBMC, the level of total viral transcripts ranged over four orders of magnitude; however, nonprocessive transcription predominated: 70% of PBMC samples had a ratio of processive to total transcripts of <0.3 and none of the samples had 100% processivity. The cell line studies revealed that, even in activated H9IIIB cells, nonprocessive transcription dominates and that latently infected cells can have different transcriptional responses to activation. This is the first study that enumerates degrees of transcriptional processivity in the circulating mononuclear cell compartment and the results suggest that limitation of Tat function may be a common phenotype throughout the course of the disease.
Topics: Cell Line; Gene Expression Regulation, Viral; Gene Products, tat; HIV-1; Humans; Leukocytes, Mononuclear; Mitogens; RNA Processing, Post-Transcriptional; RNA, Messenger; RNA, Viral; T-Lymphocytes; Transcription, Genetic; U937 Cells; tat Gene Products, Human Immunodeficiency Virus
PubMed: 10329550
DOI: 10.1006/viro.1999.9647 -
Medical Science Monitor : International... Oct 2019BACKGROUND Acute myeloid leukemia (AML) is associated with a high relapse rate and poor prognosis. This study aimed to use weighted gene coexpression network analysis...
Weighted Gene Coexpression Network Analysis Identifies Cysteine-Rich Intestinal Protein 1 (CRIP1) as a Prognostic Gene Associated with Relapse in Patients with Acute Myeloid Leukemia.
BACKGROUND Acute myeloid leukemia (AML) is associated with a high relapse rate and poor prognosis. This study aimed to use weighted gene coexpression network analysis (WGCNA) of gene coexpression networks to identify candidate prognostic biomarker genes in patients with AML and to investigate the expression of these genes in the human U937 cell line in vitro. MATERIAL AND METHODS RNA-seq data were retrieved from the Cancer Genome Atlas (TCGA) and included bone marrow samples and survival data of patients with AML (N=151), patients who did not relapse after treatment (N=119), and patients with relapse (N=40). Differentially expressed genes were identified, WGCNA was used to detect functional modules, and survival analysis was performed. The Cell Counting Kit-8 (CCK-8) assay investigated the proliferation of U937 cells transfected with short hairpin RNAs (shRNAs), shCRIP1, shHIST1H1C, and shHIST1H1E. RNA-seq analysis identified gene expression following CRIP1 knockdown. RESULTS Eighty-two genes were associated with both relapse and prognosis in patients with AML. There were two prognosis-related gene modules in the coexpression network. In the coexpression network, the histone cluster 1 H1 family member gene, HIST1H1C had the maximum relapse fold change, HIST1H1E had the lowest survival p-value, and the cysteine-rich intestinal protein 1 (CRIP1) gene had the most edge numbers and was significantly associated with poor prognosis (P=0.0165786). RNA-seq data showed that there was a significant difference in gene expression after CRIP1 knockdown in U937 cells. CONCLUSIONS WGCNA of gene coexpression networks identified CRIP1 as a potential prognostic biomarker gene in patients with AML.
Topics: Carrier Proteins; Cell Proliferation; China; Computational Biology; Databases, Genetic; Female; Gene Expression Profiling; Gene Regulatory Networks; Humans; LIM Domain Proteins; Leukemia, Myeloid, Acute; Male; Prognosis; RNA, Long Noncoding; RNA, Small Interfering; Recurrence; U937 Cells
PubMed: 31577790
DOI: 10.12659/MSM.918092 -
Ultrasonics Sonochemistry Jul 2016In this study, we report on the potential use of platinum nanoparticles (Pt-NPs), a superoxide dismutase (SOD)/catalase mimetic antioxidant, in combination with 1MHz...
In this study, we report on the potential use of platinum nanoparticles (Pt-NPs), a superoxide dismutase (SOD)/catalase mimetic antioxidant, in combination with 1MHz ultrasound (US) at an intensity of 0.4 W/cm(2), 10% duty factor, 100 Hz PRF, for 2 min. Apoptosis induction was assessed by DNA fragmentation assay, cell cycle analysis and Annexin V-FITC/PI staining. Cell killing was confirmed by cell counting and microscopic examination. The mitochondrial and Ca(2+)-dependent pathways were investigated. Caspase-8 expression and autophagy-related proteins were detected by spectrophotometry and western blot analysis, respectively. Intracellular reactive oxygen species (ROS) elevation was detected by flow cytometry, while extracellular free radical formation was assessed by electron paramagnetic resonance spin trapping spectrometry. The results showed that Pt-NPs exerted differential effects depending on their internalization. Pt-NPs functioned as potent free radical scavengers when added immediately before sonication while pre-treatment with Pt-NPs suppressed the induction of apoptosis as well as autophagy (AP), and resulted in enhanced cell killing. Dead cells displayed the features of pyknosis. The exact mode of cell death is still unclear. In conclusion, the results indicate that US-induced AP may contribute to cell survival post sonication. To our knowledge this is the first study to discuss autophagy as a pro-survival pathway in the context of US. The combination of Pt-NPs and US might be effective in cancer eradication.
Topics: Apoptosis; Humans; Lymphoma; Metal Nanoparticles; Platinum; U937 Cells
PubMed: 26964942
DOI: 10.1016/j.ultsonch.2015.12.013 -
The Journal of Allergy and Clinical... May 2014Although several novel agents are currently in clinical trials for eosinophilic disorders, none has demonstrated efficacy in reducing blood and tissue eosinophilia in... (Clinical Trial)
Clinical Trial
The eosinophil surface receptor epidermal growth factor-like module containing mucin-like hormone receptor 1 (EMR1): a novel therapeutic target for eosinophilic disorders.
BACKGROUND
Although several novel agents are currently in clinical trials for eosinophilic disorders, none has demonstrated efficacy in reducing blood and tissue eosinophilia in all subjects. Additional approaches are clearly needed.
OBJECTIVE
We sought to explore the potential of the human eosinophil surface receptor epidermal growth factor-like module containing mucin-like hormone receptor 1 (EMR1) as a therapeutic target for eosinophilic disorders.
METHODS
EMR1 expression was assessed in blood and bone marrow specimens from eosinophilic and healthy subjects, cell lines, CD34(+) cells differentiated in vitro, and tissue biopsy specimens by using flow cytometry, quantitative PCR, and immunostaining. Eosinophil targeting by a novel, humanized, afucosylated anti-EMR1 IgG1 was evaluated in vitro by using a natural killer cell-mediated killing assay and in vivo in cynomolgus monkeys.
RESULTS
Analysis of blood and bone marrow cells from healthy and eosinophilic donors and in vitro-differentiated CD34(+) cells confirmed restriction of human EMR1 surface and mRNA expression to mature eosinophils. Tissue eosinophils also expressed EMR1. Although EMR1 was highly expressed on eosinophils from all subjects, surface expression was negatively correlated with absolute eosinophil counts (r = -0.46, P < .001), and soluble plasma levels correlated positively with absolute eosinophil counts (r = 0.69, P < .001), suggesting modulation of EMR1 in vivo. Nevertheless, afucosylated anti-EMR1 mAb dramatically enhanced natural killer cell-mediated killing of eosinophils from healthy and eosinophilic donors and induced a rapid and sustained depletion of eosinophils in monkeys.
CONCLUSION
EMR1 expression is restricted to mature blood and tissue eosinophils. Targeting of eosinophils with afucosylated anti-EMR1 antibody shows promise as a treatment for eosinophilic disorders.
Topics: Antibodies, Monoclonal, Murine-Derived; Bone Marrow Cells; CD4-Positive T-Lymphocytes; Calcium-Binding Proteins; Eosinophilia; Eosinophils; Female; Gene Expression Regulation; Humans; Immunoglobulin G; K562 Cells; Male; Membrane Glycoproteins; Mucins; Receptors, G-Protein-Coupled; U937 Cells
PubMed: 24530099
DOI: 10.1016/j.jaci.2013.11.041 -
Haematologica Dec 2014CXC chemokine receptor 4 (CXCR4) is an essential regulator for homing and maintenance of hematopoietic stem cells within the bone marrow niches. Analysis of clinical... (Comparative Study)
Comparative Study
CXC chemokine receptor 4 (CXCR4) is an essential regulator for homing and maintenance of hematopoietic stem cells within the bone marrow niches. Analysis of clinical implications of bone marrow CXCR4 expression in patients with acute myeloid leukemia showed not only higher CXCR4 expression was an independent poor prognostic factor, irrespective of age, white blood cell counts, cytogenetics, and mutation status of NPM1/FLT3-ITD and CEBPA, but also showed CXCR4 expression was inversely associated with mutations of CEBPA, a gene encoding transcription factor C/EBPα. Patients with wild-type CEBPA had significantly higher CXCR4 expression than those with mutated CEBPA. We hypothesized that CEBPA might influence the expression of CXCR4. To test this hypothesis, we first examined endogenous CXCR4 expression in 293T and K562 cells over-expressing wild-type C/EBPα p42 and demonstrated that CXCR4 levels were increased in these cells, whilst the expression of the N-terminal mutant, C/EBPα p30, diminished CXCR4 transcription. We further showed p42 was bound to the CXCR4 promoter by the chromatin immunoprecipitation assays. Induction of p42 in the inducible K562-C/EBPα cell lines increased the chemotactic migration. Moreover, decreased expression of C/EBPα by RNA interference decreased levels of CXCR4 protein expression in U937 cells, thereby abrogating CXCR4-mediated chemotaxis. Our results provide, for the first time, evidence that C/EBPα indeed regulates the activation of CXCR4, which is critical for the homing and engraftment of acute myeloid leukemia cells, while p30 mutant impairs CXCR4 expression.
Topics: Adolescent; Adult; Aged; Aged, 80 and over; Blotting, Western; Bone Marrow; CCAAT-Enhancer-Binding Proteins; Case-Control Studies; Chemotaxis; Cohort Studies; Female; Flow Cytometry; Follow-Up Studies; Gene Expression Regulation, Neoplastic; Hematopoietic Stem Cells; Humans; K562 Cells; Leukemia, Myeloid, Acute; Male; Middle Aged; Mutation; Neoplasm Recurrence, Local; Neoplasm Staging; Nucleophosmin; Prognosis; Promoter Regions, Genetic; RNA, Messenger; RNA, Small Interfering; Real-Time Polymerase Chain Reaction; Receptors, CXCR4; Reverse Transcriptase Polymerase Chain Reaction; Survival Rate; U937 Cells; Young Adult
PubMed: 25193961
DOI: 10.3324/haematol.2014.107821